Sex lethal


Table of contents

Transcriptional Regulation

Repression of Sex-lethal by Extramachrochaete involves the inhibition of the formation of Daughterless/Sisterless-b heterodimers. 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). 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. 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 (Erickson, 1993). SIS-a and SIS-b form functional heterodimers (Hoshijima, 1995).

The X-linked gene runt plays a role in the regulation of Sex lethal. Reduced function of runt results in female-specific lethality and sexual transformation of XX animals that are heterozygous for Sxl. The presence of a loss-of-function runt mutation masculinizes triploid intersexes. In contrast, runt duplications cause a reduction in male viability by ectopic activation of Sex-lethal. Runt is needed for the initial step of Sex-lethal activation, but does not have a major role as an X-counting element (Torres, 1992). scute is another X linked activator of Sxl.

In D. melanogaster, a set of 'X:A numerator genes,' which includes sisterlessA (sisA ), determines sex by controlling the transcription of Sex-lethal (Sxl). sisA was characterized from D. pseudoobscura and D. virilis and the timing of sisA and Sxl expression was studied with single cell-cycle resolution in D. virilis, both to guide structure-function studies of sisA and to help understand sex determination evolution. D. virilis sisA is shown to shares 58% amino acid identity with its D. melanogaster ortholog. The identities confirm sisA as an atypical bZIP transcription factor. Although D. virilis sisA can substitute for D. melanogaster sisA, the protein is not fully functional in a heterologous context. A single copy of a D. virilis sisA expressing chimeric transgene fully rescues females homozygous for the hypomorphic, female-specific lethal allele sisA1; however, the chimeric transgene is not fully effective at rescuing hemizygous sisA1 females, a more stringent test of sisA function (Erickson, 1998).

The protein-coding sequences of sisA display a commonly observed pattern: highly conserved regions separated by completely diverged sequences. Conservation of the bZIP domain confirms the validity of this motif assignment, despite the occurrence of non-canonical residues in key positions. The N-terminal region of the protein was also found to be conserved, notwithstanding the fact that the N-terminal 15 amino acids can be deleted with only modest effects on in vivo function. An intriguing feature of the D. melanogaster protein is a run of six glycine-serine pairs hypothesized to serve as a flexible spacer linking two functional domains. Although this run is not conserved, it occurs in a region that is notable for its variable length and lack of conservation, consistent with such a spacer role. The level of amino acid sequence conservation for SisA protein within the genus (55-68% identity) is comparable to that seen with the Drosophila sex determination proteins SisB (Scute) and Tra-2: less than that for Runt and Sxl (80-90%), and is considerably greater than that for Rra (36%). Thus sisA, like most other sex-signal genes of Drosophila, does not display the remarkably rapid evolution observed for the mammalian sex-signal gene, SRY (Erickson, 1998).

Temporal and spatial features of sisA and Sxl early promoter expression are strikingly conserved. D. virilis sisA transcripts are apparent in nuclear cycle 8. At this point, the nuclei are migrating outward to the cortex. Although differences were noted among nuclei at the time of onset, all nuclei begin to transcribe sisA within one cell cycle. Initially, sisA transcripts are found primarily within nuclei. Unambiguous cytoplasmic signals arise only later during cycle 10. Other Drosophila genes that turn on at a comparably early stage display similar behavior. Nuclei near the posterior pole express lower levels of SISA mRNA than do nuclei elsewhere. All somatic and yolk nuclei continue to express sisA in the subsequent cleavage cycles. In contrast, expression is extinguished in the germline precursors, the pole cells, during cycle 9. Transcripts are readily apparent in prepole cell nuclei as the pole buds form, but disappear midway through bud formation. Transcription of sisA is maintained during the subsequent cycles of the syncytial blastoderm. For somatic nuclei, the level of SISA mRNA peaks in late cycle 12 or early cycle 13. By this point, the sisA regulatory target, SxlPe, has become active. In some embryos, focused dots of sisA hybridization can be seen in the nuclei. Such dots represent nascent transcripts. After cycle 13, nuclear dots disappear, signaling the cessation of sisA transcription. The cytoplasmic hybridization signals begin to disappear from the periphery of the embryo first, but by early to mid cycle 14, the last traces of SISA mRNA have disappeared from all surface nuclei. As had been the case with D. melanogaster, no subsequent somatic or germ-cell transcription of sisA is observed in D. virilis embryos. In D. virilis as in D. melanogaster, sisA transcripts persist in the yolk nuclei until the demise of those nuclei 10 to 12 hours after fertilization. The primary yolk nuclei derive from 70-100 nuclei that fail to migrate with the majority of nuclei toward the periphery of the embryo during cycles 8 and 9. Instead they fall back into the central yolk mass and eventually become polyploid. During cycle 14, sisA transcript levels in these nuclei increase abruptly and remain high thereafter. During normal embryogenesis, some nuclei drop from the periphery into the surface of the yolk mass to become 'secondary' yolk nuclei, perhaps as a consequence of errors in replication or division. Although not highly polyploid, these nuclei also express high levels of sisA. Probable secondary yolk nuclei can be seen in cycle 14 near the periphery and at the anterior pole of the embryo. Their behavior suggests that persistence of sisA transcription is likely to be a consequence of environmental differences between the central yolk mass and the yolk-free periphery, rather than polyploidy per se. In D. melanogaster, the early onset of sisA transcription, the extinction of sisA transcription in pole cells and the abrupt cessation of sisA transcription during nuclear cycle 14 are all reflections of this gene's participation in sex determination through its regulation of Sxl. The persistence of sisA transcription in D. melanogaster yolk nuclei suggests that the gene might also have functions that are not sex-specific. All four of these unusual features have been conserved over the >40 Myr separating D. melanogaster and D. virilis. Expression of sisA and Sxl is as tightly coupled in D. virilis as it is in D. melanogaster (Erickson, 1998).

One aim of this study was to use DNA sequence conservation to identify potential regulatory regions that allow genes to function as X:A numerator elements. The heptomeric sequence CAGGTAG, which is conserved in all three sisA genes examined here, is a promising candidate. It appears in pairs less than 200 bp upstream of the transcription start site, not only for sisA, but also for sisB (scute) and Sxl. These three genes have in common an unusually early onset of expression that allows the embryo to establish X-chromosome dosage compensation by the time general transcription begins. Perhaps this sequence provides for such an early start of transcription. Taken together, these data indicate that the same primary sex determination mechanism exists throughout the genus Drosophila (Erickson, 1998).

Brother and Big brother were isolated as Runt-interacting proteins and are homologous to CBFb, which interacts with the mammalian CBFa Runt-domain proteins. In vitro experiments indicate that Brother family proteins regulate the DNA binding activity of Runt-domain proteins without contacting DNA. Functional interactions between Brother proteins and Runt domain proteins have been demonstrated in Drosophila. A specific point mutation in Runt has been shown to disrupt interaction with Brother proteins but does not affect DNA binding activity. The point mutation was introduced into Runt by a PCR based site-directed mutagenesis. The mutant is dysfunctional in several in vivo assays. Interestingly, this mutant protein acts dominantly to interfere with the Runt-dependent activation of Sex-lethal transcription. To investigate further the requirements for Brother proteins in Drosophila development, an examination was carried out of the effects of expression of a Brother fusion protein homologous to the dominant negative CBFb::SMMHC fusion protein that is associated with leukemia in humans. This Bro::SMMHC fusion protein interferes with the activity of Runt and a second Runt domain protein, Lozenge. The effects of lozenge mutations on eye development are suppressed by expression of wild-type Brother proteins, suggesting that Brother/Big brother dosage is limiting in this developmental context. Results obtained when Runt is expressed in developing eye discs further support this hypothesis. These results firmly establish the importance of the Brother and Big brother proteins for the biological activities of Runt and Lozenge, and further suggest that Brother protein function is not restricted to enhancing DNA-binding (Li, 1999).

Repression of Sex lethalPe expression was observed in female embryos injected with Runt[G163R] mRNA. This dominant negative activity indicates that the Runt[G163R] protein interacts with some other factor(s) in the Drosophila embryos in a manner that interferes with the activity of the wild-type Runt protein. In contrast to this, no dominant negative interference is observed when runt[CK], a Runt derivative that is specifically impaired for DNA-binding, is used in this assay. If the Runt[G163R] protein is interfering by competing for interaction with some other limiting protein factors then Runt[CK] protein would also be expected to behave as a dominant negative. Taken together, these results suggest that DNA binding is required for the dominant negative activity of Runt[G163R]. This is somewhat surprising as the prevailing view, primarily from in vitro experiments, has been that the central function of the Bro/Bgb and CBFbeta proteins is to enhance DNA-binding by the Runt domain proteins. The data in this paper strongly suggest that the Bro proteins have other functions in addition to enhancement of DNA binding by Runt. What then might be the other functions of the Bro/Bgb proteins? One possibility is that Bro induces a conformational change in Runt that is required for transcriptional activation. Runt/Bro complexes induce a bend in DNA that is greater than that observed by the binding of Runt alone. Perhaps DNA-bending is critical for interactions between Runt and other transcription factors on the Sex lethalPe promoter. An alternative possibility is that Bro/Bgb may be a bridge between Runt and other proteins that are critical for transcription regulation. In this model Runt[G163R] would compete for binding to the early promoter region of Sex lethalPe-lacZ but when bound would fail to activate transcription because other Bro-interacting proteins are not recruited. In a two-hybrid screen for Bro-interacting proteins a number of proteins have been identified that appear to be members of the Trithorax group of transcriptional regulators (G. Golling, personal communication to Li, 1999) have been identified. Trithorax group proteins have been implicated as having widespread roles in transcription activation in Drosophila development and it is attractive to speculate that recruitment of such proteins by Runt and Bro contributes to the activation of Sxl transcription. It is clear from the results presented here that interactions between Runt domain proteins and Bro/Bgb/CBFbeta proteins are important for the functions of these conserved transcriptional regulators. Experiments that further address the functions of the Bro/Bgb and CBFbeta proteins will be essential for understanding the mechanisms that account for the pivotal regulatory roles of these proteins in diverse developmental contexts (Li, 1999).

Do Hairy and Runt repress target gene transcription independently of DNA binding, or as promoter bound regulators? Hairy-related transcriptional repressors show similar basic and HLH domains, and all terminate with an identical C-terminal tetrapeptide (WRPW), mutations of which largely or completely abolish repressor activity. It has proved difficult to define the precise molecular mechanism of Hairy action during segmentation. In order to explore the ability of Hairy and Runt to act as promoter-bound transcriptional regulators, heterologous transcriptional activation domains (Act) were substituted for the WRPW repression domain (of Hairy) and the activation domain of Runt and the effects of such substitution were examined on presumed targets of Hairy and Runt. Hairy-Act was used to study sex determination. Ectopic Hairy mimics the activity of Deadpan in repressing early Sex-lethal transcription. Expression of Hairy-Act activates Sxl and causes male lethality, implying that Deadpan recognizes the Sxl promoter directly, and excludes models for Sxl regulation in which DPN functions as a passive repressor (Jiménez, 1996).

The hermaphrodite locus, has both maternal and zygotic functions required for normal female development in Drosophila. Maternal her function is needed for the viability of female offspring, while zygotic her function is needed for female sexual differentiation. Maternal her function is needed early in the hierarchy: genetic interactions of her with the sisterless genes (sis-a and sis-b), with function-specific Sex-lethal alleles suggest that maternal her function is needed for Sxl initiation. When mothers are defective for her function, their daughters fail to activate a reporter gene for the Sxl early promoter and are deficient in SXL protein expression. Dosage compensation is misregulated in the moribund daughters: some salivary gland cells show binding of the Maleless (MLE) dosage compensation regulatory protein to the X chromosome, a binding pattern normally seen only in males. Thus maternal her function is needed early in the hierarchy as a positive regulator of Sxl, and the maternal effects of her on female viability probably reflect Sxl's role in regulating dosage compensation (Pultz, 1995).

Germ cells in embryos derived from nos mutant mothers do not migrate to the primitive gonad and prematurely express several germline-specific markers. These defects have been traced back to the syncytial blastoderm stage. Pole cells in nos minus embryos fail to establish/maintain transcriptional quiescence; the sex determination gene Sex-lethal (Sxl) and the segmentation genes fushi tarazu and even-skipped are ectopically activated in nos minus germ cells. nos minus germ cells are unable to attenuate the cell cycle and instead continue dividing. Unexpectedly, removal of the Sxl gene in the zygote mitigates both the migration and mitotic defects of nos minus germ cells. Supporting the conclusion that Sxl is an important target for nos repression, ectopic, premature expression of Sxl protein in germ cells disrupts migration and stimulates mitotic activity (Deshpande, 1999b).

In wild-type embryos, transcription factors, such as Runt, Sisterless-a, and Scute, are responsible for activating the Sxl establishment promoter, Sxl-Pe. The genes encoding these positive regulators are on the X chromosome, and they are expressed in the early precellular zygote in direct proportion to the number of gene copies. Sufficient quantities of the X-linked activators are produced by 2X/2A nuclei to activate Sxl-Pe, while quantities produced by 1X/2A nuclei are insufficient to activate Sxl-Pe. Pole cells differ from the surrounding soma in that these activators are not expressed at detectable levels in the germline precursors, and Sxl-Pe remains off in both sexes. The failure to express these activators most likely reflects the global downregulation of RNA polymerase II transcription in wild-type pole cells. Thus, one mechanism that might account for the inappropriate activation of Sxl-Pe in nos- pole cells would be a general derepression of somatically active genes. As a consequence, the genes encoding the X-linked activators would be expressed, and these in turn would activate Sxl-Pe. Although it seems reasonable to believe that the ectopic expression of the X-linked activators could contribute to the activation of Sxl-Pe in nos- pole cells, it does not readily explain why Sxl-Pe is turned on not only in 2X but also in 1X pole cells. Moreover, when Scute protein expression was examined in nos- embryos, the level of Scute protein in pole cells was less than that seen in 1X/2A somatic nuclei. For this reason, it is suspected that Sxl-Pe may be activated in nos- pole cells by a mechanism that, at least in part, bypasses the normal regulation of this promoter by the X/A counting system (Deshpande, 1999b).

Although Nos protein is likely to control Sxl-Pe activity by an indirect mechanism, a number of lines of evidence indicate that Sxl is an important nos regulatory target. In wild-type, Sxl proteins are normally not expressed in the germline until after the formation of the primitive gonad, and at this stage expression is restricted to the female germline. As a consequence of the ectopic activation of Sxl-Pe, Sxl proteins are present in nos- pole cells at the blastoderm stage. It would appear that the premature appearance of Sxl proteins in the pole cells is an important contributing factor to the nos- phenotype. The migration and cell cycle defects of nos- germ cells can be alleviated by the elimination of the Sxl gene. Conversely, it is possible to induce both of these defects in wild-type germ cells by ectopically expressing Sxl protein. While the removal of Sxl mitigates some of the defects of nos- germ cells, it should be noted that these cells are still abnormal. They fail to establish/maintain transcriptional quiescence, and they cannot form a functional adult germline. This finding indicates that Sxl is not the only target for nos regulation (Deshpande, 1999b).

Why does ectopic expression of Sxl protein (either in the absence of nos or in the presence of the Sxl transgene) disrupt germ cell migration and induce cell cycle defects? Sxl encodes an RNA-binding protein that functions in the soma as both a splicing and translational regulator. Since the Sxl protein is predominantly localized in the cytoplasm of nos- pole cells, it is imagined that Sxl also functions to regulate the translation of mRNAs encoding proteins critical to migration or cell cycle control. An important goal for future study will be the identification of these Sxl targets (Deshpande, 1999b).

Drosophila sex is determined by the action of the X:A chromosome balance on transcription of Sex-lethal (Sxl), a feminizing switch gene. Loss-of-function mutations in denominator elements of the X:A signal were obtained by selecting for dominant suppressors of a female-specific lethal mutation in the numerator element, sisterlessA (sisA). Ten suppressors were recovered in this extensive genome-wide selection. All were mutations in deadpan, a pleiotropic locus previously discovered to be a denominator element. Detailed genetic and molecular characterization is presented of this diverse set of new dpn alleles including their effects on Sxl. Although selected only for impairment of sex-specific functions, all are also impaired in nonsex-specific functions. Male-lethal effects were anticipated for mutations in a major denominator element, but viability of males lacking dpn function is reduced no more than 50% relative to their dpn- sisters. Moreover, loss of dpn activity in males causes only a modest derepression of the Sxl 'establishment' promoter (Sxlpe), the X:A target. By itself, dpn cannot account for the masculinizing effect of increased autosomal ploidy, the effect that gives rise to the concept of the X:A ratio; nevertheless, if there are other denominator elements, these results suggest that their individual contributions to the sex-determination signal are even less than those of dpn. The time course of expression of dpn and of Sxl in dpn mutant backgrounds suggests that dpn is required for sex determination only during the later stages of X:A signaling in males to prevent inappropriate expression of Sxlpe in the face of increasing sis gene product levels (Barbash, 1996).

Sxl transcriptional regulation: Nanos downregulates transcription and modulates CTD phosphorylation in the soma of early Drosophila embryos

nanos (nos) specifies posterior development in the Drosophila embryo by repressing the translation of maternal hb mRNA. In addition to this somatic function, nos is required in the germline progenitors, the pole cells, to establish transcriptional quiescence. nos has been shown to be required to keep turned off in the germline of both sexes the Sex-lethal establishment promoter, Sxl-Pe. nos also functions to repress Sxl-Pe activity in the surrounding soma. Sxl-Pe is inappropriately activated in the soma of male embryos from nos mothers, while Sxl-Pe can be repressed in female embryos by ectopic Nos protein. nos appears to play a global role in repressing transcription in the soma since the effects of nos on promoter activity are correlated with changes in the phosphorylation status of the carboxy terminal domain (CTD) repeats of the large RNA polymerase II subunit. Finally, evidence is presented indicating that the suppression of transcription in the soma by Nos protein is important for normal embryonic development (Deshpande, 2005).

During the rapid nuclear division cycles in cleavage stage Drosophila embryos, RNA polymerase II transcription is largely shut down and only a few genes are actively transcribed. RNA polymerase II transcription in somatic nuclei is upregulated soon after they migrate to the periphery of the embryo at stage 9 and by nuclear cycle 10 and 11 many of the key segmentation genes are already actively transcribed. While RNA polymerase II activity is substantially augmented when the nuclei reach the periphery of the embryo in the soma, the opposite occurs in the germline pole cell nuclei. When these nuclei migrate into the posterior pole plasm and pole cells are formed, transcription is shut down rather than activated. Previous studies have implicated the posterior determinants nos and pum in establishing/maintaining transcriptional quiescence in pole cells. In embryos derived from mothers mutant for either nos or pum, RNA polymerase II transcription is not properly downregulated in the pole cells and several genes that are normally active only in somatic nuclei are ectopically expressed (Deshpande, 2005).

Since only nos mRNA localized at the posterior pole is translated, pole cells have the highest levels of Nos. However, translation of the localized message generates a Nos gradient that extends to the center of the embryo. An obvious question is whether this Nos gradient also affects RNA polymerase II activity in somatic nuclei. Indeed, transcription of Sxl-Pe is upregulated in somatic nuclei when Nos protein is removed, and is repressed when Nos protein is ectopically expressed. The role of Nos protein in repressing transcription is not restricted to the sex determination pathway since the activity of other promoters also appears to be increased in the absence of nos function (Deshpande, 2005).

Several mechanisms could potentially explain the ectopic activation of Sxl-Pe in the soma and germline of nos mutant embryos. The most obvious is that this promoter is turned-on by maternal Hb expressed in the absence of nos. However, Sxl-Pe is upregulated in nos embryos even when maternal Hb is eliminated. In addition, ectopic expression of Hb from a transgene lacking NREs seemed to repress rather than activate Sxl-Pe. Another possibility is that the zygotic expression of one or more of the X-linked numerators is elevated in nos embryos, upsetting X chromosome to autosome counting. However, since none of the known numerators has a recognizable NRE in the 3′UTR of its message, it seems unlikely that these genes are direct targets for translational repression by Nos protein. In addition, it is not at all clear why numerator genes (which are transcribed in the zygote) would be subject to translational repression by Nos, while autosomal denominator genes such as deadpan (which turns off Sxl-Pe) would not (Deshpande, 2005).

For this reason, the idea is favored that Sxl-Pe is activated in nosm embryos at least in part because RNA polymerase II activity is upregulated. Support for this idea comes from analysis of CTD phosphorylation. When RNA polymerase is transcriptionally engaged the CTD domain is phosphorylated on serine 2 and 5. In wild-type pole cells, phospho-ser2 cannot be detected, while there is only little phospho-ser5. In contrast, phospho-ser2 is found in nosm pole cells, while the level of phospho-ser5 is increased. nos-dependent alterations in CTD phosphorylation are also evident in the soma. When Nos is absent, the level of ser2 and ser5 CTD phosphorylation is elevated, while both types of CTD phosphorylation are reduced by ectopic Nos protein (Deshpande, 2005).

Additional evidence that nos has a global effect on transcription comes from the finding that nos regulates the methylation of histone H3 in the germline of worms and flies. In both organisms, the methylation of histone H3 on lysine 4 (H3meK4) is upregulated in the soma when zygotic transcription commences in early embryogenesis. In contrast, little or no methylation H3 K4 is observed in the transcriptionally quiescent germline. Inhibition of H3 K4 methylation in germ cells requires nos and H3meK4 is markedly upregulated in nos germ cells. In light of these findings, K4 methylation was examined in the soma of nosm embryos. As might be expected from the effects of nos on CTD phosphorylation, somatic H3meK4 is elevated compared to wild type (Deshpande, 2005).

Since phosphorylation of serines 2 and 5 are correlated with transcription, the nos-dependent alterations in CTD phosphorylation are consistent with the idea that nos has a global impact on RNA polymerase II activity. If this is the case, an important question is whether CTD phosphorylation is the cause or the consequence of nos induced changes in the activity of the transcriptional apparatus. Because actively transcribing RNA polymerase has a hyperphosphorylated CTD domain, any mechanism, which leads to a general increase (or decrease) in transcription, would likely alter the level of CTD phosphorylation. This makes it difficult to distinguish between cause and effect. In contrast, besides being a characteristic feature of elongating polymerase, CTD phosphorylation has been linked to the last steps in the initiation process, promoter clearance and the formation of an elongation competent RNA polymerase complex. Moreover, there is growing evidence that these steps in the transcription cycle are subject to regulation (Deshpande, 2005).

The fact that CTD phosphorylation may be a key control point in the transcriptional cycle raises the possibility that nos exerts its effects on polymerase activity by inhibiting the translation of some factor which promotes CTD phosphorylation. In nos mutants, the level of this factor would increase, leading to a general derepression of transcription. Conversely, the level of this factor would decrease by ectopic Nos, reducing overall transcription (Deshpande, 2005).

While the results clearly show that Sxl-Pe is inappropriately turned on in the soma of male embryos and upregulated in the soma of female embryos in the absence of nos activity, it was initially surprising to find that there is usually not a very pronounced posterior-anterior activation gradient. In fact, the smaller Sxl-Pe0.4 kb promoter is clearly activated not only in the posterior but also in the anterior of nos embryos, while the larger Sxl-Pe3.0, usually shows at most only a very shallow posterior–anterior gradient of β-galactosidase expression. Since the Nos gradient does not extend beyond the midpoint of the embryo, and the repressive effects of Nos on hb mRNA translation are restricted to this posterior domain, one might have expected that the activation of Sxl-Pe in would be tightly restricted to the posterior half of nos mutant embryos. However, proteins of average size would be expected to diffuse (in water or even in cytoplasm) through the volume of a fly embryo over a time scale of minutes, and the establishment of gradients like those seen for Nos or Bcd are likely to require special mechanisms including a localize source of product, as well as the sequestration (e.g. nuclear localization) and degradation of the product. If the Nos target for inhibiting general RNA Pol II activity is translated from a uniformly distributed maternal mRNA and is able to equilibrate through the embryo during the time between the onset of the very rapid nuclear divisions and the formation of the cellular blastoderm, only a shallow gradient of this factor in the soma might be expected at any one time in the presence or absence of Nos. In contrast, in pole cells, where repression of this factor by Nos would presumably be required to impose transcriptional quiescence, the formation of the cell membrane would prevent factor synthesized in the soma from influencing polymerase activity. This would enable Nos in the pole cells to reduce the level of this factor below the threshold required for transcriptional activation (Deshpande, 2005).

As observed in many species, establishing transcriptional quiescence in the newly formed pole cells during early embryogenesis is a critical step in the development of the Drosophila germline. However, it is not immediately obvious what role nos mediated down regulation of polymerase activity would have in the development of the soma. Obviously, hyperactivation of Sxl-Pe in nosm embryos could inappropriately switch on the Sxl autoregulatory feedback loop in males. However, analysis of Sxl accumulation in post-blastoderm stages suggests that only very few nosm 1X/2A embryos actually make the wrong choice in sexual identity. There is also little evidence of a sex-bias in adult progeny of nos hb germline clone mothers. The fact that the Sxl autoregulatory loop is usually not activated in male nosm embryos, which are hemizygous for Sxl, is not altogether surprising. In females that have only a single wild type Sxl gene, activation of the autoregulatory loop is severely compromised by conditions which diminish Sxl-Pe activity. Since the amount of Sxl produced by Sxl-Pe in nosm male embryos is much less than that in wild-type females, the autoregulatory loop should be activated infrequently (Deshpande, 2005).

While nosm males largely escape the effects of activating Sxl-Pe, the increased polymerase activity appears to have other consequences. Previous studies have shown that removal of maternal hb suppresses the posterior defects of nosm embryos. However, it has been shown that only about 40% of the hb+/hb embryos from nos hb mothers survive to adults. Likewise, it was found that only 60% of the embryos produced by hb nos mothers hatch as first instar larva, and that an even lower number survive to the adult stage. Taken together, these experiments argue that nos has important functions in the soma besides blocking translation of maternal hb mRNA. It seems possible that the segmentation/developmental defects evident in progeny of hb nos clone mothers could arise from the upregulation of various patterning genes in the absence of nos activity. It is presumed that in wild-type embryos the activity of the transcriptional apparatus and of target zygote promoters is appropriately adjusted to compensate for the repressive effects exerted by Nos. Because the transcriptional apparatus is hyperactivated in the absence of Nos function, this balance is perturbed and many genes are overexpressed (Deshpande, 2005).

Drosophila JAK/STAT pathway reveals distinct initiation and reinforcement steps in early transcription of Sxl

X-linked signal elements (XSEs) communicate the dose of X chromosomes to the regulatory-switch gene Sex-lethal (Sxl) during Drosophila sex determination. Unequal XSE expression in precellular XX and XY nuclei ensures that only XX embryos will activate the establishment promoter, SxlPe, to produce a pulse of the RNA-binding protein, SXL. Once XSE protein concentrations have been assessed, SxlPe is inactivated and the maintenance promoter, SxlPm, is turned on in both sexes; however, only in females is SXL present to direct the SxlPm-derived transcripts to be spliced into functional mRNA. Thereafter, Sxl is maintained in the on state by positive autoregulatory RNA splicing. Once set in the stable on (female) or off (male) state, Sxl controls somatic sexual development through control of downstream effectors of sexual differentiation and dosage compensation. Most XSEs encode transcription factors that bind SxlPe, but the XSE unpaired (upd) encodes a secreted ligand for the JAK/STAT pathway. Although STAT directly regulates SxlPe, it is dispensable for promoter activation. Instead, JAK/STAT is needed to maintain high-level SxlPe expression in order to ensure Sxl autoregulation in XX embryos. Thus, upd is a unique XSE that augments, rather than defines, the initial sex-determination signal (Avila, 2007).

The question of how embryos differentiate between precise 2-fold differences in X-linked signal element (XSE) doses is central to understanding how genetic constitution defines sexual fate. Current X-chromosome-counting models posit that the female fate is set when XSE proteins exceed a threshold concentration and activate SxlPe. The XSE threshold is set by interactions between the XSEs and other proteins in the embryo. Some XSEs interact with maternally supplied proteins to form dose-sensitive transcription factors, such as Scute/Daughterless, that bind SxlPe, but XSE doses are also assessed with reference to maternally and zygotically expressed repressors. Three XSE proteins, SisA, Scute, and Runt, are viewed as acting similarly by binding directly to and activating SxlPe. The fourth XSE, unpaired (upd, also called outstretched or sisC), encodes a secreted ligand that signals through the JAK kinase (hopscotch) to activate the Stat92E transcription factor. Although upd meets the criteria of an XSE, its effects on sex determination are weaker than those of sisA, scute, and runt, and changes in its gene dose have only moderate effects on Sxl. To understand how this comparatively dose-insensitive XSE regulates sex, when and where upd, JAK, and STAT act on the Sxl switch was examined (Avila, 2007).

Using in situ hybridization, the early embryonic expression pattern of upd was defined. No evidence was found for maternally supplied transcripts and it was observed that upd mRNA was first detectable in nuclear cycle 13. The fact that the first upd transcripts are present throughout the embryo, including at the poles, is consistent with the distribution of phosphorylated Stat92E. As cellularization progresses past early cycle 14, the upd pattern resolves into indistinct stripes that developed into a 14 stripe pattern during gastrulation. These results show that upd expression begins later than that of the other XSEs (sisA in cycle 8; scute in cycle 9) and also, paradoxically, that it begins after the onset of transcription of its target, Sxl, in cycle 12 (Avila, 2007).

To understand how upd functions in Sxl activation and how it differs from other XSEs, upd mutations were examined for their effects on SxlPe by using in situ hybridization and on Sxl protein levels by using immunostaining with SXL antibody. Significantly, the RNA probes detected nascent Sxl transcripts, allowing monitoring of both the spatial and temporal responses of SxlPe on a cell-cycle by cell-cycle basis (Avila, 2007).

updsisC1, a loss-of-function mutation that appears to specifically affect sex determination was examined, because it has no observable effect on later upd functions. Consistent with the fact that upd has a modest effect on SxlPe, it was found that two-thirds of homozygous updsisC embryos expressed SxlPe in a manner indistinguishable from that of the wild-type. A small proportion of embryos, 15%, had within their middle sections several clusters of 5-15 nuclei that did not express SxlPe, whereas the remaining 18% had severe defects, with SxlPe expression being absent from most of the central regions of the embryos. Despite early aberrations in SxlPe activity, immunostaining revealed no lasting defect in the expression of SXL, because updsisC1 embryos that reached germband extension stained in a 1:1 male:female ratio. To determine the effects of a complete loss of zygotic upd activity, updYC43, a probable null mutation, and the deficiency Df(1)ue69, which deletes upd and the upd-like gene, upd3, were examined. With respect to SxlPe, it was found that upd-null-mutant females were more severely affected than were updsisC1 embryos. At cellularization, the defects ranged from embryos containing large clusters of nuclei that did not express SxlPe in the central part of embryo to those in which the entire central region failed to express the promoter. The poles, however, expressed SxlPe normally. Immunostainings of updYC43 and Df(1)ue69 embryo collections revealed that these alleles had strong but incompletely penetrant effects on the later distribution of SXL. The fact that an estimated 40% of mutant female embryos stage 6 and older failed to express SXL in their central regions is consistent with the observed defects in SxlPe activity. The remainder eventually expressed normal levels of SXL in all their tissues, indicating that most upd mutant females were able to compensate for reduced SxlPe activity and ultimately engaged autoregulatory Sxl mRNA splicing. Two upd-like genes, upd2 and upd3, map adjacent to upd. Loss of zygotic upd2 had no effect on SxlPe, and the effects of Df(1)ue69 (upd3-,upd-) appeared identical to those of updYC43 when analyzed in a common genetic background. This shows that XSE activity in this region of the X is due to upd alone (Avila, 2007).

Except for the ligands, each component of the JAK/STAT pathway is maternally deposited into the embryo. To eliminate JAK/STAT activity completely, the dominant female-sterile technique was used to generate females lacking maternal hopscotch (hop) or Stat92E, which encode the only JAK kinase and STAT in Drosophila. It was expected that by removing maternal hop, STAT would remain unphosphoryated, allowing a determination of the effects of the loss of the entire pathway on SxlPe (Avila, 2007).

When Sxl expression was examined in cycle 14 embryos derived from hopC111 germline clones, it was found that SxlPe was active in the anterior and posterior regions of the embryos but almost completely inactive in the central region of the embryos. In contrast to the results with upd mutants and deficiencies, all of which exhibited considerable embryo-to-embryo variation, loss of maternal hop had nearly identical effects on SxlPe in every embryo. This more potent effect of maternal hopC111 as compared to upd mutants suggests that zygotic Upd might not be the only activator of JAK in the precellular embryo (Avila, 2007).

The findings with hopC111 were confirmed by using the Stat92E06346 mutation. Cycle 14 embryos derived from Stat92E06346 germline clones also lacked nearly all SxlPe expression in their central regions, but they were even more strongly affected than hopC111 females because SxlPe activity was also reduced in the termini. These findings are contrary to predictions of a linear JAK/STAT pathway going from zygotic Upd through receptor and kinase to activated STAT. Instead, the progressive weakening of SxlPe by removal of upd and Stat92E suggests that there is hop-independent control of Stat92E function in sex determination. The possibility of cross-talk between signaling systems is supported by the finding that the torso receptor-tyrosine-kinase pathway activates STAT92E in the embryo termini (Avila, 2007).

Although the hopC111 and Stat92E06346 mutations had large effects on SxlPe during cycle 14, the period of maximum SxlPe expression, it was found that these mutations had little effect on SxlPe at earlier stages. In wild-type females, SxlPe is first activated in cycle 12. Expression increases throughout cycle 13 and reaches a peak in the first minutes of cycle 14. In embryos from hopC111 mothers, SxlPe was expressed as in the wild-type during cycles 12 and 13. However, upon entry into cycle 14, SxlPe activity ceased in the middle sections of the embryos. A similar phenomenon was observed in embryos carrying strong upd mutants and in those derived from Stat92E06346 germline clones. These results show that JAK/STAT, and thus upd XSE function, is not needed for the initial activation of SxlPe. Instead, upd must function as a different kind of XSE: one dispensable for the initial assessment of X-chromosome dose, but needed to maintain SxlPe activity in the final stage of the X-counting process (Avila, 2007).

When the progeny of hopC111 mutant mothers were examined for Sxl protein, it was found that defects in SxlPe expression led to a permanent failure to express SXL in the central regions in 35% of female embryos. This suggests that the loss of SxlPe activity in cycle 14 can reduce the level of early Sxl to below the threshold normally required to activate autoregulatory mRNA splicing. Although 35% of female embryos were defective for later Sxl expression, most females that completed gastrulation expressed Sxl uniformly. This striking discordance between the effects of hop (and upd and Stat92E) mutants on SxlPe activity and ultimate Sxl levels suggests that stable Sxl autoregulation can be established even when SxlPe function has been seriously compromised. Although some rescuing Sxl mRNA or protein may have diffused from the poles, an alternative explanation is that expression of SxlPe during cycles 12 and 13 might often have provided sufficient Sxl to trigger autoregulation once the maintenance promoter, SxlPm, had been activated (Avila, 2007).

SxlPe is thought to have two main functional elements: a proximal 390 bp X-counting region responsible for sex-specific activation, and a more distal (to -1.4 kb) element that elevates Sxl transcription. Three predicted STAT-binding sites are located in these elements at positions -253, -393, and -428 bp. To test their roles, consensus TTC sequences were changed to TTT because such changes block binding by STAT92E and the mammalian homologs STATs 5 and 5a. In situ hybridizations revealed that the mutation in the proximal STAT site, S1, greatly reduced the number of nuclei expressing SxlPe-lacZ, creating a patchy staining pattern and lower overall mRNA levels. Mutations in S1 and S2, or in all three sites together, caused a strong but variable loss of SxlPe-lacZ expression in most nuclei, resulting in dramatically reduced accumulation of lacZ mRNA. Although the S1, S2, S3 mutant appeared to have a slightly stronger effect than the double mutant, both transgenes exhibited phenotypes reminiscent of those seen in embryos derived from Stat92E06346 germline clones. These results show that STAT92E acts through the consensus binding sites at SxlPe (Avila, 2007).

SxlPe is remarkable for both its rapid response and exquisite sensitivity to X-chromosome dose. In male embryos, it is always off. In female embryos, SxlPe is strongly expressed, but only during a 35-40 min period from mid cycle 12 until about 10-15 min into cycle 14. Given these time constraints, many have assumed that all XSEs would function to establish the initial on or off state of SxlPe. However, it was found that upd behaved very differently than sisA and scute, both of which are required for SxlPe activation and expression. Loss of upd or the JAK/STAT pathway had little or no effect on SxlPe during cycles 12 or 13. Instead, JAK/STAT mutations blocked SxlPe expression late in the process, during cycle 14. This observation is interpreted as revealing that SxlPe is regulated in two mechanistically distinct phases: the first controlling the initial response to X-chromosome dose, and the second acting to maintain or reinforce the initial decision (Avila, 2007).

The relatively late actions of upd and hop offer explanations for several puzzling aspects of upd's function in sex determination. First, upd is considered a weak XSE. This is both because Sxl is comparatively insensitive to upd dose and because loss of upd or JAK/STAT function doesn't eliminate Sxl expression. Both effects are consistent with expectations of a two-step, initiation and maintenance, model for SxlPe function. JAK/STAT mutations would not be expected to eliminate all Sxl function in a two-step model because the STAT-independent initiation step would produce Sxl mRNA and protein. The exact gene dose of upd would not be particularly important for sex because excess active STAT could not induce SxlPe without the prior actions of the initiating XSEs and because even a single dose of upd+ could provide enough active STAT to augment an earlier decision to become female. Thus, the proposed STAT maintenance function explains both the failure of the constitutively active hoptum-l allele to induce ectopic SxlPe expression in males and the ability of hoptum-l to further stimulate SxlPe activity in females. Likewise, the requirement for STAT site S2, located just distal to the 390 bp X-counting region of SxlPe, and the finding that upd is first expressed after Sxl can be explained if STAT's role is to bolster transcription from SxlPe in embryos that already have counted two Xs. Although neither essential for SxlPe expression nor highly dose sensitive, upd, hop, and Stat92E nonetheless play important roles at SxlPe. In their absence, the period of SxlPe activity is cut short, reducing the concentration of Sxl and preventing a large fraction of embryos from engaging the maintenance mode of Sxl expression (Avila, 2007).

How might STAT92E function in a two-step model? One possibility is that STAT might antagonize the late-acting repressor Dpn. Alternatively, the STAT transcription factor might augment, stabilize, or replace earlier-acting XSE activator complexes as their concentrations diminish in cycle 14. BAP60, a core component of the Brahma chromatin-remodeling complex, has been shown to interact with two components of the sex-determination signal. If STAT92E also interacts with the Brahma complex, it might maintain SxlPe chromatin in an active state, facilitating the restoration of transcription after the 13th mitosis (Avila, 2007).

Understanding the commonalities and unique mechanisms STATs employ in their multitude of roles is a fundamental goal of research on this ubiquitous signaling pathway. It is also essential for understanding why the pathway has so often been co-opted into new roles during evolution. STATS seem primarily permissive rather than instructive. They are rarely the primary signals defining cell fate. In these respects, comparison of the even-skipped (eve) stripe 3 enhancer and SxlPe reveals interesting parallels. Both SxlPe and eve stripe 3 are regulated by the balance between several activators and repressors. The responses of both elements to JAK/STAT signaling are extremely rapid, occurring within the dynamic environment of the precellular embryo. Stat92E is important for each, but its roles augment the actions of other factors, rather than being responsible for defining the initiating signals (Avila, 2007).

With respect to the evolution of the sex signal, it has been proposed that a diffusible JAK/STAT signal might have been recruited to allow non linear signal amplification or, alternatively, that a diffusible ligand might render SxlPe less sensitive to random fluctuations in cell-autonomous XSE protein concentrations. Although the weak dose dependence of upd argues against signal amplification, a buffering function is consistent with existing data. These findings suggest another possibility. STAT proteins respond rapidly to a range of regulatory signals; it may be this ability to act within a matter of minutes that brought JAK/STAT into the temporally dynamic X-chromosome-counting process (Avila, 2007).

lilliputian, the sole Drosophila member of the FMR2/AF4 (Fragile X Mental Retardation/Acute Lymphoblastic Leukemia) family of transcription factors, is widely expressed with roles in segmentation, cellularization, and gastrulation during early embryogenesis with additional distinct roles at later stages of embryonic and postembryonic development. This study identified lilli in a genetic screen based on the suppression of a lethal phenotype that is associated with ectopic expression of the transcription factor encoded by the segmentation gene runt in the blastoderm embryo. In contrast to other factors identified by this screen, lilli appears to have no role in mediating either the establishment or maintenance of engrailed (en) repression by Runt. Instead, it was found that Lilli plays a critical role in the Runt-dependent activation of the pair-rule segmentation gene fushi–tarazu (ftz). The requirement for lilli is distinct from and temporally precedes the Runt-dependent activation of ftz that is mediated by the orphan nuclear receptor protein Ftz-F1. A role is described for lilli in the activation of Sex-lethal (Sxl), an early target of Runt in the sex determination pathway. However, lilli is not required for all targets that are activated by Runt and appears to have no role in activation of sloppy paired (slp1). Based on these results it is suggested that Lilli plays an architectural role in facilitating transcriptional activation that depends both on the target gene and the developmental context (Vanderzwan-Butler, 2006).

This study uncovered Lilli's role in Runt-dependent transcriptional regulation based on the identification of lilli mutations as dose-dependent suppressors of the lethality produced by threshold levels of NGT-driven Runt expression. In contrast to all of the other Runt-interacting genes and deficiency intervals identified as suppressors in this genetic screen, a reduction in maternal lilli dosage has no effect on either the establishment or maintenance of Runt-dependent en repression. The target of Runt that provides the most dramatic and clearest evidence for a functional interaction between runt and lilli is the pair-rule gene ftz. It is notable that the ftz expression is not discernibly altered by the relatively low levels of NGT-driven Runt used in the genetic screen. This raises a question regarding the basis for lilli acting as a dose-dependent suppressor of the lethality associated with ectopic Runt expression. One explanation is that there are subtle changes in ftz expression at the threshold levels of NGT-driven Runt used in the viability assays that contribute to lethality. A second possibility is that there are other targets of Runt and Lilli that contribute to the lethality associated with ectopic Runt expression. Although Sxl would seem to be one obvious candidate for such a target, the developmental window for Sxl activation occurs prior to the stage during which the NGT-drivers are useful for manipulating gene expression. Indeed, it has not been possible to detect activation of Sxl by NGT-driven Runt, even at levels that are tenfold higher than the levels used in the genetic screen. Finally, it is possible that the effect on viability is in part due to a non-specific effect of Lilli on GAL4-dependent activation of Runt. There is some evidence for non-specificity, especially with Df(2L)C144; however, there is also a clear suppression of lethality with other lilli alleles that do not show a comparable reduction in NGT-driven UAS-lacZ expression. Thus it seems likely that the identification of lilli is due to a combination of specific and non-specific effects on the lethality produced by NGT-driven Runt expression. If this interpretation is correct, then it also seems likely that other deficiency intervals that were eliminated from further consideration due to apparent non-specific effects may in fact have specific and interesting effects that would be revealed by more directly assaying the effects of these mutations on the responses of different targets to NGT-driven Runt expression (Vanderzwan-Butler, 2006).

These observations confirm and extend findings regarding a role for Lilli in the transcriptional activation of the pair-rule gene ftz (Tang, 2001). Lilli does not appear to have any role in regulating other pair-rule genes, and the effects of eliminating maternal Lilli on segment-polarity gene expression have been interpreted as an indirect effect due to the loss of Ftz (Tang, 2001). Thus ftz appears to stand out as the sole gene in the segmentation pathway that shares a requirement for both Lilli and Runt. The previous work from Tang did identify two other targets for Lilli in the early Drosophila embryo, serendipity-α (sry-α) and huckebein (hkb). There is no evidence that either of these genes is regulated by Runt. Thus, just as there are targets of Runt in the segmentation pathway whose regulation is Lilli-independent, there are also targets of Lilli that do not involve interactions with Runt (Vanderzwan-Butler, 2006).

This work adds Sxl as an additional candidate target for Lilli. The elimination of maternal Lilli interferes with the activation of the SxlPE:lacZ reporter gene in all somatic cells of the female embryo. This global effect stands in contrast to the more localized role of Runt which is only required for Sxl activation in cells within the pre-segmental region of female embryos. The failure in Sxl activation observed in the absence of maternal Lilli is similar to the phenotype of embryos that are mutant for either sisA or sisB. Indeed the possibility is considered that the primary defect in lilli germline clone embryos was the failure to activate either of these two X-chromosome linked numerators. Expression of sisA was found to be normal in lilli germline clone embryos (VanderZwan, 2003). The low level of sisB expression in wild-type embryos made it difficult to unambiguously determine whether lilli was important for sisB activation. To further investigate the role of Lilli in Sxl activation the expression of the SxlGOF:lacZ reporter gene was examined. Both Runt and SisB contribute to the ectopic expression of this reporter in male embryos. The elimination of maternal Lilli has a more severe effect on the expression of the SxlGOF:lacZ reporter than is observed in embryos that are mutant for either runt or sisB (VanderZwan, 2003). The most straightforward interpretation of these results is that Lilli is directly involved in the transcriptional activation of the early embryonic Sxl promoter, in this case in cooperation with the four different X-linked factors that are responsible for the sex-specific expression of Sxl in female embryos (Vanderzwan-Butler, 2006).

The inclusion of Sxl gives four putative direct targets of Lilli in the Drosophila embryo. These four genes, Sxl, ftz, sry-α and hkb are normally activated at very early stages, and in all four cases this activation is reduced, if not abrogated, in the absence of maternally provided lilli. The notion that Lilli functions primarily in activation is consistent with observations on the properties of the mammalian homologs FMR2 and LAF4 (Hillman, 2001). However, early activation is clearly not the sole identifying characteristic of Lilli's targets. Indeed, for three of these targets, there are other genes in the same developmental pathway that are activated at the same time that do not require Lilli. In the cellularization pathway, Lilli is required for expression of sry-α but has no role in the activation of bottleneck (bnk) or nullo (Tang, 2001). In the segmentation pathway, the gap gene hkb is expressed in the anterior and posterior poles in response to signaling by the terminal pathway. Elimination of maternal Lilli greatly reduces hkb expression, but has no obvious effect on tailless (tll), another gap gene that is activated at the same stage in response to the terminal signaling pathway (Tang, 2001). Finally, the requirement for maternally provided Lilli that is observed for ftz is not shared with the pair-rule segmentation genes eve, hairy and runt (Tang, 2001). This last observation is perhaps most important as the wealth of information that exists on pair-rule gene regulation provides a useful framework for further considering the potential attributes of Lilli-dependent targets (Vanderzwan-Butler, 2006).

Elimination of maternal Lilli reduces, but does not eliminate ftz expression. The reduced expression that remains is similar to what is obtained in embryos that lack Runt, and is presumed to be in response to other activating factors. The complications presented by these other factors are bypassed in experiments in which Runt is over-expressed, either by heat-shock or by NGT-driven expression. Indeed, the inability of ftz to respond to ectopic Runt in the absence of maternal Lilli provides very compelling evidence for the importance of Lilli in ftz activation (Vanderzwan-Butler, 2006).

Lilli is acutely required for mediating Runt-dependent activation of ftz during the blastoderm stage, and this requirement precedes the temporal requirements for Ftz-F1. Ftz-F1 was initially identified as a factor that interacts with sequences within the ftz 'zebra element', a 669-bp, promoter proximal element that drives early expression in response to gap and pair-rule gene transcription factors. However, subsequent studies revealed that Ftz-F1 plays a more significant role in mediating Ftz-dependent auto-regulation by the so-called 'upstream element' during the early stages of germ-band extension. The earlier requirement for Lilli strongly suggests it contributes to the early 'zebra element'-dependent activation of ftz in response to activating inputs from Runt (Vanderzwan-Butler, 2006).

What is the role of Lilli in mediating Runt-dependent activation? Directed yeast two-hybrid assays fail to detect direct interactions between the full length Runt and Lilli proteins. This observation suggests that other factors contribute to the functional interactions described above. A notable conserved feature that Lilli shares with its mammalian homologs is an HMG-box. This structural DNA-binding motif interacts with the minor groove of DNA and modulates DNA structure by bending. These properties have been interpreted to reflect an architectural role for HMG-box proteins in facilitating the formation of higher order chromatin structures that contribute to the regulation of gene expression. It seems likely that chromatin architecture is important for ftz zebra element function. Although the 'zebra element' was one of the very first cis-regulatory elements in the segmentation pathway to be described, there is not yet a clear understanding of the rules that govern its activity. This is in contrast to the relatively simple combinatorial rules that have been elucidated for several of the stripe-specific elements of the pair-rule genes eve, hairy, and runt. It is proposed that the difficulty in identifying discrete regulatory modules within the zebra element reflects the importance of chromatin architecture in conferring high-fidelity regulation of the zebra element in response to inputs from Runt and other gap and pair-rule transcription factors (Vanderzwan-Butler, 2006).

It is interesting to note that the cis-regulatory element responsible for the initial activation of Sxl shares several properties with the ftz zebra element. The minimal DNA element necessary to faithfully recapitulate the strong, early sex-specific activation of the Sxl promoter is 1.7 kb in size. As found for the ftz zebra element, smaller reporter gene constructs do not function properly, although sub-elements that confer sex-specific regulation, and augment this activation have been identified. The on/off regulation of Sxl is in response to a twofold difference in the activity of four different DNA-binding transcription factors. It is easy to imagine that chromatin architecture may be critical in sensing this twofold difference in a robust and reproducible manner. There is one further similarity shared by Sxl and ftz that is intriguing. The initial Lilli-dependent activation of both genes is followed by a second phase of gene expression that involves distinct cis-regulatory components. In the case of ftz, the switch is from regulation by the zebra element to regulation by the upstream element, whereas for Sxl the switch is from expression at the SxlPe promoter to expression at SxlM a different promoter that is activated in all somatic cells of both male and female embryos . Perhaps the unique requirements for Lilli reflect architecture-dependent regulatory elements that retain the ability to be rapidly re-organized in a developmentally dynamic manner. Further studies on the mechanisms by which Lilli participates in the activation of ftz and Sxl in the early Drosophila embryo should provide new insights on the role of chromatin architecture in developmentally regulated gene expression (Vanderzwan-Butler, 2006).

Sex lethal is part of the Hedgehog signaling complex

Sex-lethal (Sxl), the Drosophila sex-determination master switch, is on in females and controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. Sxl protein has been shown to be part of the Hh cytoplasmic signaling complex and Hh promotes Sxl nuclear entry (Vied, 2001; Horabin, 2003). In the wing disc anterior compartment, Patched (Ptc), the Hh receptor, acts positively in this process. This study shows that the levels and rate of nuclear entry of full-length Cubitus interruptus (Ci), the Hh signaling target, are enhanced by Sxl. This effect requires the cholesterol but not palmitoyl modification on Hh, and expands the zone of full-length Ci expression. Expansion of Ci activation and its downstream targets, particularly decapentaplegic the Drosophila TGFß homolog, suggests a mechanism for generating different body sizes in the sexes; in Drosophila, females are larger and this difference is controlled by Sxl. Consistent with this proposal, discs expressing ectopic Sxl show an increase in growth. In keeping with the idea of the involvement of a signaling system, this growth effect by Sxl is not cell autonomous. These results have implications for all organisms that are sexually dimorphic and use Hh for patterning (Horabin, 2005) (Horabin, 2005).

Drosophila Hh is synthesized as a 45 kDa precursor that is shortened to a mature form with two lipid modifications; palmitic acid at the N terminus and cholesterol at the C terminus. Maturation involves autoproteolytic processing under the control of the C-terminal domain of Hh. To test whether either of the lipid modifications plays a role in Hh promoted Sxl nuclear entry, female wing discs expressing Hh with only a single modification were examined. HhN encodes the N-terminal region of Hh that is palmitoylated but, because it does not undergo autoproteolytic processing, does not contain the cholesterol moiety. This form of Hh is functional for Ci activation and full-length Ci is detected distantly anterior of the AP boundary. Where HhN levels are maximal, there is a reduction of full-length Ci, most likely from the activation of en, which inhibits Ci transcription. HhN does not increase Sxl nuclear levels, however. The normal high nuclear levels in the posterior compartment and graded nuclear localization in the anterior compartment (Horabin, 2003) are detected, with no change in the cells expressing HhN (Horabin, 2005).

The alternative single modification [cholesterol without palmitoyl (C84S-Hh)], by contrast, is active with respect to Sxl. The dppGAL4 driver was used to drive expression of C84S-Hh. Relative to endogenous Hh, about threefold less of the nuclear export inhibitor Leptomycin B (LMB) was required to detect nuclear Sxl in these discs, suggesting that the nuclear Sxl is effected primarily by the ectopic Hh (Horabin, 2005).

C84S-Hh has been shown to dominantly destabilize Ci, decreasing the expression of Hh target genes. Patterning of the wing is compromised and the size of the region between veins L3 and L4 is reduced. C84S-Hh is also unable to rescue the embryonic segmentation phenotype caused by loss of Hh. C84S-Hh destabilizes Ci, but only in males. Females show the opposite effect, increasing the levels of full-length Ci (Horabin, 2005).

This sex specificity, coupled with the observation that Sxl is present in the Hh cytoplasmic complex, suggests that Sxl may be acting to stabilize Ci on Hh signaling. If this is the case, expressing Sxl in males should increase the levels of full-length Ci. Indeed, male discs expressing Sxl (MS3 isoform), as well as C84S-Hh under the control of dppGAL4, now show higher levels of full-length Ci and the protein is more nuclear, as seen in females. Taken together, these results suggest that when the cholesterol moiety is present on Hh, Sxl enhances the production of full-length Ci (Horabin, 2005).

Curiously, the presence of Sxl does not temper the wing patterning defect caused by the ectopic expression of C84S-Hh; the reported narrowing between wing veins L3 and L4 is the same in the two sexes. The form of Ci that Sxl stabilizes through C84S-Hh must not be the form responsible for Hh patterning (Horabin, 2005).

The data presented here show that when Sxl is present, the Hh signal is augmented. This is seen as an increase in full-length Ci in whole-mount tissue, and in Western blots which give a more quantitative sense of protein levels. In addition to elevating the levels of full-length Ci, several of the Hh downstream targets, including ptc, dpp and some of the downstream targets of Dpp, show an increase in expression. Conversely, removal of Sxl in female cells shows a reduction in the strength of the Hh signal (Horabin, 2005).

Sxl also enhances the nuclear entry rate of Ci, with either endogenous Hh or Hh that has only the cholesterol modification. In females, when Sxl is co-expressed with Hh with only the cholesterol modification, the amount of LMB required to detect nuclear Ci is reduced (by almost sixfold), further supporting the idea that Sxl affects Ci nuclear entry rate on Hh signaling (Horabin, 2005).

Hh enhancement of Sxl nuclear entry also depends on the cholesterol and not the palmitoyl modification. Given that Ci and Sxl are in a complex in the cytoplasm and both respond to the Hh cholesterol modification, it is tempting to speculate, although the data presented does not address this issue, that the two proteins may also enter the nucleus as a complex. This may be the method by which Sxl stabilizes Ci, diverting it from rapid proteolysis, particularly the highly activated form that is functionally detectable but has not been identified biochemically (Horabin, 2005).

Stabilization of full-length Ci by Hh with only the cholesterol modification in females is in contrast to what occurs in males. this form of Hh can destabilize Ci as well as compromise the Hh signal, but only in males. The effect of the cholesterol moiety contrasts with the palmitoyl that potentiates Hh in activating Ci for patterning. This is generally also true in vertebrates, where the cholesterol modification appears to have less of a role in patterning and a more significant role in the release and extracellular transport of the Hh ligand (Horabin, 2005).

In both sexes, ectopic expression of Sxl shows an increase in intensity of ptc expression, indicating it is possible to further elevate the Hh response. Other than en, which was difficult to score in these experiments, ptc requires the highest levels of Ci activation for its transcription (Horabin, 2005).

In females, the ectopic Sxl elevates ptc expression in the cells near the AP boundary, but the depth of the cells showing this highest level of Ci activation is reduced. A reduction in the number of cells transcribing ptc, when compared with the wider but less intense width of ptc transcription in the control half of the disc, suggests a restriction in Hh diffusion. Elevated ptc transcription is expected to produce more Ptc at the membrane, which should sequester more Hh close to the AP boundary. This result shows that Sxl can both enhance the Hh response and effectively alter the Hh gradient (Horabin, 2005).

In males, the increase in ptc transcription induced by Sxl both intensifies and widens the ptc expression zone. This suggests that the activation of Ci is at a lower peak in males than in females, and its enhancement by ectopic Sxl does not reach the same maximum that additional Sxl in females produces (Horabin, 2005).

Ectopic expression of Sxl in the dpp expression zone has been shown to adversely affect female wing development, narrowing the region between veins L3 and L4. This defect was taken to suggest that the relative concentrations of both Ci and Sxl are important for their normal function (Horabin, 2003). The data presented in this study support this conclusion while providing an explanation for the apparent decrease in effectiveness of the Hh signal. When additional Sxl is expressed, the slope of the Hh gradient becomes steeper. Since Hh directly patterns the L3 to L4 wing vein region, a steeper gradient of Hh will reduce the area patterned because the normal Hh patterning minimum is reached more rapidly. The L3 to L4 intervein region should correspondingly become narrower. No adult males expressing Sxl were recovered (presumably because of upsets in dosage compensation) so their wings could not be scored (Horabin, 2005).

Depending on the expression driver used, ectopic Sxl is not only lethal to males but also females. This is perhaps not altogether surprising given that Sxl can modulate the signal strength of a molecule crucial to the development of numerous tissues. The in vivo concentration of Sxl is, most likely, tightly controlled. It has been shown that Sxl negatively regulates translation of its own mRNA. Combined with its positive autoregulatory splicing feedback loop, which ensures that essentially all of the Sxl mRNA is spliced in the productive female mode in females, this dual negative and positive autoregulation implies a homeostasis that keeps the concentration of Sxl in a predetermined fixed range. The potent effect of Sxl on the Hh signal makes the requirement for this dual regulation more readily understood (Horabin, 2005).

Mutations in Sxl that produce sex transformed females generally result in animals that are small and male-like in size. Females transformed by mutations in tra appear as males but maintain the female size, indicating that sexual dimorphic body size is controlled by Sxl (Horabin, 2005).

The enhanced levels of full-length Ci suggest that Sxl promotes disc growth. Indeed, when ectopic Sxl is being expressed in the dorsal half, many of the discs, both male and female, show an overgrowth phenotype with the dorsal half of the wing pouch frequently expanded and distorted. This growth effect is non autonomous, indicating that it is affected by a system that signals beyond the cells expressing Sxl. This is consistent with the idea that Hh signaling is augmented to result in the overgrowth. The experiments described here do not rule out the possibility that Sxl may additionally regulate growth autonomously (Horabin, 2005).

Hh with only the cholesterol modification has the greater impact on Sxl and its stabilization of full-length Ci. However, the Ci that is stabilized does not appear to accomplish Hh patterning. This raises the mechanistic question of how Sxl achieves growth of the entire disc (Horabin, 2005).

Simply reducing the levels of the repressor form of Ci (which is accomplished by increasing the levels of full-length Ci) should increase the expression of the growth factor dpp. This is because dpp is affected by Ci at two levels: absence of the Ci repressor ameliorates repression to give low levels of dpp expression, while activated full-length Ci further elevates dpp transcription. Indeed, while the wing patterning defect caused by the ectopic expression of C84S-Hh narrows the region between wing veins L3 and L4 equally in the two sexes (due to its dominant-negative effect on endogenous Hh), the overall sexual dimorphic size difference is maintained. Consistent with this idea, co-expressing Sxl and Hh with only the cholesterol modification produces an overgrowth phenotype in discs, indicating Sxl can promote disc growth through this form of Hh (Horabin, 2005).

The growth induced by Dpp has been described as 'balanced', involving both mass accumulation as well as cell cycle progression. The net effect is that cell size does not change, nor does the ploidy. This is in contrast to growth induced by hyperactivation of Ras, Myc or Phosphoinositide 3 kinase, which increase growth but do not induce a progression through the G2/M phase of the cell cycle and, as a result, increase cell size (Horabin, 2005).

It is proposed that in the wild-type gradient of Hh with both its lipid modifications, Sxl augments the overall Hh signal to increase both full-length as well as activated full-length Ci. The two Hh targets (Ci and Sxl) respond differentially to the various components of the pathway (Horabin, 2003). Since Sxl is able to alter signal strength, the final outcome of the Hh signal must reflect the balance in activities of the components, modulated by the lipid moieties recognized, the membrane proteins used (Ptc versus Smo) and the proteins present in the Hh cytoplasmic complex. The studies reported here provide a strong rationale for why Sxl resides within the Hh cytoplasmic complex (Horabin, 2005).

Sxl not only elevates expression of dpp and its downstream targets to induce growth, but is able to elevate ptc expression. Enhancing ptc suggests that the Hh signal is 'corrected' for the enlarged patterning field, since short-range patterning has to be controlled by Hh. By enhancing dpp, Sxl indirectly also enhances the long-range patterning system of the disc. Augmenting the Hh signal would thus appear an elegant solution for increasing overall size without changing the basic body plan or pattern. Since Sxl is expressed in all female tissues from very early in development and this expression is maintained for the rest of the life cycle, Sxl is constantly available to upregulate the Hh signal. This augmentation must be kept within check, however, because, as argued above, too high an increase can change the overall slope of the Hh gradient, effectively changing the final patterning of the tissue (Horabin, 2005).

The Hh pathway can also control body size in mammals. ptc1 mutations in mice provide an overgrowth phenotype with large body size, while increasing ptc1 expression decreases body size. Humans with basal cell nevus syndrome, an autosomal-dominant condition caused by the inheritance of a mutant ptc allele, have been reported to have multiple developmental abnormalities and, relevant to this study, larger body size. Whether the mechanism described in this study is global to sexually dimorphic organisms that use Hh for patterning remains to be seen (Horabin, 2005).

Sex-lethal protein mediates polyadenylation switching in the female germline

The Drosophila master sex-switch protein Sex-lethal (Sxl) regulates the splicing and/or translation of three known targets to mediate somatic sexual differentiation. Genetic studies suggest that additional target(s) of Sxl exist, particularly in the female germline. Surprisingly, detailed molecular characterization of a new potential target of Sxl, enhancer of rudimentary [e(r)], reveals that Sxl regulates e(r) by a novel mechanism-polyadenylation switching-specifically in the female germline. Sxl binds to multiple Sxl-binding sites, which include the GU-rich poly(A) enhancer, and competes for the binding of Cleavage stimulation factor 64 kilodalton subunit (CstF64: involved in binding and processing mRNA for polyadenylation) in vitro. The Sxl-binding sites are able to confer sex-specific poly(A) switching onto an otherwise nonresponsive polyadenylation signal in vivo. The sex-specific poly(A) switching of e(r) provides a means for translational regulation in germ cells. A model is presented for the Sxl-dependent poly(A) site choice in the female germline (Gawande, 2006).

Since all known examples of Sxl regulation involve its binding to uridine-rich sequences, a search was performed of the entire Drosophila genome for potential high-affinity Sxl-binding sites. The Sxl consensus (UUUUUGUU(G/U)U(G/U)UUU(G/U)UU) was used used for the search; a search using a shorter Sxl-binding site (U8) yielded a significantly larger and experimentally unmanageable number of hits in the genome and therefore the search was not pursued further. Seven of the candidates showed sex-specific mRNA isoforms. This study describes the detailed characterization of one of the seven candidates, e(r). The GADFLY annotation database showed two e(r) transcripts (CT5770 and CT29800) resulting from an alternatively spliced exon and two alternative polyadenylation sites. Multiple potential Sxl-binding sites were found in e(r): one adjacent to the 3' splice site of exon 2 and three downstream of the proximal polyadenylation site (Gawande, 2006).

To determine whether alternative splicing of exon 2 was the basis for the sex-specific isoform of e(r), RNase protection analysis was performed. Exon 2 was found to be alternatively spliced, but was not spliced in a sex-specific manner. Next, an RNA blot was probed with the fs-UTR probe, which corresponds to the sequence between the two polyadenylation sites of e(r) and includes potential Sxl sites. This probe hybridized to the longer isoform that was present in females, but not to the shorter isoform present in both sexes. The shorter isoform is referred to as e(r)-non-sex-specific [e(r)-nss] and the longer isoform will be referred to as e(r)-female-specific (e(r)-fs). Presence of non-sex-specific and female-specific isoforms of e(r) is reminiscent of the known Sxl target, tra, although it is the sex-specific splicing regulation that contributes to the synthesis of the two isoforms of tra. It is concluded that use of two poly(A) sites, rather than alternative splicing, accounts for the sex-specific size difference of the e(r) transcripts (Gawande, 2006).

This study demonstrates that Sxl regulates e(r) expression in vivo by a novel mechanism-polyadenylation switching-which allows translation regulation in the female germline. A model that explains the Sxl-mediated regulation of e(r) must answer the following questions. First, what is the molecular basis for the default polyadenylation pattern in male flies? Second, how does Sxl mediate poly(A) switching in females? Third, why does the poly(A) switching of e(r) occur primarily in the female germline (Gawande, 2006)?

This study shows that three key factors account for the default poly(A) pattern of e(r) in male flies: differences in the binding affinities of CstF-64 for the two alternative GU-rich elements; order of the two polyadenylation sites; and cis competition between the two poly(A) signals. First, the longer GU-rich enhancer element downstream of the proximal cleavage/polyadenylation site provides at least in part a basis for the increased apparent binding affinity for CstF-64, most likely by providing multiple registers for binding, and thus confers competitive advantage to the proximal poly(A) site (Gawande, 2006).

Second, the order of the two poly(A) sites is also important. Contrary to expectation that the proximal site is inherently stronger than the distal polyadenylation (DP) site, DP males exclusively use the DA site. This demonstrates that the arrangement of the two sites is important for the default poly(A) site choice and excludes the possibility that the distal site is inherently weak. The usage of the DA site is consistent with the known RNA-binding properties of CstF-64 and sequences of natural poly(A) sites. Moreover, in this system, the promoter-proximal site is always used by default in males. Both transcription and splicing influence polyadenylation. Since an EGFP reporter, unlike the endogenous transcript, lacks an intron, coupling between the transcription and polyadenylation machineries best explains why the promoter-proximal site is always preferred in males (Gawande, 2006).

Third, mutation of the Sxl-binding site or the proximal GU-rich element compromises CstF64 binding and activates the distal site in males. This is what would be expected if the Sxl-binding site is also a polyadenylation element (or the CstF64-binding site) and if Sxl blocks the proximal poly(A) site, which is consistent with the Sxl blockage model proposed for splicing regulation. Since CstF-64 binds directly to the GU-rich enhancer element to facilitate recruitment of the polyadenylation machinery to the poly(A) signal, reduced affinity of CstF-64 to the mutant proximal GU-rich element provides a basis for the activation of the distal site in males. This shows that the longer GU-rich element associated with the proximal site is also important for the default poly(A) site choice. It is possible that the non-canonical poly(A) element (UAUAAA instead of AAUAAA) and its sequence context confer increased dependence on the long GU-rich element for polyadenylation at the proximal site. This feature could make the proximal GU-rich element and CstF-64 binding an attractive target for Sxl regulation (Gawande, 2006).

The simplest explanation for the combined results is that in the female germline Sxl competes for the binding of CstF-64 to the proximal GU-rich element and represses polyadenylation at the proximal site, leading to activation of the distal site; Sxl does not bind to the distal GU-rich element. The proximal GU-rich element is much longer than is necessary for polyadenylation or CstF-64 binding, most likely to confer competitive advantage (by providing multiple registers for CstF-64 binding to the proximal site in males and to accommodate Sxl binding in females. Moreover, Sxl and CstF-64 have similar sequence preference (GU-rich sequences devoid of cytidines) . Therefore, it is not surprising that the sequence requirement of the proximal GU-rich element for polyadenylation in males and for poly(A) switching in females could not be uncoupled, as was performed using a three nucleotide U-to-C substitution for Sxl-mediated splicing regulation of tra, which involves competition between Sxl and U2AF; U2AF prefers uridine- and cytidine-rich sequences devoid of guanosines. Specificity of Sxl for the proximal but not the distal GU-rich element and the ability to convert a poly(A) site that is nonresponsive to poly(A) switching to a site that supports female-specific switching provide an explanation for why switching occurs in wt and D123D' females, but not in DP and DD' females. Moreover, Sxl-dependent poly(A) site switching of e(r) as well as splice site switching of Sxl and msl2 transcripts displays a need for multiple Sxl-binding sites for efficient regulation. The ability of Sxl to discriminate between alternative GU-rich sequences is the crux of the sex-specific poly(A) switching. GU-rich sequences belong to a class of regulatory RNA motifs that are defined by base composition rather than an exact sequence. Such simple repetitive sequences offer several advantages for gene regulation: for example, binding affinity but not specificity for RNA-binding proteins can change as a function of the length of the sequence; two proteins (e.g., Sxl and CstF-64) with different binding site length requirements can recognize the same sequence; a regulatory protein such as Sxl can regulate multiple processes, polyadenylation and splicing, by antagonizing CstF-64 and U2AF65, respectively, because of the possibility of overlapping binding specificities; and single nucleotide changes are less disruptive (Gawande, 2006).

Table of contents

Sex lethal: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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