torso

Promoter Structure

The promoter contains a TATA box upstream from the start site (Sprenger, 1989).

Malignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. The requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) has been investigated using a genetic approach in Drosophila. The JAK/STAT pathway plays little or no role in signaling by wild-type Tor. STAT, encoded by marelle (mrl; Stat92E), is essential for the gain-of-function mutant Tor (Tor^GOF) to activate ectopic gene expression. These findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation (Li, 2002).

How does Tor^GOF RTK activate Mrl? There are at least three possible mechanisms through which STAT activation by RTK could occur. RTK could directly bind and activate STAT proteins. Alternatively, STAT could be indirectly activated by the RTK, either via JAK or MAPK. Genetic evidence allows the possibilities that Tor^GOF activates Mrl via JAK or MAPK to be ruled out. (1) Whether or not removal of Hop activity modifies the tor^GOF phenotype was examined. Surprisingly, a hop null mutation does not suppress tor^GOF, indicating that unlike Mrl, Hop is not required for ectopic tll expression. (2) Removal of mrl does not suppress rl^Sevenmaker (rl^Sem), which encodes a GOF mutant form of Drosophila MAPK, suggesting that Mrl is not essential for the effects of GOF mutation in MAPK. To test for a physical interaction between Mrl and Tor^GOF, Tor was immunoprecipitated from wild-type and tor^GOF embryos, respectively, with anti-Tor antibody, and the presence of Mrl was examined in the immune complexes. A specific band corresponding to Mrl was detected in the immunoprecipitates. The Tor-Mrl association, however, is only observed in the presence of vanadate (a general tyrosine phosphatase inhibitor), suggesting that this interaction takes place only when the cytoplasmic protein phosphorylation status is preserved, or when Tor and/or Mrl have been activated by the presence of vanadate. Altogether, these results are consistent with a direct activation of Mrl by Tor^GOF, possibly following recruitment of Mrl to phosphotyrosine residues on the Tor RTK via SH2-phosphotyrosine peptide interaction (Li, 2002).

Since Mrl activation is required for ectopic tll expression induced by Tor^GOF, whether Mrl-binding sites (TTCNNNGAA) are present in the regulatory region of the tll gene was examined. A search in the tll regulatory region revealed two putative Mrl-binding sites with the consensus TTCNNNGAA located at –2357 (site 1) and –2462 (site 2) upstream of the tll transcription start site. These two sites are able to bind Mrl, although site 2 shows a much lower affinity. Interestingly, the two Mrl sites are located 105 bp apart in the tll regulatory region. This configuration is reminiscent of that existing in the eve stripe 3 enhancer, where cooperative binding of two Mrl homodimers has been demonstrated. To assess the functional relevance of the two Mrl sites in tll expression, transgenes containing the 5.9 kb regulatory fragment upstream of the tll transcription start site fused to the lacZ gene were introduced into flies. This 5.9 kb fragment had been shown previously to drive lacZ expression in a pattern almost identical to that of the endogenous tll gene. Accordingly, lacZ expression is greatly expanded in a tor^GOF background. A 5.9 kb fragment with the two Mrl binding sites mutated, showed wild-type activity for lacZ expression in wild-type embryos, suggesting that these Mrl-binding sites are dispensable for tll expression under normal Tor signaling. However, in a tor^GOF background, the mutant 5.9 kb fragment shows greatly diminished ability to drive lacZ expression in an expanded domain compared to the situation when the Mrl binding sites are wild type. These results are consistent with the genetic results that Mrl is required for the full activity of gain-of-function, but not wild-type Tor (Li, 2002).

To account for the involvement of Mrl in tll regulation, it is proposed that a hyperactivated RTK requires a downstream pathway that is not essential for wild-type RTK under normal physiological situations. In wild-type embryos, Tor is activated only in the two terminal regions and defines the spatial limits of tll expression domains by relieving the transcriptional repressors bound to the tll promoter. Mrl is not an essential factor for tll activation in the terminal regions, although it remains to be determined whether Mrl contributes to the activation of tll expression redundantly with other yet unidentified factors. In tor^GOF mutant embryos, Tor^GOF is constitutively active in all regions of the embryo and causes ectopic tll expression. In this case, Mrl activation is indispensable for the ectopic tll expression in the central regions of the embryo. The differential requirement for Mrl in central and terminal regions might be due to the lack of other activators of tll and/or the presence of additional repressors in the central region of the embryos. Consistent with this idea, in the absence of Tor signaling (such as in tor mutant embryos), tll can be induced by uniformly expressing activated forms of downstream signaling components (such as Ras^V12 or 14-3-3). The resulting induction of tll expression happens preferentially in the terminal regions. Thus tll expression could be determined by the balance between repressors and activators that can bind to the tll promoter (Li, 2002).

These findings may explain some of the conflicting observations on the role of STAT in RTK signaling in mammals. For example, thanatophoric dysplasia type II (TD II) dwarfism in humans is caused by mutations that lead to constitutive activation of a human RTK FGF receptor 3 (FGFR3). Similar to Tor^GOF activating Mrl, it has been shown that an activated mutant FGFR3 specifically activates STAT1 in both human patient tissues and mouse models. The activated STAT1 in this case induces expression of the cell-cycle inhibitor p21^WAF1/CIP1, resulting in growth inhibition of bone tissues. However, STAT1 is not known to be required for bone development. STAT1 knockout mice have perfect bones, although they exhibit defective immune systems. This might be explained by a redundancy among different STAT proteins. Alternatively, STAT1 may not be required for normal FGFR3 signaling in bone development. The presence of several STAT genes in mammals makes it technically difficult to distinguish between the above two possibilities using the mouse as a genetic model. In contrast, the presence of a single JAK and a single STAT gene in Drosophila allows the relationship between RTK and JAK/STAT signaling to be examined, without being limited by gene redundancy. These observations in Drosophila suggest that the TD II syndrome in humans could be explained if STAT1 is not normally required for FGFR3 signaling, but it becomes essential only for the activating mutant FGFR3 (Li, 2002).

Targets of Activity

There are least two separate but synergistically interacting promoter regions that mediate transcriptional activation of tailless (tll) by the terminal system. This functional synergism between regulatory elements may play a role in the translation of the Torso morphogen gradient into the sharp boundary of tll gene activity (Liaw, 1993).

Activation of the Torso RTK at the poles of the embryo sets off a phosphorylation cascade that leads to the spatially specific transcription of the tailless (tll) gene. The TOR response element (TOR-RE) has been mapped to an 11-bp sequence. The gene activator GAGA and the repressor Grainyhead/NTF-1 bind to the TOR-RE. NTF-1 can be phosphorylated by rolled, also known as MAPK (mitogen-activated protein kinase), a protein in the Ras pathway activated by Torso. Thus activation of the TOR RTK at the poles of the embryos leads to inactivation of a repressor and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo (Liaw, 1995).

Anterior repression of orthodenticle is carried out by Huckebein which in turn receives input for the torso system, from Dorsal and from Bicoid. Dorsal functions in the anterior repression of otd expression. The repression function of Dorsal is mediated, at least in part, through Huckebein, since anterior hkb expression is lost in dorsal mutants. Contrary to early models of embryonic pattern formation, high levels of Bicoid are not required for otd activation or for the establishment of anterior head structures (Gao, 1996).

sloppy paired, in addition to behaving like a segment polarity gene and as a pair rule gene, acts like a gap gene in the head. All three maternal systems active in the cephalic region are required for proper slp expression. High levels of the terminal system (Torso) inhibit slp through bicoid. Low levels of terminal system activity seem to potentiate bcd as an activator of slp in more posterior positions. Dorsal, the morphogen of the dorsoventral system, and the head-specific gap gene empty spiracles, act as repressor and corepressor respectively in the regulation of slp (Grossnicklaus, 1994).

The anterior, the dorsoventral and terminal systems, are required for the activation of crocodile expression and for the spatial control of the anterior cap domain, while the posterior system is not required for the regulation of the croc expression pattern. In the absence of bcd activity, croc fails to be expressed in the anterior cap domain. Although Bcd acts in a concentration-dependent manner, croc expression can only be expanded ventrally in the absence of dorsal activity. In fact, the lack of dorsal activity causes a strong reduction of the croc expression domain to a single spot, corresponding in position to the peak of bicoid activity at the anterior pole. Conversely, Dorsal activity along the entire dorsoventral axis, as in embryos laid by Toll mutant females, causes an expansion of the croc expression domain towards the dorsal-most position. In embryos lacking tor activity, croc expression is abolished in the dorsal region. However, if tor is activated ectopically due to a dominant tor mutation, the croc expression domains are expanded significantly on the ventral side. Thus Dorsal postively regulates croc and Bcd requires Dl to set the spatial limit of the croc anterior expression domain (Häcker, 1995).

In the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The maternal gene torso is required for the development of the unsegmented region of the head (acron). otd expression responds to the activity of the maternal tor gene at the anterior pole of the embryo (Finkelstein, 1990).

The closely linked POU domain genes pdm-1 and pdm-2 are first expressed early during cellularization in the presumptive abdomen; the initially broad domain soon resolves into two stripes. This expression pattern is regulated by the same mechanisms that define gap gene expression domains. The borders of pdm-1 expression are set by the terminal system genes torso and tailless, and the gradient morphogen encoded by hunchback. (Cockerill, 1993).

The three maternal systems (anterioposterior (bicoid); terminal (torso); dorsoventral (dorsal) control the early expression of Goosecoid. The GSC stripe never appears in bicoid mutants, the stripe is shifted anteriorly in torso mutants and the ventral repression of the stripe is abolished in dorsal mutants (Goriely, 1996).

Transcription of Bicoid target genes is repressed at the anterior pole owing to the activity of torso . Both activation by BCD and repression by TOR can be reproduced by a minimal promoter containing only BCD-binding sites upstream of a transcriptional start site. Repression requires the D-raf kinase and is associated with phosphorylation of BCD protein. Repression requires neither tailless nor huckebein, previously thought to constitute the sole zygotic output of the TOR signaling system. Thus phosphorylation of BCD down-regulates transcriptional activation by BCD (Ronchi, 1993).

The Torso receptor tyrosine kinase cascade represses expression of Bicoid targets at the most anterior tip of the embryo. Neither the homeodomain (HD) nor the activation domain of BCD is targeted by Torso in the anterior repression of BCD targets. When a BCD mutant protein whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein, whose activation domain has been replaced by a generic activation domain, indicates that the activation domain of BCD is also not specifically required for its down-regulation by Torso. It is propose that Torso does not affect the ability of BCD to bind DNA, but instead directs modification of BCD or of a potential BCD co-factor, which renders the BCD protein unable to activate transcription (Bellaiche, 1996).

capicua (cic) is involved in gene repression in Drosophila terminal and dorsoventral patterning. Tor signaling proceeds via the Ras/Raf/MAPK pathway to regulate expression of the zygotic genes tll and hkb, which are specifically expressed at each pole of the embryo. These genes encode transcription factors that initiate the developmental programs leading to differentiation of head and tail structures. Tor signaling does not activate terminal gene expression directly; rather, it functions by antagonizing at the poles a uniformly distributed repressor activity, allowing other maternal factors to activate transcription locally. Evidence for this view comes from the identification of regulatory elements in the tll promoter (called tor response elements, tor-REs) that confer terminal-specific expression and that, when mutated, cause severe derepression of tll transcription (Jimenez, 2000 and references therein).

Additional evidence for the regulation of tll and hkb by relief of repression derives from the role of the Groucho (Gro) corepressor in this process. Gro is a nuclear WD-repeat protein that does not bind DNA but interacts with a variety of DNA-bound transcriptional repressors. These associations recruit Gro to target promoters, bringing about transcriptional repression. Gro has been shown to participate in terminal development by restricting the expression of tll and hkb to the embryonic termini: embryos deprived of maternal Gro function show derepression of tll and hkb toward the middle of the embryo (Jimenez, 2000 and references therein).

What is the actual target of Tor signal inactivation at the embryonic poles? The Drosophila Yan Ets-like repressor factor is known to be degraded in response to RTK signaling during eye development. Thus, it is possible that the target of Tor signaling is similarly inactivated at the embryonic poles. The Gro protein is uniformly distributed in the blastoderm embryo and does not show down-regulation at the termini. Also, Gro corepressor activity during sex determination is not inhibited by Tor signaling, arguing that Gro is not the target of the Tor signal. To monitor the pattern of Cic distribution in embryos, a polyclonal antibody was raised against an HMG box-containing fragment of the protein. This antibody reveals a distinctive nuclear signal in wild-type but not cic¹ blastoderm embryos, confirming both that Cic is a nuclear protein and that the cic¹ allele is a strong loss-of-function mutation. Remarkably, Cic is distributed asymmetrically in blastoderm embryos, being present in nuclei from the presumptive trunk but absent at each embryonic pole. Because CIC mRNA is uniformly distributed in the embryo, the exclusion of the protein from the poles argues that Cic is under negative post-transcriptional regulation by the Tor signal transduction pathway (Jimenez, 2000).

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterized Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity (Janody, 2000).

The results indicate that early anterior expression of a labral determinant, cnc, and of two foregut determinants, wg and hh, is repressed when zygotic expression of hb is allowed to persist at the anterior pole of the Drosophila blastoderm embryo. Expression of cnc, wg and hh is under the positive regulation of bcd and torso but no zygotic gene has yet been implicated in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and that torso-induced anterior repression of hb is necessary for their positive control by torso. To determine whether the positive control of cnc, wg and hh by torso could be the result of a double negative control involving hb, expression of these genes was analysed in hb zygotic mutant embryos derived from torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb at the pole, expression of these genes should be recovered in hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is not recovered in hb minus embryos derived from torso females whereas it is normal in hb minus embryos. This indicates that, although necessary, the anterior repression of hb is not sufficient to mediate Torso positive control on cnc, wg and hh early anterior expression (Janody, 2000).

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). Since these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. A well-conserved sequence repeat, (GTTT)₄ has been identified that is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)₄-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers (Andrioli, 2002).

The eve2Delta(GTTT)₄-lacZ transgene is repressed at the anterior tip, even in gt mutants, suggesting that yet another mechanism prevents activation in this region. This mechanism could work through another localized repressor activity, or by modifying Bcd, the major activator of eve 2. Consistent with the latter possibility, it has been previously shown that Bcd-dependent activation of hb and orthodenticle (otd) is downregulated by the Tor phosphorylation cascade at the anterior tip. To test whether tor controls the ability of Bcd to activate eve 2, the eve2Delta(GTTT)₄-lacZ transgene was crossed into embryos lacking tor activity. This causes a significant derepression at the anterior tip, suggesting that tor-mediated modification of bcd activity is important for preventing activation in this region. A similar derepression is not detected with the wild-type eve2-lacZ transgene in tor mutants, suggesting that Tor-mediated repression is dependent on the (GTTT)₄-binding activity. In summary, these results suggest that multiple activities are required for anterior repression of eve 2, and that three different mechanisms prevent activation in different anterior regions (Andrioli, 2002).

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