Promoter Structure

The regulation of tailless is complex. It has to be activated in both anterior and posterior. Three different maternal morphogen gradients regulate expression of tailless. There are at least two separate but synergistically interacting regions that mediate tll expression by means of 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. Activation of tailless in the anterior is through Bicoid (Liaw, 1993 and Pignoni, 1992).

In addition to regions mediating activation by the terminal system, regions mediating both activation and repression by Bicoid, and repression by Dorsal have been identified. Binding sites of BCD protein in a 0.5 kb region could be crucial for the BCD-dependent activation of tll expression in the anterior stripe (Liaw, 1993).

Activation of the Torso RTK at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of tailless (tll). Studies of the TOR response element (tor-RE) in the tll promoter indicates that the key activity modulated by the TOR RTK pathway is a repressor. The tor-RE has been mapped to an 11-bp sequence. The proteins GAGA (Trithorax-like) and Grainyhead bind to the tor-RE. Grainyhead can be phosphorylated by Rolled, also known as MAPK (mitogen-activated protein kinase), a member of the ras pathway. tll expression is expanded in embryos lacking maternal grainhead activity. These results make Grainyhead a likely target for modulation by the TOR RTK pathway in vivo. Thus activation of the TOR RTK at the poles of the embryos leads to inactivation of the repressor and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo (Liaw, 1995).

The expression of tailless in the head is not detectably regulated by the head gap genes buttonhead or orthodenticle, or by the proneural gene lethal of scute or by tailless itself. Approximately 6 kb of sequenced upstream regulatory DNA can drive lacZ expression in a pattern that mimics the full tailless embryonic expression pattern. Within this sequence multiple modules are identified responsible for different aspects of the tailless pattern. The ability to drive expression in the HL domain is localized to three different regions. In addition to identifying additional torso response elements that mediate early blastoderm polar expression, it is shown that the complex brain expression pattern is driven by a combination of modules; thus expression at a low level throughout the brain and at a high level in the dorsal medial portion of the brain and in the optic lobe, as well as neuroblast-specific repression are mediated by different DNA regions (Rudolph, 1997).

GAGA is usually considered a gene activator, but here it may act to repress. Other evidence points to Groucho, a protein that doesn't bind to DNA, but nevertheless interacts with transcription factors, as a regulator of tailless (Parouch, 1994).

During embryogenesis, the activated Torso receptor tyrosine kinase (TOR RTK) pathway activates tailless (tll) expression by a relief-of-repression mechanism. Various lines of evidence have suggested that multiple factors are required for this repression. Tramtrack69 (TTK69) binds to two sites within tll cis-regulatory DNA, TC2 and TC5. TTK69 is phosphorylated by mitogen activated protein kinase. These two sites are similar to the TTK69 consensus binding sites identified in the eve gene (GGTCCTGC) and in the fushi tarazu (ftz) zebra element (AnGTCCTnGCA). TTK69 binding sequences from both eve and ftz regulatory regions share a core sequence, TCCT. Three additional sites containing the core sequence were found in the tll-MRRe fragment, designated TC1, TC3 and TC4. In embryos lacking maternal ttk69 activity, the expression of both endogenous tll and lacZ driven by the tll minimal regulatory region (tll-MRR) are expanded. Further, in wild-type embryos, the tll-MRR mutated in TC5 drives uniform lacZ expression before late stage 4. It is concluded that TTK69 is required for early (before the end of stage 4) repression of tll transcription (Chen, 2002).

In the absence of activation of the TOR RTK pathway, tll is repressed throughout the embryo. Several lines of evidence presented in this study indicate that TTK69 plays a role in this repression, and that its repressive effect is down-regulated by TOR RTK signaling. First, TTK69 activity is required to repress both endogenous tll expression and lacZ expression mediated by the tll-MRR. Second, TTK69 binds to two sites in the tll MRRe, TC2 and TC5. Third, mutation in TC5 causes the tll-MRR to drive uniform early expression of lacZ. Finally, TTK69 is phosphorylated in vitro by MAPK, a target of the TOR pathway. These data indicate that TTK69 functions early during the period when tll is repressed in the central domain of the embryo, i.e. before and during embryonic stage 4 (Chen, 2002).

TTK69 has been shown to act as a transcriptional repressor in a number of contexts during Drosophila development. TTK69 was first identified by its binding to a repression element in the upstream enhancer of the ftz gene, and subsequently shown to act as a transcriptional repressor to regulate expression of additional pair-rule genes, namely eve, odd-skipped, runt and hairy. Later in development, TTK69 acts as an inhibitory regulator to down-regulate the cellular response to neuralizing signals. In the developing eye, TTK69 inhibits photoreceptor cell fate by interacting with and enhancing the activity of the general repressor Yan. The function of TTK69 as a repressor of tll activity in the early embryo is thus consistent with other described roles of the protein during Drosophila development. There are presently at least five known proteins ¯ Gaga, Grainyhead, Groucho, Capicua, and TTK69 ¯ that appear to play a role in the TOR RTK-modulated repression of tll (Chen, 2002).

Protein interaction studies suggest that at least some of these proteins are part of a larger complex that represses tll. Cic and Gro have been shown to interact with each other in vitro. In addition to interacting with each other directly, some of the described tll repressors are likely to be associated with each other through their interactions with other proteins. Both Gro and TTK69 have been shown to interact with histone deacetylase RPD3; in addition, TTK69 interacts with the co-repressors CtBP and Sin3A. Therefore, in addition to the transcriptional regulators with a described role in tll repression, RPD3, CtBP and Sin3A may also be involved in the tor-modulated repression of tll, and are possible members of a multiprotein tll repression complex (Chen, 2002).

Several features of the organization of binding sites in the tll-MRRe lend further support to the idea of a tll repression complex. First, at either end of the tll-MRRe, there is a tor-RE flanked on one side by a demonstrated TTK69 binding site and on the other by a TTK69 core binding site (although these core sites did not bind TTK69 in the in vitro studies, they might do so in vivo, in the presence of other transcriptional regulators), suggesting that adjacent binding sites may promote functional interactions between TTK69 and transcription factors binding to tor-RE (tor-REB). Secondly, tll-MRR with base substitution in either tor-REa or TC5 drives uniform lacZ expression during early stage 4, indicating that both tor-REa and TC5 are required for repression. Thirdly, the tor-REb containing region, including TC2, has partial repression activity; whether this repression activity is a function of tor-REb, TC2, or both sites remains to be determined (Chen, 2002).

Assembly of the repression complex must involve binding of proteins (possibly including GAGA and/or NTF-1) to tor-RE, since repression cannot be established in the absence of this site. Since tll expression is observed only at the poles of the embryo 90 min after fertilization, the repression complex must be assembled by this time. TTK69 is likely to play an accessory role in establishing the complex, but is not absolutely required (since repression can be established later in the absence of TTK69). TTK69 presumably binds to TC5 and may also interact with other proteins, such as GAGA or other BTB domain proteins (since the BTB domain proteins tend to form multimeric aggregates). Consistent with the detection of TTK69 uniformly throughout stage 3, but not in stage 5 embryos, TTK69 is assumed to be displaced from the repression complex by late stage 4 and degraded (Chen, 2002).

Multiple lines of genetic and biochemical evidence converge to support the idea that TTK69 is regulated by RTK pathway activity. When ttk69 is ectopically expressed in the early embryo, the level of TTK69 is reduced at the poles of the embryo, where the TOR pathway is active. Genetic analysis in the developing eye has shown that repression activity of TTK69 is regulated by an RTK pathway. Further, uniform expression in the developing eye of a constitutively active ras, rasV12, causes a dramatic reduction in TTK69 levels, suggesting that TTK69 is degraded where ras is active. The demonstration that TTK69 is phosphorylated by MAP kinase provides further in vitro evidence to support the notion that the activity of TTK69 is modulated by RTK signaling. Phosphorylation of TTK69 might lead to its degradation by a pathway similar to that described for its isoform TTK88. Degradation of TTK88 is initiated when it complexes with Phyllopod (Phyl) and Seven in absentia (Sina); Sina interacts with a ubiquitin conjugating enzyme UBCD1, resulting in ubiquitination of TTK88 and its targeting for proteolytic destruction. The conjugation of TTK69 with dSMT3, a ubiquitin-like protein, has been shown to be correlated, both in vitro and in vivo, with the extent of TTK69 phosphorylation. It is concluded that the activation of tll expression at the poles of the embryo is mediated in part by MAP kinase inactivation of TTK69 repression (Chen, 2002).

The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. The human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor Grainyhead binding to dring, the fly homolog of dinG. Electrophoretic mobility shift assays demonstrate that the Grh-dring complex forms on regulatory elements of genes whose expression is repressed by Grh but not on elements where Grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes (Tuckfield, 2002).

Strong evolutionary conservation of amino acid sequence exists between the mammalian and Drosophila members of the Grainyhead-like family. The likelihood of a similar conservation of function led the idea of the existence of a Drosophila homolog of dinG. Database searches identified a sequence that has been termed dring (FlyBase term: Sex combs extra), which has 44% identity and 61% similarity to the dinG amino acid sequence and 50% identity and 68% similarity in the domain of the dinG protein which interacts with the GRH-like family. To determine whether the Drosophila factor Dring could interact with Grh, radiolabeled in vitro-transcribed and translated Grh was generated for GST chromatography assays. Grh was shown to be specifically retained on a GST-Dring matrix but not on GST alone, confirming the evolutionary conservation of this interaction (Tuckfield, 2002).

DinG can interact with CP2 and repress transcription from a CP2-dependent promoter. These data were generated in the context of a concatemerized consensus CP2 binding site. No physiological target genes of CP2-mediated repression have been identified in mammalian systems. In contrast, the regulatory regions in the dpp and tll genes involved in Grh-mediated repression have been clearly defined in vivo. In view of the significant homology between Grh and CP2 in the DNA binding domain, whether the CP2-dinG complex could form on the Grh-responsive element in the dpp promoter was examined. A probe containing the DRE-B region of the dpp promoter was studied in an EMSA in the presence of nuclear extract from the mammalian cell line JEG-3. Addition of this extract to the DRE-B probe resulted in the formation of a DNA-protein complex. This complex was ablated by the addition of either anti-CP2 or anti-dinG antiserum. To extend this observation, whether the GRH-DRING complex could assemble on the regulatory regions in the dpp and tll genes that are critical for GRH-mediated repression was examined. Probes containing the DRE-B region of the dpp promoter and the tor-RE element in the tll promoter were studied in an EMSA with Drosophila embryo extract in the presence and absence of anti-Grh antiserum or anti-dinG antiserum (which cross-reacts with the Drosophila DRING protein). The be2 element of the Ddc promoter (where Grh functions as a transcriptional activator) was also studied. A complex consisting of at least Grh and Dring formed on both the dpp and tll elements. In both settings, the complex was ablated (or shifted out of the gel) by anti-Grh and anti-dinG antisera. In contrast, the complex formed on the Ddc promoter was ablated by the addition of anti-Grh antiserum but remained unchanged in the presence of anti-dinG antiserum (Tuckfield, 2002).

The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient that is critical for patterning the anterior-posterior (AP) body plan in Drosophila. Previous work identified several target genes that respond directly to Bcd-dependent activation. Positioning of these targets along the AP axis is thought to be controlled by cis-regulatory modules (CRMs) that contain clusters of Bcd-binding sites of different 'strengths.' A combination of Bcd-site cluster analysis and evolutionary conservation has been used to predict Bcd-dependent CRMs. Tested were 14 predicted CRMs by in vivo reporter gene assays; 11 showed Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the most anterior part of the embryo, whereas others extend into middle and posterior regions. However, no strong correlation is detected between AP position of target gene expression and the strength of Bcd site clusters alone. Rather, binding sites for other activators, including Hunchback and Caudal correlate with CRM expression in middle and posterior body regions. Also, many Bcd-dependent CRMs contain clusters of sites for the gap protein Krüppel, which may limit the posterior extent of activation by the Bcd gradient. It is proposed that the key design principle in AP patterning is the differential integration of positive and negative transcriptional information at the level of individual CRMs for each target gene (Ochoa-Espinosa, 2005).

In reporter gene assays, 11 of the 14 tested fragments directed expression patterns in wild-type embryos that recapitulate all or part of the endogenous patterns of the associated genes. These experiments identified several elements that control segmentation genes, including three new gap gene CRMs. Two CRMs were found in the genomic region that lies 5' of the gap gene gt. One CRM (gt23) is initially expressed in a broad anterior domain and then refines into two stripes. A second CRM (gt1) is expressed later in a small dorsal domain very near the anterior tip. Double stain experiments indicated that the timing and spatial regulation of both patterns are indistinguishable from the anterior expression domains of the endogenous gt gene. A CRM 3' of the gap gene tll was identified that drives expression similar to the anterior tll domain (Ochoa-Espinosa, 2005).

Four novel CRMs were identified near known pair rule genes. One CRM was detected in the 3' region of hairy and drives expression of a small anterior dorsal domain similar to the hairy 0 stripe of the endogenous gene. Another CRM is located 3' of the paired gene and directs expression of an early broad domain that coincides with the later position of the native paired stripes 1 and 2. Two more CRMs (slpA and slpB) were identified in the slp locus, which contains the two related genes, slp1 and slp2. Both slpA and slpB faithfully reproduce parts of the early slp1 and slp2 expression patterns (Ochoa-Espinosa, 2005).

Four other CRMs were identified near the genes bowl, CG9571, D/fsh, and bl/Mir7. In three cases (bowl, CG9571, and D/fsh), the newly identified CRMs direct patterns similar to their associated endogenous genes. The final CRM (bl/Mir7) is located in the sixth intron of the bl gene and directs a strong anterior domain of expression. However, the endogenous bl gene is expressed nearly ubiquitously , which makes it an unlikely target of regulation by this CRM. One potential target of this element is the microRNA gene (Mir7), which is located 7 kb downstream in the eighth intron of bl. Four of the CRMs reported here (gt1, gt23, slpA, and D/fsh) were also identified in a recent genome-wide search for new patterning elements based on clusters of combinations of different binding sites including Bcd. The fragments used in that study were significantly larger in size but show very similar patterns to those in this study (Ochoa-Espinosa, 2005).

The Torso signaling pathway modulates a dual transcriptional switch to regulate tailless expression

The Torso (Tor) signaling pathway activates tailless (tll) expression by relieving tll repression. None of the repressors identified so far, such as Capicuo, Groucho and Tramtrack69 (Ttk69), bind to the tor response element (tor-RE) or fully elucidate tll repression. In this study, an expanded tll expression pattern was shown in embryos with reduced heat shock factor (hsf) and Trithorax-like (Trl) activities. The GAGA factor, GAF encoded by Trl, bound weakly to the tor-RE, and this binding was enhanced by both Hsf and Ttk69. A similar extent of expansion of tll expression was observed in embryos with simultaneous knockdown of hsf, Trl and ttk69 activities, and in embryos with constitutively active Tor. Hsf is a substrate of mitogen-activated protein kinase and S378 is the major phosphorylation site. Phosphorylation converts Hsf from a repressor to an activator that works with GAF to activate tll expression. In conclusion, the GAF/Hsf/Ttk69 complex binding to the tor-RE remodels local chromatin structure to repress tll expression and the Tor signaling pathway activate tll expression by modulating a dual transcriptional switch (Chen, 2009).

This study has shown that the interaction of GAF with Hsf and Ttk69 plays a critical role in regulation of tll expression. At locations where the Tor pathway is inactive, GAF, Hsf, and Ttk69 constitute a protein complex that binds to the tor-RE tightly. The protein complex recruits other co-repressors and chromatin remodeling factors containing Rpd3 to the organization of a high-order local chromatin structure. tll expression is off in this scenario (Chen, 2009).

Both Hsf and Ttk69 have been shown to be substrates of Mapk. At locations where the Tor pathway is active, activated Mapk phosphorylates both Hsf and Ttk69. The phosphorylated Ttk69 is degraded. The phosphorylation converts Hsf into an activator that leads to an increase in tll. In addition, the expanded lacZ expression patterns in hsf4 embryos at the nonpermissive temperature suggested that other activators, such as Stat, are apparently required for full activation of tll (Chen, 2009).

A protein–protein interaction network, including GAF, Hsf, Rpd3 and Ttk69, is required for tll repression. Studies show that Capicuo interacts with Groucho, which associates with Rpd3 and Sin3A to repress tll expression. Furthermore, CtBP is essential for Ttk69 to suppress neuronal cell fate. Therefore, these factors may also be recruited to repress tll expression (Chen, 2009).

Multiple GAFs and Ttk69s bind to the flanking regions of the tor-RE. The results from the DNaseI footprinting experiments showed that GAF binds to four sites in the tll-MRR, including the tor-RE. DNA sequences in footprints a and b match the consensus sequence bound by GAF, 3.5 GA repeats. Although DNA sequence in footprint c does not match the consensus sequence, this site contains a GAGA tandem repeat with 1-bp spacing. These three sites are well protected by GAF from DNaseI digestion, without or with little influence by Hsf. Therefore, GAF oligomer binds to these sites with high affinity and likely assists its own binding to the tor-RE. Similarly, binding of Even-skipped protein to low-affinity sites was assisted by its own binding to high-affinity sites at a distance (Chen, 2009).

Ttk69 acts as a co-repressor to increase GAF/Hsf binding to the tor-RE. In another EMSA experiment, a probe containing both the tor-RE and TC5 was used. Ttk69 binds to TC5 and assists GAF/Hsf/Ttk69 binding to the tor-RE. This is consistent with a previous report that base substitution a TCCT element (TC5) at the 3' flanking region of the tor-RE clearly affects the initiation of tll repression. Additionally, the binding of Ttk69 to TC2 might facilitate tll repression (Chen, 2009).

Results from the DNaseI footprintings showed different patterns over the tor-RE protected by either GAF/Hsf or GAF alone. Unexpectedly, these different tor-RE complexes showed the same mobility in results from EMSA experiments, which might be explained by the altered binding property of GAF in the presence of Hsf. BTB domains in GAF and human Promyelocytic Leukemia Zinc Finger (PLZF) proteins have been shown to belong to the Ttk subfamily, and BTB homodimer is a unit of PLZF oligomer. Since GAF oligomer presumably assisted itself in binding to the tor-RE, GAF homodimer could bind the 5'-end as well as the 3'-end of the tor-RE. When Hsf was added in addition to GAF, the interaction of GAF with Hsf influenced GAF binding to the tor-RE, leading to the alteration of the footprinting pattern. However, it remains unclear whether Hsf contacts DNA when it exists in the DNA–protein complex. Nevertheless, the results from shift-western blotting clearly demonstrated the presence of both GAF and Hsf in the DNA–protein complex (Chen, 2009).

Displacement of GAF monomer by Ttk69 to form a GAF/Hsf/Ttk69 complex might explain the unchanged band-shift patterns when the three proteins were added to the binding mixture. Molecular weights between GAF and Ttk69 are slightly different and BTB domains in both GAF and Ttk69 proteins belong to the same subfamily. Molecular modeling with crystal structure of the BTB domain in human BACH1 (PDB code: 2ihc) as a template was used to test the displacement hypothesis. Results showed that buried areas and free energies (ΔGs) of engaged interfaces among GAF and Ttk69 homodimers and GAF/Ttk69 heterodimer were similar, suggesting that formation of a GAF/Ttk69 heterodimer is possible. This explanation is partially supported by the detection of both Ttk69 and Hsf in the DNA–protein complex. In addition, Ttk69 has been shown to inhibit GAF activation in the absence of Ttk69 binding sites, and addition of Ttk69 significantly increases GAF binding to the 173-bp probe that contains multiple GAF binding sites. However, results from EMSA show that mobility of the DNA–protein complex is slightly affected. In conclusion, these data plus the existence of GAF, Hsf and Ttk69 in the DNA–protein complex indicate that the interaction of GAF with Hsf and Ttk69 form a protein complex binding to the tor-RE tightly (Chen, 2009).

In summary, multiple factors bind to the tor-RE and its flanking regions to form a large repression complex, consistent with the finding that the 240-bp cis-regulatory region of tll, but not the tor-RE itself, silences from a heterologous promoter (Chen, 2009).

Several studies on gene repression, i.e., bacterial bipA and eno genes, fly decapentaplegic and human and GP91phox genes, have revealed that multiple low-affinity sites are bound by a repressor to regulate gene expression. Likewise, multiple weak binding sites that cluster within a short range of a cis-regulatory region are reported to facilitate the cooperative binding of factors, leading to a sharp definition in expression patterns. Reduction of repressor concentration leads to a loss in the definition at the edge of expression domains. In this study, the binding affinity of GAF to the tor-RE is low, and multiple tor-REs are present in the tll cis-regulatory region. This explains the poorly-defined boundary of the tll expression patterns in embryos that have reduced hsf and Trl activities (Chen, 2009).

Footprint b was marginally affected by supplementing Hsf. The DNA sequence in this site contains one binding site each for GAF and Hsf (−130 GAGAGAG and −115 GAATCCTGCGGAA), located in regions O and P, respectively. The sequence for GAF binding matches the consensus sequence. Although this putative Hsf binding sequence does not match the consensus sequence, it has been shown that Hsf is able to bind a sequence with two GAAs spaced by 7 bp. Interestingly, in contrast to the role of tor-RE, deletion of either region O or P from the tll-MRR results in a drastic reduction of lacZ mRNA levels, but no changes in the expression patterns. These results not only further support the notion that Hsf and GAF are required for tll activation, but also provide an explanation for (1) the different tll expression patterns in embryos with reduced hsf and Trl activities by either removing one copy of the genes or using RNAi to knockdown activities of the genes, and (2) the different tll expression levels resulting from either base substitutions in the tor-RE or reduction of gene activities (Chen, 2009).

These results showed a moderate effect of Hsf on GAF binding to footprint c. In addition to the GAGA repeat, this site also contains two GAA repeats with the 7-bp spacing. Results from the DNaseI footprinting experiments showed that GAF bound to this site less efficiently. Furthermore, deletion of this site from the tll-MRR results in a low level and slightly expanded lacZ expression pattern. In addition, footprint d at the 5'-end of the tor-RE was a weak site bound by GAF and the footprint pattern protected by GAF was significantly affected by Hsf. Base substitutions to this site severely damaged tll repression. These data supported the notion that the low and high affinity sites bound by GAF are the major contributors to tll repression and activation (Chen, 2009).

All signaling pathways regulate, at least in part, specific factors to activate the expression of target genes. In most well-studied signal pathways, the signals directly activate factors. In some cases, the signals switch on expression of their target genes from a repressed state. For example, in the absence of Notch, Wnt, Hedgehog or nuclear receptor signaling, the expression of target genes is repressed. There is a common mechanism among these cases. A specific factor or complex, such as Su(H)/CBF1 in the Notch pathway, Lef/Tcf in the Wnt pathway, Ci/Gli in the Hh pathway or the nuclear receptors themselves, bind to a specific DNA sequence to prevent target genes from being transcribed. When the signaling pathway is active, the repression is relieved by the processed receptor, activated activator, co-activators or by the nuclear receptor itself. Results from this study indicated that both GAF-associated proteins, Hsf and Ttk69, constitute a dual al switch for tll expression that includes degradation of the Ttk69 co-repressor and conversion of the Hsf repressor into an activator after Mapk phosphorylation, where Mapk is a downstream effector of the Tor pathway (Chen, 2009).

Transcriptional Regulation

Activation of the receptor tyrosine kinase (RTK) Torso defines the spatial domains of expression of the transcription factors Tailless and Huckebein in the posterior. Torso regulates tailless and huckebein through the elements of the Ras pathway. Transcription of tll during early embryogenesis is apparently activated via a phosphorylation cascade containing Torso, and the fly's serine-threonine kinase Raf homolog. Corkscrew, a tyrosine phosphatase in the Torso pathway, also helps regulate tailless transcription. The MEK homolog (downstream of raf1) is involved in both positive and negative regulation of tailless (Tsuda, 1993). Raf, required for expression of tailless and huckebein can be activated by RTK in a Ras-independent pathway (Hou, 1995).

The terminal portions of the Drosophila body pattern are specified by the localized activity of the receptor tyrosine kinase Torso (Tor) at each pole of the early embryo. Tor activity elicits the transcription of two 'gap' genes, tailless (tll) and huckebein (hkb), in overlapping but distinct domains by stimulating the Ras signal transduction pathway. Quantitative variations in the level of Ras activity can specify qualitatively distinct transcriptional and morphological responses. Low levels of Ras activity at the posterior pole direct tll but not hkb transcription; higher levels drive transcription of both genes. Correspondingly, low levels of Ras activity specify a limited subset of posterior terminal structures, whereas higher levels specify a larger subset. When a constitutively active 1X RasV12 gene is expressed in torso mutant embryos, brachyenteron (byn) is expressed in a small terminal cap, whereas the domain of expression in 2X RasV12 is much broader. Because both the activation and repression of terminal byn expression is known to depend, respectively, on tll and hkb, it is surmised that higher levels of Ras activity are required at the posterior of wild-type (ras+) embryos to drive sufficiently high levels of Hkb expression to repress byn expression (a phenomenon not observed with even 2X RasV12 ectopic expression). 1X RasV12 forms the least terminal of the posterior terminal structures: the eighth abdominal dentical band and the posterior spiracles. The extent of restoration is considerably greater in 2X RasV12 embryos: these form additional terminal structures such as the anal tuft and anal pads.The response to Ras activity is not uniform along the body. Instead, levels of Ras activity that suffice to drive tll and hkb transcription at the posterior pole fail to drive their expression in more central portions of the body, apparently due to repression by other gap gene products. The levels of Huckebein and/or Kruppel through the embryo might be responsible for a failure to express hkb in response to moderate RasV12 activity. It is concluded that tll and hkb transcription, as well as the terminal structures, are specified by two inputs: a gradient of Ras activity, which emanates from the pole, and the opposing influence of more centrally deployed gap genes, which repress the response to Ras (Greenwood, 1997).

Tailless acts as a repressor of Kruppel and knirps in the central domain of the recently fertilized embryo. Groucho acts throughout the embryo to repress the repressor of Kruppel and knirps, allowing the expression of these gap genes in the central domain of the embryo. Patterning of the non-segmental termini of the Drosophila embryo depends on signaling via the Torso receptor tyrosine kinase. Activation of Torso at the poles of the embryo triggers expression of the terminal zygotic gap genes tailless (tll) and huckebein (hkb). The Groucho (Gro) corepressor acts in this process to confine terminal gap gene expression to the embryonic termini. Embryos lacking maternal gro activity display ectopic tll and hkb transcription; in turn, tll then leads to lack of abdominal expression of the Kruppel and knirps gap genes. torso signaling permits terminal gap gene expression by antagonizing Gro-mediated repression. Groucho-mediated repression of tailless is relieved by the torso pathway suggesting that Groucho is the nuclear target for MAP kinase signaling. It is suggested that Groucho functions as a corepressor along with an unknown protein unrelated to Hairy, since Groucho mediated repression takes place in the absence of known Hairy-related bHLH proteins. Thus, the corepressor Gro is employed in diverse developmental contexts and, probably, by a variety of DNA-binding repressors (Paroush, 1997).

BCD and the terminal system are both required to activate the anterior-dorsal stripe of tll expression that correlates with formation of the acron. In the absence of bcd, the anterior cap of tll expression established by the terminal system persists and an ectopic telson forms at the anterior; in the absence of terminal system activity, only an abnormal anterior stripe forms (Pignoni, 1992).

Mutations in torso and trunk that express low levels of the respective protein have differential affects on the expression of tailless and huckebein. For example a reduced amount of TRK can trigger signaling of TOR to levels required to activate tll but not hkb. For a given number of TOR receptors, an increase in the amount of TRK results in the appearance of more structures of the most posterior segment (A8) (Furriols, 1996).

The role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK) has been examined. Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. Using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, it has been demonstrated that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. When terminal activity is progressively removed, there is a corresponding progressive malformation and eventual loss of terminal cuticular structures. The first terminal cuticular elements that are malformed or lost require the highest terminal activation (e.g., the anal tuft and posterior spiracles visualized by the presence of Filzkorper material). The next elements that are malformed or lost require intermediate levels of terminal signal (e.g., the abdominal 8 (A8) denticle belt and the posterior spiracles). Finally, the last elements that are malformed or lost require the lowest levels of terminal activity (e.g., posterior A7). While in the absence of D-raf activity, no activated MAPK (dp-ERK) is observed at the posterior pole. In csw null mutant embryos, where the tll and Hb expression domains are present though mispositioned, reduced levels of dp-ERK reactivity are observed. Collectively, these results reveal that a precise transcriptional response translates into a specific cell identity (Ghiglione, 1999).

Two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. For example, analysis of Torextracellular-Egfrcytoplasmic reveals that the posterior domain of Hunchback does not retract from the posterior pole, but rather remains as a terminal cap. Further, the anterior border of this posterior Hb domain is shifted posteriorly. Altogether, these results illustrate how a gradient of MAPK activity controls differential gene expression and thus, the establishment of various cell fates. The roles of quantitative mechanisms in defining RTK specificity are discussed. It is possible that in some instances, the generation of differing magnitudes of activity from the cytoplasmic domains of specific RTKs might be dependent on the specific affinities of the downstream signal transducers to the receptor. Csw binds through one of its SH2 domains to only one phosphotyrosine on Tor. Perhaps a higher or lower affinity of Csw to this site, or addition of another site that would also engage the second SH2 domain of Csw, would increase or decrease signal output. Presumably, in each individual cell there exists a mechanism built into the enhancer elements of the promoters of both tll and hkb that acts to read directly the magnitude of Tor signaling. In the tll promoter, a Tor-response element that mediates the repression of tll has been identified, indicating that the Tor signal activates tll by a mechanism of derepression. A putative candidate for this repressor activity is encoded by the transcription factor Grainyhead. Grainyhead binds to the Tor-response element and can be directly phosphorylated by MAPK in vitro: a decrease in Gh activity has been shown to cause tll expansion in early embryos. Further, the transcriptional corepressor Groucho is required for terminal patterning. Further characterization of how Gh and/or Gro activities are regulated by activated MAPK should clairify how differing levels of phosphorylation translate into derepression of terminal target genes (Ghiglione, 1999).

Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Csw functions throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, cswlf, has allowed a genetic approach to identify loci required for Csw function. To investigate the effect of the cswlf lesion during embryonic RTK signaling, females bearing cswlf germline clones were mated to hemizygous cswlf males and the resulting cswlf/cswlf female and cswlf/Y male progeny were examined. That cswlf is the weakest of the series of csw alleles is supported by the analysis of the expression patterns of tll and hkb, transcription factors whose expression directs the formation of larval terminal structures and is dependent on the activity of the Torso RTK. All of the previously identified csw alleles reduce, to varying degrees, the posterior expression of both tll and hkb; however, the cswlf mutation does not affect the expression of either tll or hkb (Firth, 2000).

During early embryogenesis in Drosophila, Caudal mRNA is distributed as a gradient with its highest level at the posterior of the embryo. This suggests that the Caudal homeodomain transcription factor might play a role in establishing the posterior domains of the embryo, which undergo gastrulation and give rise to the posterior gut. By generating embryos lacking both the maternal and zygotic mRNA contribution, caudal has been shown to be essential for invagination of the hindgut primordium and for further specification and development of the hindgut. Mature embryos lacking cad activity (maternal and/or zygotic contributions) were examined to assess the requirement for cad in establishing the structures that arise from the posterior ~15% of the blastoderm embryo, namely the posterior midgut, Malpighian tubules and hindgut (Wu, 1998).

The stages of gastrulation can be observationally followed by using expression of brachyenteron byn as a marker for the hindgut primordium. In the wild-type embryo, byn is expressed in a ring at the circumference of the amnioproctodeal plate. The edges of this ring come together as the posterior midgut primordium invaginates during stages 6 and 7; the ring of the hindgut primordium then sinks inward during stage 8 and is completely internalized by the end of stage 9. The zygotically expressed cad stripe and the posterior wg stripe are also expressed in the bordering ring (i.e., the hindgut primordium) of the invaginating amnioproctodeal plate. Strikingly, in cad-deficient embryos, the byn-expressing ring of hindgut primordium draws together, but fails to invaginate, remaining on the outside of the embryo. Thus, although internalization of the Malpighian tubule and posterior midgut primordia is normal in cad-deficient embryos, the gastrulation movements necessary for internalization of the hindgut primordium do not occur in embryos lacking cad activity (Wu, 1998).

The absence of the hindgut primordium from cad-deficient embryos suggests that Caudal regulates genes required for establishing and/or maintaining the hindgut primordium. tailless, forkhead, byn, bowl and wingless are likely targets for cad regulation, since all are required for some aspect of hindgut development: the hindgut is missing from both tll and fkh embryos, and severely reduced in wg, byn and bowl embryos. bowl, also called bowel, codes for a zinc finger transcription factor related to odd-skipped. Since maternally provided Caudal, which persists only through the blastoderm stage, is sufficient for essentially normal hindgut formation, the fact that all of these genes are expressed at the posterior of the embryo during the blastoderm stage means that they are potential targets for regulation by Caudal. The effect of absence of maternal and/or zygotic cad activity on the expression of these genes was assessed by in situ hybridization with appropriate probes. For tll, byn and bowl, absence of cad activity does not result in a detectable effect on expression. As described below, however, cad activity is essential for expression of fkh and wg. Both maternal and zygotic cad contributions are necessary for posterior wg expression. During early stage 5, just prior to its expression in 14 stripes that are required to establish the segmental pattern, wg is expressed in two domains at the anterior, and in a broad posterior stripe. This terminal wg stripe is located at approximately 8-12% EL, overlapping with the posterior of the zygotic cad stripe and with the position of the hindgut and Malpighian tubule primordia in the blastoderm fate map. Expression of the wg terminal stripe has been shown to be independent of other segmentation genes, but has not been otherwise characterized. All embryos from mutant cad germline mothers (even those expressing zygotic cad) fail to express the terminal stripe of wg. These results demonstrate that maternal cad activity is essential for the transcription of wg in the terminal stripe. Among embryos from wg heterozygous parents, approximately one-quarter (presumably those lacking only the zygotic component of cad expression) lack the terminal wg stripe. Thus both maternal and zygotic cad activities are required for expression of the terminal wg stripe (Wu, 1998).

The expression of the early cap of fkh also requires cad activity; approximately half of the embryos from mutant cad germline females mated to cad heterozygous males (i. e., cad m-z - embryos) show a dramatic reduction in both the size and intensity of the posterior cap of fkh expression. However, if cad is supplied either maternally or zygotically, fkh expression is normal. Thus expression of the posterior cap of fkh requires cad activity, which can be provided either maternally or zygotically. Later, by stage 10, fkh expression is as strong in cad-deficient as in wild-type embryos, indicating that this later expression is independent of cad activity. Since tll and hkb are also required to activate early fkh expression but are not themselves regulated by cad, cad must act combinatorially with these two genes to promote early fkh expression (Wu, 1998).

cad also regulates wg in combination with other genes. In addition to the demonstrated requirement for cad, expression of the posterior wg stripe requires positive input from fkh and tll, since the stripe is absent from the respective mutant embryos. Since embryos lacking either maternal or zygotic cad fail to express the posterior wg stripe, but still express fkh and tll, cad must act combinatorially with fkh and tll to promote formation of the posterior wg stripe. Expression of the terminal stripe thus requires the combinatorial action of cad, tll and fkh; the posterior limit of the stripe is known to be defined by repression by hkb (Wu, 1998).

The failure of the hindgut to become internalized in caudal-deficient embryos raises the question of whether cad might regulate a zygotically expressed gene required for the invagination of the amnioproctodeal plate. One gene known to be required for gastrulation is fog; fog mutant embryos lack not only the posterior midgut, but, as revealed by anti-Crb staining, the Malpighian tubules and hindgut as well. In the blastoderm stage embryo, fog expression is first activated in the region that will become the ventral furrow; shortly thereafter, expression is initiated in a posterior cap, in the region that will become the amnioproctodeal invagination. In cad-deficient embryos, fog expression in the prospective ventral furrow is normal, but is significantly reduced in the posterior cap. Thus, cad is required for the normal level of expression of fog in the prospective amnioproctodeal plate; decreased fog expression in cad-deficient embryos is likely responsible for the failure of the hindgut primordium to be internalized during gastrulation. Since fkh or wg mutant embryos do not display detectable defects in gastrulation, fog is the only gene presently known to mediate the effects of cad on gastrulation. In fog mutant embryos, none of the posterior gut primordia invaginate, while in cad-deficient embryos the posterior midgut and Malpighian tubule primordium do invaginate; thus, consistent with the in situ hybridization results, a low level of fog activity is present at the posterior of embryos lacking cad (Wu, 1998).

In addition to cad, three other genes (fkh, byn and wg,), which are required at the posterior of the Drosophila embryo for formation of the hindgut, are related to genes found throughout the metazoa, known as HNF-3 (alpha, beta, and gamma), Brachyury (also known as T) and Wnt, respectively. In many cases, these homologs are expressed in portions of the 'blastopore equivalent' at the posterior of the embryo, that overlap with domains of expression of cad (Cdx). In C. elegans, a Wnt homolog is expressed, and required for proper posterior development, in the same posterior blastomere where the cad homolog pal-1 functions. In sea urchin, HNF-3 and Brachyury homologs are expressed in the vegetal plate just prior to gastrulation. In fish and frog, Caudal, Brachyury and Wnt (Wnt8 and Wnt11) are initially expressed around most or all of the blastopore lip while HNF-3 expression is dorsally localized. As gastrulation proceeds, the expression of these genes becomes more restricted and non-overlapping, with HNF-3 and Brachyury expression becoming localized to the notochord and Wnt8 expression retreating from the dorsal position and becoming exclusively ventral. Patterns of expression of HNF-3 and Brachyury consistent with this general description have been found in ascidians, amphioxus, chick and mouse. Required roles for some of these genes have been demonstrated by analysis of mutants: mouse HNF-3beta knockouts reveal requirements in the formation of the node, notochord and head process; fish no tail and mouse T mutants reveal a requirement for Brachyury in migration of mesoderm through the primitive streak and in formation of the notochord. There is thus a constellation of conserved genes -- cad (Cdx), fkh (HNF-3), wg (Wnt8 and Wnt11) and byn (Brachyury) -- whose overlapping expression patterns in the blastopore equivalent suggests function in a related process. The phenotypes of the available mutations in these genes suggest that the common function is to specify cell fate at the blastopore; in most cases, essential parts of this fate are internalization and forward migration, two of the cellular movements that occur during gastrulation (Wu, 1998 and references).

The striking conservation in expression (and likely in function) of cad suggests that the regulation of posterior terminal development in Drosophila by Caudal may represent a more ancient regulatory mechanism than the tor receptor and the two genes that it activates: tll and hkb. Of these three genes, a vertebrate homolog is known only for tll; the function of this vertebrate gene, Tlx, is related to that of Drosophila tll not in the posterior, but rather in the anterior, in the establishment of the brain. Thus the Torso receptor pathway and its activation of tll and hkb has probably been superimposed relatively recently (in evolutionary terms) upon a more ancient, Caudal-regulated network of gene activity controlling gastrulation and gut formation. The fact that the same four genes are expressed at both the blastopore equivalent of chordates and at the amnioproctodeal invagination of Drosophila suggests that these two highly dynamic domains are homologous. Given the regulatory hierarchy that is present in Drosophila, it is proposed that in embryos of the proximate ancestor to arthropods and chordates, the posterior was defined by a posterior-to-anterior gradient of Cad activity. Cad is thought to have then activated expression of downstream network of genes in control of invagination (gastrulation) and gut specification. Cad expression in the archenteron probably continued during evolution and played an essential developmental role, since this structure differentiated into the gut. Going beyond the bilaterian ancestor to chordates and arthropods, it is worth considering that this nexus of gene expression may have evolved even more basally in the metazoa. The foregoing, by homologizing the insect amnioproctodeal invagination with the echinoderm and vertebrate blastopore, does not fit with the classical definition of protostomes and deuterostomes. This view categorizes arthropods as protostomes, in which the mouth is derived from the primary invagination of gastrulation; chordates are categorized as deuterostomes, where the mouth arises from a secondary invagination. More recently, comparisons of gastrulation patterns in many different species, as well as construction of molecularly based cladograms, have called into question the utility of these classically defined groups. While there continues to be uncertainty in understanding of 'protostome' and 'deuterostome' phyla, the significant conclusion of the information presented here is that there may be a homology between the blastopore of vertebrates and the amnioproctodeal (posterior) invagination of insects (Wu, 1998 and references).

capicua is involved in gene repression in Drosophila terminal and dorsoventral patterning. Given the similarities between torso gain of function mutations (torgof) and cic1 phenotypes, the expression patterns of tailless and huckebein were examined in capicua1 (cic1) embryos. Expression of both genes expands toward the center of such embryos, predominantly in the posterior domain. The expanded expression of tll and hkb is very similar to that observed in torgof and groucho mutant embryos. The expression of a lacZ transgene under the control of a tor-RE from the tll promoter that drives terminal-specific transcription has also been examined. In cic1 mutant embryos, expression of this construct is derepressed toward the middle of the embryo. Together, these results suggest that the cic gene is normally required to restrict tll and hkb expression to the embryonic poles (Jimenez, 2000).

cic could affect tll and hkb expression by restricting Tor signaling to the embryonic poles (e.g., by limiting the domain of Tor receptor activation, or the domain of Tor signal transduction inside of the embryo). Alternatively, cic could function, like gro, as a repressor of tll and hkb downstream of the Tor pathway. To help distinguish between these possibilities, epistasis analyses were performed using loss-of-function mutations in tor, Draf, and Dsor (encoding a Drosophila MAPK kinase homolog) that normally cause a phenotype complementary to that of cic1, that is, absence of terminal structures. Embryos from females homozygous for cic1 and tor are identical to those from cic1 females alone. Likewise, cic1 females carrying loss-of-function clones of Draf or Dsor in the germ line produce embryos that display the cic phenotype. Thus, cic acts genetically downstream of Draf and Dsor. In addition, the domain of Tor signal activity was examined directly using a monoclonal antibody against the active, diphosphorylated form of Drosophila MAPK (known as Erk) and a normal pattern of Erk activation was found in cic1 embryos. This shows that derepression of tll and hkb in cic1 mutant embryos is not due to an expanded domain of Tor signaling, suggesting that cic is part of the activity that represses tll and hkb in the central region of the embryo and is inhibited by Tor signaling at the embryonic poles (Jimenez, 2000).

The similar effects of cic and gro on terminal patterning raise the possibility that cic is necessary for Gro corepressor activity in general. However, two lines of evidence argue against this idea: (1) Gro participates in many developmental processes, whereas the role of cic appears restricted to terminal and dorsoventral patterning; (2) Gro-dependent repression by Hairy in a sex determination assay does not require cic function. These results indicate that cic does not generally affect Gro activity (Jimenez, 2000).

During early Drosophila and C. elegans development, the germ cell precursors undergo a period of transcriptional quiescence. Germ cell-less (Gcl), a germ plasm component necessary for the proper formation of 'pole cells', the germ cell precursors in Drosophila, is required for the establishment of this transcriptional quiescence. While control embryos silence transcription prior to pole cell formation in the pole cell-destined nuclei, this silencing does not occur in embryos that lack Gcl activity. The failure to establish quiescence is tightly correlated with failure to form the pole cells. Furthermore, Gcl can repress transcription of at least a subset of genes in an ectopic context, independent of other germ plasm components. These results place Gcl as the earliest gene known to act in the transcriptional repression of the germline. Gcl's subcellular distribution on the nucleoplasmic surface of the nuclear envelope (Jongens, 1994) and its effect on transcription suggest that it may act to repress transcription in a manner similar to that proposed for transcriptional silencing of telomeric regions (Leatherman, 2002).

gcl is required to repress transcription during the establishment of the germ cell lineage. To determine if this activity is dependent or independent of other germ plasm components, the effect of ectopically localizing Gcl on transcription was examined. Replacement of the 3'UTR of the gcl transcript with the 3'UTR of bicoid results in the anterior localization of gcl mRNA and protein to the anterior pole of the embryo. In these 'hgb' embryos, a slightly variable but consistent decrease was found in the intensity of H5 staining (H5 is a monoclonal antibody that recognizes a phosphorylated form of RNA polymerase II that is associated with active transcription) in the anterior nuclei compared to control embryos throughout the syncytial blastoderm stage, and this decrease indicates that Gcl is sufficient to repress transcription ectopically. However, the anterior expression of Gcl clearly does not lead to complete silencing of the anterior nuclei, since some H5 staining persists (Leatherman, 2002).

The reduced H5 staining observed in the anterior of the hgb embryos could be due to global partial repression of all genes, or it could result from a specific subset of genes being severely repressed while others are unaffected. To distinguish between these possibilities, the expression was examined of specific genes whose expression pattern includes the anterior of the embryo, including sisA, sisB (scute), tailless, huckebein, hunchback, and knirps. These genes are all independently activated by maternally contributed factors, so any effects on their transcription are likely to be direct rather than a consequence of an earlier defect. By using in situ hybridization, it was found that the early anterior expression domains of sisA, sisB, tailless, and huckebein are severely repressed in all of the hgb embryos examined, but no effect was seen on hunchback and knirps expression. These data suggest that the transcriptionally repressive effect of Gcl is not global, but rather specific to a subset of genes. Gcl is also present in a variety of tissues later in development, at times when transcription is active, which further suggests a non-global mode of silencing (Leatherman, 2002).

Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline: Regulation of tailless transcription

In Drosophila, the germline precursor cells, i.e. pole cells, are formed at the posterior of the embryo. As observed for newly formed germ cells in many other eukaryotes, the pole cells are distinguished from the soma by their transcriptional quiescence. To learn more about the mechanisms involved in establishing quiescence, a potent transcriptional activator, Bicoid (Bcd), was ectopically expressed in pole cells. Bcd overrides the machinery that downregulates transcription, and activates not only its target gene hunchback but also the normally female specific Sex-lethal promoter, Sxl-Pe, in the pole cells of both sexes. Unexpectedly, the terminal pathway gene torso-like is required for Bcd-dependent transcription. However, terminal signaling is known to be attenuated in pole cells, and this raises the question of how this is accomplished. Evidence is presented indicating that polar granule component (pgc) is required to downregulate terminal signaling in early pole cells. Consistently, pole cells compromised for pgc function exhibit elevated levels of activated MAP kinase and premature transcription of the target gene tailless (tll). Furthermore, pgc is required to establish a repressive chromatin architecture in pole cells (Deshpande, 2004).

The germline of Drosophila is derived from a special group of cells called pole cells that are formed during early embryonic development. The Drosophila embryo initially develops as a syncytium of rapidly dividing nuclei that undergo multiple rounds of synchronized mitotic cycles. Prior to the tenth division cycle, several nuclei migrate into the specialized cytoplasm or pole plasm at the posterior of the embryo. These nuclei cellularize precociously and these newly formed cells divide two or three times to produce ~30-35 germline precursor cells. The remaining nuclei migrate to the surface of the embryo at nuclear division cycle 10-11. They then undergo several more synchronous divisions and cellularize at the end of nuclear cycle 14 to form the cellular blastoderm (Deshpande, 2004 and references therein).

In addition to their earlier cellularization and slower rate of mitosis, pole cells differ in their transcriptional activity. Somatic nuclei substantially upregulate RNA polymerase II transcription after they migrate to the surface of the embryo. The activation of zygotic gene expression is essential for these nuclei to respond appropriately to the maternal pathways that assign positional information along the axes of the embryo. By contrast, pole cell nuclei shut down RNA polymerase II transcription when they enter the pole plasm and they then remain transcriptionally quiescent until much later stages of embryogenesis. Transcriptional quiescence is a hallmark of germline precursor cells in many organisms. For example, in C. elegans, RNA polymerase II transcription is repressed in the germ cell lineage by the product of the pie-1 gene. Transcriptional inactivity appears to be crucial in establishing germ cell identity as mutations in pie-1 switch the fate of these cells to that of a somatic lineage (Deshpande, 2004 and references therein).

A number of maternally derived gene products are likely to contribute to transcriptional quiescence in the pole cells of Drosophila. One of these is Germ cell less (Gcl), a component of the germ plasm that is necessary for the formation of pole cells. gcl appears to be involved in the establishment of transcriptional quiescence and in embryos lacking gcl activity, newly formed pole buds are unable to silence the transcription of genes such as sisterless-a and scute. Conversely, when Gcl protein is ectopically expressed in the anterior of the embryo it can downregulate the transcription of terminal group genes such as tailless (tll) and huckebein (Leatherman, 2002). A second maternally derived gene product involved in transcriptional quiescence is Nanos. In the soma, Nanos, together with Pumilio, plays a key role in posterior determination by blocking the translation of maternally derived hunchback (hb) mRNA. Nanos (Nos) also plays a role in down-regulating transcription in pole cells, and in embryos produced by nos mutant mothers: genes that are normally active only in somatic nuclei are inappropriately transcribed in pole cells. These include the pair-rule genes fushi tarazu and even skipped, and the somatic sex determination gene Sex-lethal (Deshpande, 2004 and references therein).

The global effects of nos and gcl mutations on RNA polymerase II activity in pole cells are analogous to those seen in pie-1 mutants in C. elegans. In pie-1 mutants, genes that are normally expressed only in somatic lineages are turned on in the germ cell lineage. In wild-type C. elegans embryos, the inhibition of transcription in the germ cell lineage is correlated with a marked reduction in phosphorylation of the CTD repeats of the large subunit of RNA polymerase II (Seydoux, 1997). The CTD repeats are phosphorylated when polymerase is transcriptionally engaged. PIE-1 protein may prevent transcription by inhibiting this modification. As in C. elegans, the RNA polymerase II CTD repeats are underphosphorylated in the pole cells of wild-type Drosophila embryos. In the pole cells of gcl and nos mutant embryos, however, the level of CTD phosphorylation is elevated (Leatherman, 2002; Deshpande, 2004 and references therein).

Previous studies have shown that when a heterologous transcriptional activator, GAL4-VP16, is expressed in pole cells, it is unable to activate transcription of target gene(s) (Van Doren, 1998). This finding suggests that even if a potent activator were to be produced in pole cells, it would not be able to overcome the inhibition of the basal transcriptional machinery by gcl, nos and other factors. However, since GAL4-VP16 is a chimera of a yeast DNA-binding domain and a mammalian activation domain, an alternative possibility is that co-factors essential for its activity may be absent or inactive in Drosophila pole cells. For these reasons, tests were performed to see whether a transcription factor that is normally present and active in the somatic cells of early Drosophila embryos can promote the transcription of target genes when inappropriately expressed in pole cells. The homeodomain protein Bicoid (Bcd), which activates the zygotic transcription of hb and other genes specifying anterior development, was tested. A Bcd protein gradient is generated in precellular blastoderm embryos from the translation of maternal mRNA localized at the anterior pole. Although Bcd is not present in the posterior of wild-type embryos, increasing the bcd gene dose results in expansion of the gradient toward the posterior and a concomitant change in the pattern of zygotic gene expression. This result suggests that co-factors crucial for Bcd function are likely to be ubiquitous (Deshpande, 2004 and references therein).

Ectopic expression of Bcd in pole cells can induce the transcription of the bcd target gene hb. In addition to activating hb transcription, Bcd protein perturbs the migration of the pole cells to the primitive somatic gonad and causes abnormalities in cell cycle control. These effects on germ cell development resemble those observed in embryos from nos mutant females. Moreover, as in the case of nos- pole cells, the Sxl promoter Sxl-Pe is also turned on in pole cells by Bcd in a sex-nonspecific manner. Surprisingly, transcriptional activation in pole cells by Bcd requires the activity of the terminal signaling system. This observation is unexpected, since previous studies have established that the transcription of a downstream target gene of the terminal pathway, tailless (tll) is shut down completely in pole cells. Moreover, the doubly phosphorylated active isoform of MAP kinase ERK, which serves as a sensitive readout of the terminal pathway, is nearly absent in pole cells. Taken together, these findings argue that the activity of terminal signaling pathway in pole cells of wild-type embryos must be substantially attenuated, but not shut off completely. What mechanisms are responsible for downregulating terminal signaling in the presumptive germline? Evidence indicates that polar granule component (pgc) functions to attenuate the terminal pathway in newly formed pole cells. pgc encodes a non-translated RNA that is localized in specialized germ cell-specific structures called polar granules (Nakamura, 1996). Loss of pgc function in newly formed pole cells results in the ectopic phosphorylation of ERK and the activation of the ERK dependent target gene tll. pgc is required to block the establishment of an active chromatin architecture in pole cells (Deshpande, 2004).

Thus Bcd protein expressed from a bcd-nos3'UTR transgene (the 3' UTR of nos serves to localize the bcd message to pole cells) can activate the transcription of its target gene hb in pole cells, overcoming whatever mechanisms are responsible for transcriptional quiescence. In addition to activating transcription of hb, Bcd has other phenotypic effects. It prevents the pole cells from properly arresting their cell cycle and disrupts their migration to the somatic gonad. Because similar defects in pole cell development can be induced by the inappropriate expression of Sxl protein in these cells, one plausible hypothesis is that Bcd not only activates the hb promoter, but also turns on the Sxl establishment promoter, Sxl-Pe. Consistent with this idea, the Sxl-Pe:lacZ reporter is turned on in the pole cells of male and female bcd-nos 3' UTR embryos and Sxl protein accumulates in these cells. Although previous studies indicate that Sxl-Pe is responsive to Bcd, it is somewhat surprising that Sxl-Pe is not only inappropriately turned on in pole cells by Bcd, but that it is activated in both sexes. This suggests that Bcd activation of Sxl-Pe in pole cells must proceed by a mechanism that bypasses the X/A chromosome counting system which controls Sxl-Pe activity in the soma. It is interesting to note that the activation of Sxl-Pe in pole cells in the absence of nos function also seems to depend upon a mechanism(s) that circumvents the X/A chromosome counting system (Deshpande, 2004).

That Bcd protein depends upon other ancillary factors to turn on transcription in pole cells is demonstrated by the requirement for tsl function in the activation of both the hb and Sxl-Pe promoters. tsl is a component of the maternal terminal signaling pathway that activates the zygotic genes, tll and huckebein (hkb), at the poles of the embryo. In addition, the terminal pathway has opposing effects on the expression of bcd-dependent gap genes. At the anterior pole, where terminal signaling activity is highest, Bcd targets such as hb and orthodenticle (otd) are repressed. At a distance from the anterior pole, where both the concentration of Bcd protein and the strength of the terminal signaling cascade is much lower, the terminal pathway has an opposite, positive effect on hb and otd expression. Two mechanisms are thought to account for the positive effects of the terminal pathway on bcd target genes: (1) Bcd is a direct target for phosphorylation by the terminal signaling cascade; (2) regulatory regions of bcd target genes have sites for other transcription factors whose activity can be directly modulated by the terminal system (Deshpande, 2004).

The concentration of Bcd protein produced by the bcd-nos 3' UTR transgene in pole cells is much less than it is at the anterior pole. Similarly, the activity of the terminal signaling cascade in pole cells is much reduced compared with that in the somatic nuclei at the anterior and posterior poles. Thus, in both of these respects, the conditions in the bcd-nos 3' UTR pole cells would appear to most closely approximate those in the region of the embryo where the terminal signaling cascade potentiates rather than inhibits Bcd activity. This would explain why activation of transcription in pole cells by Bcd depends on the terminal signaling pathway and why in this particular instance this pathway does not antagonize the activity of the ectopically expressed Bcd protein (Deshpande, 2004).

The fact that the terminal pathway can function in pole cells, yet does not turn on its target gene tll indicates that the activity of this pathway is attenuated in the germline. It seems likely that several different mechanisms may be responsible for preventing pole cells from responding to the terminal pathway and turning on tll transcription. One mechanism appears to be an inhibition of the signaling cascade itself. In the posterior and anterior soma of pre-cellular blastoderm embryos, the terminal signaling cascade directs the phosphorylation of the MAP kinase ERK. While phosphorylated ERK can also be detected in wild-type pole cells, the amount of activated kinase is much less than in the surrounding soma. Consistent with this observation, potentiating the terminal system using either a gain-of-function torso receptor mutant or by expressing elevated levels of the receptor in pole cells using a torso transgene (which has the nos 3' UTR) had only a small effect on the activity of a tll-lacZ reporter in the germline. By contrast, gain-of-function torso mutation substantially upregulates the tll reporter in the soma (Deshpande, 2004).

To identify factors that could be involved in repressing the terminal pathway in pole cells, three genes, nos, gcl and pgc, were examined that are known to play an important role in the early development of the germline and have been implicated in transcriptional quiescence. Of these three, only pgc appears to have significant effects on the terminal signaling pathway in pole cells. The expression of a tll reporter is turned on in pole cells of embryos deficient in pgc activity. That this is due at least in part to a failure to properly attenuate the terminal signaling pathway in the germline is suggested by the fact that the level of activated ERK is greatly elevated in pgc pole cells compared with wild type. Although these findings implicate pgc in downregulating the terminal pathway, how this is accomplished and whether pgc has a direct rather than an indirect role in this process remains to be determined. In addition, these studies indicate that pgc has functions in addition to attenuating this signaling cascade: (1) it was found that there are abnormalities in the formation of pole cells in pgc embryos and Vasa-positive 'cells' are observed in cycle 9-10 embryos at abnormal locations; (2) the loss of pgc activity may lead to the inappropriate activation of genes in addition to tll. Two markers for global transcriptional activity, CTD phosphorylation and histone H3 K4 methylation, are present in pole cells of pgc embryos (Deshpande, 2004).

The results also suggest that multiple and interrelated levels of regulation are responsible for ensuring transcriptional quiescence in the pole cells. For example, Sxl-Pe can be upregulated by the terminal pathway in the soma and requires this pathway to be activated by Bcd in pole cells. However, this promoter is not activated in pole cells in the absence of pgc function. Thus, the activation of the terminal signaling cascade in pole cells is not sufficient in itself to induce Sxl-Pe. This suggests that mechanisms are in place in pgc pole cells that would override any effects of activated ERK on Sxl-Pe activity. Similarly, although loss of nos activity leads to the activation of Sxl-Pe in pole cells, and the upregulation of tll in the posterior soma, the tll promoter is not turned on in nos pole cells. It is presumed that tll is not activated in pole cells because it requires the terminal system that still remains attenuated in nos pole cells. Redundancy is also suggested by the finding that although the loss of gcl leads to the expression of the X chromosome counting genes sis-a and scute in pole cells (Leatherman, 2002), Sxl-Pe is not activated, suggesting that nos function is sufficient to keep Sxl-Pe off in gcl mutant pole cells even though several X chromosome counting genes are activated. Similarly, no obvious effect was observed of nos mutations on scute expression in pole cells. This implies that gcl and nos may be responsible for repressing the transcription of different sets of genes (Deshpande, 2004).

Finally, although transcription is upregulated in pgc pole cells between nuclear cycles 9/10-13, a high level of transcriptional activity is not maintained in the pole cells that are present by the time the cellular blastoderm is formed. The tll reporter is turned off, and both CTD phosphorylation and histone H3 K4 methylation disappear. One possible interpretation of this finding is that pgc has an early function in establishing transcriptional quiescence, but is not required after nuclear cycle 13 because of the activity of other factors such nos or gcl. However, since the number of pole cells at cellularization is reduced compared with the number present earlier, it also possible that the only pole cells that remain are the ones in which the amount of pgc activity is sufficient to establish some degree of transcriptional repression. Further studies with bona fide null alleles will be required to resolve this question, and to understand how pgc functions during pole cell formation and germ cell determination (Deshpande, 2004).

Capicua integrates input from two maternal systems in Drosophila terminal patterning

In Drosophila, the maternal terminal system specifies cell fates at the embryonic poles via the localised stimulation of the Torso receptor tyrosine kinase (RTK). Signalling by the Torso pathway relieves repression mediated by the Capicua and Groucho repressors, allowing the restricted expression of the zygotic terminal gap genes tailless and huckebein. This study reports a novel positive input into tailless and huckebein transcription by maternal posterior group genes, previously implicated in abdomen and pole cell formation. Absence of a subset of posterior group genes, or their overactivation, leads to the spatial reduction or expansion of the tailless and huckebein posterior expression domains, respectively. The terminal and posterior systems converge, and exclusion of Capicua from the termini of posterior group mutants is ineffective, accounting for reduced terminal gap gene expression in these embryos. It is proposed that the terminal and posterior systems function coordinately to alleviate transcriptional silencing by Capicua, and that the posterior system fine-tunes Torso RTK signalling output, ensuring precise spatial domains of tailless and huckebein expression (Cinnamon, 2004).

Terminal gap gene expression must be tightly regulated for the correct specification of terminal cell fates at the nonsegmented poles. Clearly, the Tor pathway plays a key role in driving tll and hkb transcription, given that terminal gap genes are not expressed at the posterior end of terminal group mutants, and as a result terminal structures such as the terminal filzkorper (FK) do not form. In this paper, a novel biological role is unraveled for the maternal posterior system, showing that members of this group, in particular Nos, positively regulate transcription of the zygotic subordinate genes of the terminal system. Torso response elements (TREs) in the tll upstream regulatory region, which are derepressed in cic mutants, also respond to alterations in maternal osk dosage, and the Cic repressor is not excluded from the termini of posterior group mutants. These results are consistent with the posterior system feeding into the Tor signalling pathway, upstream of or at the level of the Cic repressor. It is suggested that the concerted activities of both the terminal and posterior systems, in their spatially overlapping zones of action, generate accurate domains of terminal gap gene expression at the posterior (Cinnamon, 2004).

It was originally proposed that the four maternal systems that pattern the early Drosophila embryo act largely independently of each other. Recent work, however, demonstrated interactions between the Tor pathway and the anterior and D/V systems. For example, tll has been shown to respond to the anterior determinant Bicoid (Bcd) even when Tor signalling is genetically blocked. Indeed, cis-acting DNA elements responsive to these three maternal systems have been found in the tll upstream regulatory region. The current results now link the terminal and posterior systems, previously thought to be independent of each other, in terminal gap gene regulation, reinforcing the idea that maternal systems that pattern the early embryo act in a coordinated manner (Cinnamon, 2004 and references therein).

Why has the positive input, by posterior group genes into terminal patterning, been largely overlooked to date? Classical segmentation studies mostly involved phenotypic analyses at the cuticular level. For this reason, and when taking into account the primary contribution of the terminal system, the delicate input by the posterior group has gone unnoticed. Thus, the unextended FK that develops in posterior group mutant background, which may arise from decreased terminal gap gene expression, had largely been attributed to pleiotropic effects arising from abdominal defects. It has been possible to detect the relatively subtle changes in tll and hkb gene expression patterns only by investigating terminal gap gene regulation at the molecular level. In fact, at least one other molecular study had previously reported reduced terminal gap gene expression in osk mutant embryos (Cinnamon, 2004 and references therein).

One emerging concept is that, for the refinement of the expression levels and spatial extents of RTK signalling targets, it is also imperative to integrate accurately information originating from other, non-RTK sources. In many cases this integration occurs at the level of target gene enhancers, with various effectors of distinct signalling pathways binding to specific DNA elements to regulate transcription. For example, D-Pax2 expression in the cone and pigment cells of the developing eye is regulated by effectors of the EGFR RTK pathway, such as Pointed P2 and Yan, and also by the Notch signalling component Suppressor of Hairless, as well as by the transcription factor Lozenge. The current study shows that terminal gap gene expression requires not only Tor RTK pathway activity but also a contribution from the posterior system. In this instance, inputs from these two maternal coordinate systems are interpreted and linked not at the level of terminal gap gene promoters but at the level of the Cic repressor. Thus, Cic functions as an integrator of multiple regulatory inputs, with both the posterior and terminal systems acting to relieve transcriptional silencing mediated by this repressor (Cinnamon, 2004).

Surprisingly, anterior tll and hkb expression is also reduced in posterior group mutants. Similarly, others have reported prolonged bcd expression and head defects in pum mutants. It is speculated that low levels of Osk and Nos, which escape translational repression, similarly regulate terminal gap gene expression via Cic removal at the anterior. In accordance with this, the dismissal of Cic from the anterior pole of posterior group mutants is also ineffective (Cinnamon, 2004).

How does Nos, which has been assigned the role of a translational repressor, positively regulate tll and hkb transcription? The results suggest that Nos does so indirectly, by downregulating the accumulation of the Cic repressor at the termini. The exact mechanism by which the Tor pathway mediates the exclusion of Cic from terminal regions has not been established, but one model argues that phosphorylation of Cic by MAPK causes degradation of the protein, as in the case of Yan. Thus, Nos could be affecting this process in one of several possible ways, at the level or downstream of MAPK. For example, Nos could be facilitating the translocation of phosphorylated MAPK into the nucleus. In posterior group mutants, then, activated MAPK would remain in the cytoplasm rather than enter the nucleus, impeding Cic phosphorylation and degradation. Alternatively, Nos may be modulating MAPK activity, or regulating adaptor proteins that promote Cic phosphorylation by nuclear MAPK. Nos may also be controlling the translation of factors that are involved in the nuclear trafficking (import/export) or degradation of Cic, or perhaps may even be acting on the cic message itself. Future studies will distinguish between these possibilities, and may shed new light on the molecular mechanisms underlying role of Nos in other developmental processes, for example, the establishment/maintenance of transcriptional quiescence in pole cells. The positive input by the posterior group genes is viewed as evolving to modulate terminal pathway activity, merging with other varied modes of Tor regulation to ultimately ensure accurate tll and hkb expression and, consequently, precise cell fate determination (Cinnamon, 2004).

The Tor signal transduction pathway is under multiple tiers of regulation, outside and inside the nucleus. For instance, internalisation and trafficking of the activated Tor receptor to the lysosome for degradation attenuates the signal, as evident by the spatial broadening and temporal prolonging of Tor activation in mutants for hrs, a component of the endosomal recycling machinery (Lloyd. 2002). Yet another level of control is provided by the tyrosine phosphatase corkscrew, which sharpens the gradient of Tor activity. Additionally, multiple cytoplasmic adaptor proteins take part in transducing the Tor signal, conceivably buffering against surplus or deficiency in signalling (Cinnamon, 2004).

In the nucleus, tll and hkb are subjected to silencing by several repressors. Derepression of tll is observed in grainy-head and tramtrack69 (ttk69) mutants, and the proteins encoded by these genes bind tll promoter sequences. Cic and Gro appear to play a leading role in terminal gap gene silencing, given that mutations in cic and gro bring about a significant expansion of the tll and hkb expression domains. Intriguingly, however, tll expression never reaches the middle of the embryo in these mutants. tll is uniformly expressed, albeit weakly, throughout the embryo only when both the developmental corepressors Gro and CtBP are removed concomitantly. This broadened tll expression likely stems from the fact that there is a redundancy in the activities that normally restrict terminal gap gene transcription from inappropriately spreading into the central portion of the embryo; by jointly removing the Gro and CtBP coregulators, activity of the above repressors is compromised. Alternatively, CtBP might be acting in conjunction with a novel, unidentified repressor that prevents tll transcription in the middlemost region of the embryo (Cinnamon, 2004).

So what is the purpose of the input by the posterior group genes into tll and hkb transcription? Quantitative differences in Tor receptor activity have to be eventually interpreted and translated into distinct cell fates at the termini. Strong Tor activation induces both hkb and tll expression, whereas weaker Tor activation only brings about tll expression. It is surmised that the precision endowed by the Tor RTK cascade may not suffice for the complex patterning of the termini, given that mere two-fold fluctuations in Tor signalling result in defective embryonic development. For example, mutants with reduced Tor RTK activity show partial tll expression and the complete loss of hkb. These mutants consequently develop incomplete terminal structures and die at the larval stage. Conversely, overactivation of the Tor pathway leads to anterior expansion of the posterior tll expression domain, perturbing segmentation in central body parts, likely as a result of downregulation of abdominal gap genes by the Tll protein. Thus, the precise spatial confinement of terminal gap gene expression domains requires the coordinated integration of regulatory inputs, coming from two maternal systems and converging on the same effector protein, Cic (Cinnamon, 2004).

Spatially distinct downregulation of Capicua repression and Tailless activation by the Torso RTK pathway in the Drosophila embryo

Specification of the terminal regions of the Drosophila embryo depends on the Torso RTK pathway, which triggers expression of the zygotic genes tailless and huckebein at the embryonic poles. However, it has been shown that the Torso signalling pathway does not directly activate expression of these zygotic genes; rather, it induces their expression by inactivating, at the embryonic poles, a uniformly distributed repressor activity. In particular, it has been shown that Torso signalling regulates accumulation of the Capicua transcriptional repressor: as a consequence of Torso signalling Capicua is downregulated specifically at the poles of blastoderm stage embryos. Extending the current model, it is shown that activation of the Torso pathway can trigger tailless expression without eliminating Capicua. In addition, analysis of gene activation by the Torso pathway and downregulation of Capicua unveil differences between the terminal and the central embryonic regions that are independent of Torso signalling, hitherto thought to be the only system responsible for confering terminal specificities. These data provide new insights into the mode of action of the Torso signalling pathway and on the events patterning the early Drosophila embryo (de las Heras, 2006).

While the Tor pathway is normally activated only at the embryonic poles, tor constitutive mutations trigger its activation over the entire embryo in a ligand-independent manner. In these cases, expression of the tor target genes is expanded too much broader domains and embryos develop head and tail structures lacking most of the segmented trunk. According to the current model one would expect that tll domain expansion in these mutations would be accompanied by an expansion of the Cic downregulation domain (de las Heras, 2006).

Embryos from mutant females bearing the torD4021 constitutive mutation (a strong gain-of-function mutation that acts as a dominant female sterile) have been analyzed and instead it was found that Cic protein is still downregulated only at the poles, as in the wild-type embryos. Therefore, while in the wild-type the posterior tll domain is complementary to the domain of Cic accumulation, in embryos from torD4021/+females these domains overlap and tll is expressed in spite of the presence of nuclear Cic. This behaviour is not allele-specific since embryos from homozygous females for another tor constitutive mutation (torRL3) display the same kind of Cic distribution and tll expression (de las Heras, 2006).

It has been postulated that wild-type Tor receptors and Tor receptors activated by ligand-independent constitutive mutations could signal through distinct downstream effectors. Therefore, whether the persistent accumulation of Cic in embryos from tor constitutive mutant females could be due to a distinct property of these mutations was analyzed. Alternatively, the persistent Cic accumulation could reflect a difference in response between Tor activation in the middle versus the terminal embryonic regions. To test these possibilities, ligand-dependent activation of the Tor receptor was triggered over the entire embryo by general expression of the torso-like (tsl) gene. tsl is the only known gene in the Tor pathway whose expression is locally restricted. Indeed its restricted expression in a group of cells at each end of the developing oocyte is the determinant for the local activation of the Tor pathway, since its ectopic expression is sufficient to induce widespread activation of the Tor receptor. Accordingly, it was found that driving tsl expression with a tubGAL4 driver in the oocyte gives rise to an expansion of the tll expression domain and to the generation of embryos with a tor-gain-of-function phenotype, in that they develop head and tail structures and lack most of the segmented trunk. However, and similarly to what is described above for tor constitutive mutations, in these embryos Cic downregulation is not expanded to a broader domain, indicating that even ligand-induced activation of the Tor pathway is unable to inhibit Cic protein accumulation in the embryonic middle regions (de las Heras, 2006).

In the experiments described above, activation of the Tor pathway over the whole embryo did not result in an expansion of Cic downregulation. Paradoxically, activated Tor could trigger downstream targets in the middle region even though Cic was still present. These observations raise the question of whether under these circumstances Cic is still able to act as a transcriptional repressor. Alternatively, Tor signalling could impair cic activity without removing Cic protein from the nuclei. To address this issue, the contribution of cic function was analyzed in embryos from tor constitutive mutants (de las Heras, 2006).

The strong transformations associated with the ectopic activation of the Tor pathway due to torD4021 mutations and tubGAL4 driven expression of tsl make it difficult to assess the operational state of the Cic repressor under these circumstances. To overcome this difficulty use was made of the weaker torRL3 constitutive mutation and cuticular transformations, which are more sensitive to small changes in the expression of tor targets genes than what can be visualized by whole mount in situs, were scored. Besides, in the following experiments the torRL3 genotype was examined in a trunk (trk) background to eliminate ligand-induced activation. On its own, a single copy of torRL3 gives rise to a very mild phenotype, in which occasionally one abdominal segment is deleted. In contrast, removing just one copy of the cic gene does not affect the embryonic pattern. However, a single copy of the torRL3 mutation combined with the removal of just one copy of the cic gene gives rise to prominent transformations; embryos from such females display variable phenotypes but in every case they show major deletions of the embryonic segments. Accordingly, there is an expansion of the domain of tll expression, which also in that case overlaps with the domain where Cic accumulates. In this situation, whether nuclear Cic protein is still functional can be assessed by removing the remaining copy of the cic gene and comparing the two phenotypes. Indeed, embryos from trk torRL3/+; cic/cic have a much stronger phenotype that those from trk torRL3/+; cic/+. Therefore, the Cic protein present in trk torRL3/+; cic/+ embryos is still at least in part functional implying that the torRL3 mutation is able to trigger tll activation without eliminating all cic repression activity (de las Heras, 2006).

What mechanisms are activated by Tor signalling that could bypass the need for Cic downregulation to activate terminal target genes? It has been suggested that the Stat92E transcription factor plays a role as a mediator of Tor signalling elicited by a Tor constitutive mutant receptor, but not in Tor signalling promoted by ligand-dependent activation of the receptor at the poles. The role of Stat92E was assessed in the tor constitutive mutant background. A reduction was found in the transformations associated with the trk torRL3/+; cic/+ genotype by removing a single copy of the stat92E gene. Whether this could also apply in the case of ectopic activation of the Tor pathway through ligand binding was analyzed; also in this case it was found that there is a reduction of the strength of the phenotype. In this case, however, the reduction is smaller, which could be due to the fact that the original transformation generated by the tubGAL4/UAStsl combination is much stronger and/or to a weaker involvement of stat92E in ligand-induced Tor signalling. Regardless, the results suggest that there is no fundamental difference in the role of stat92E between ligand-induced or constitutive activation of the Tor receptor. In support of this conclusion there is the recent observation that Stat92E is specifically phosphorylated at the poles by ligand-induced Tor signalling. Therefore, similarly to what was observed in the embryonic middle regions, it is proposed that Tor could also induce tll activation in the poles, and this occurs by a Cic downregulation-independent mechanism via stat92E. Altogether these results suggest that Tor signalling could normally trigger tll expression at the poles of wild-type embryos by two kinds of regulatory mechanisms, relief of cic repression and positive activation of tll expression. The positive effect of Tor signalling on tll expression could have been obscured by the fact that there is also a still unidentified Tor-independent activator, since terminal fate is specified in embryos lacking both Tor signalling and Cic repression. Accordingly, it has to be noted that stat92E mutants suppress ectopic activation of tll in the middle embryonic regions but not tll activation at the poles, which suggests that the role of stat92E on Tor signalling could be somehow redundant at the poles but absolutely required when Tor signalling is triggered in the embryonic middle regions (de las Heras, 2006).

The following conclusions can be drawn from these results. First, while activation of the Tor pathway at the embryonic poles downregulates Cic, Tor signalling appears to be necessary but not sufficient to eliminate Cic protein, as it can do so only at the embryonic poles. In this regard, it has to be noted that recent results indicate that the posterior maternal system can also affect Cic downregulation. Second, impairment of Cic repressor function is not an absolute requirement for tll expression, since tll can be expressed in situations where Cic repressor is still functional. In this regard, tll expression appears to be the result of a balance between repressor and activator factors and Cic repression might be overcome provided that activation is enhanced. And finally, there are differences between the terminal and the central embryonic regions that are independent of Tor signalling, as judged by the spatially restricted capacity of the Tor pathway to inhibit Cic accumulation and by the apparently distinct regional redundancy of stat92E function in Tor-dependent patterning. These results suggest that the Tor signalling pathway is not the only system that establishes a difference between the terminal and the central regions of the Drosophila embryo (de las Heras, 2006).

STAT is an essential activator of the zygotic genome in the early Drosophila embryo

In many organisms, transcription of the zygotic genome begins during the maternal-to-zygotic transition (MZT), which is characterized by a dramatic increase in global transcriptional activities and coincides with embryonic stem cell differentiation. In Drosophila, it has been shown that maternal morphogen gradients and ubiquitously distributed general transcription factors may cooperate to upregulate zygotic genes that are essential for pattern formation in the early embryo. This study shows that Drosophila STAT (STAT92E) functions as a general transcription factor that, together with the transcription factor Zelda, induces transcription of a large number of early-transcribed zygotic genes during the MZT. STAT92E is present in the early embryo as a maternal product and is active around the MZT. DNA-binding motifs for STAT and Zelda are highly enriched in promoters of early zygotic genes but not in housekeeping genes. Loss of Stat92E in the early embryo, similarly to loss of zelda, preferentially down-regulates early zygotic genes important for pattern formation. STAT92E and Zelda synergistically regulate transcription. It is concluded that STAT92E, in conjunction with Zelda, plays an important role in transcription of the zygotic genome at the onset of embryonic development (Tsurumi, 2011).

This study describes a bioinformatics approach to investigating the mechanisms controlling transcription of the zygotic genome that occurs during the MZT; STAT92E was identified as an important general transcription factor essential for up-regulation of a large number of early 'zygotic genes'. The role of STAT92E was described in controlling transcription of a few representative early zygotic genes, such as dpp, Kr, and tll, that are important for pattern formation and/or cell fate specification in the early embryo. These studies suggest that STAT92E cooperates with Zelda to control transcription of many 'zygotic genes' expressed during the MZT. While STAT mainly regulates transcription levels, but not spatial patterns, of dpp, tll, and Kr, and possibly also other 'zygotic genes', Zelda is essential for both levels and expression patterns of these genes (Tsurumi, 2011).

The transcriptional network that controls the onset of zygotic gene expression during the MZT has remained incompletely understood. It has been proposed that transcription of the zygotic genome depends on the combined input from maternally derived morphogens and general transcription factors. The former are distributed in broad gradients in the early embryo and directly control positional information (e.g., Bicoid, Caudal, and Dorsal), whereas the latter are presumably uniformly distributed regulators that augment the upregulation of a large number of zygotic genes. Other than Zelda, which plays a key role as a general regulator of early zygotic expression, the identities of these general transcriptional activators have remained largely elusive. It has been shown that combining Dorsal with Zelda- or STAT-binding sites supports transcription in a broad domain in the embryo. The demonstration of STAT92E as another general transcription factor sheds light on the components and mechanisms of the controlling network in the early embryo. Moreover, STAT92E and Zelda may cooperate to synergistically regulate zygotic genes. The results thus validate the bioinformatics approach as useful in identifying ubiquitously expressed transcription factors that may play redundant roles with other factors and thus might otherwise be difficult to identify (Tsurumi, 2011).

The conclusion that STAT92E is important for the levels but not the spatial domains of target gene expression in the early embryo is consistent with several previous reports. It has been shown that in Stat92E or hop mutant embryos, expression of eve stripes 3 and 5 are significantly reduced but not completely abolished. In addition, JAK/STAT activation is required for the maintenance of high levels, but not initiation, of Sxl expression during the MZT. Moreover, it has previously been shown that STAT92E is particularly important for TorsoGOF-induced ectopic tll expression but not essential for the spatial domains of tll expression in wild-type embryos under normal conditions. On the other hand, Zelda may be important for both levels and spatial patterns of gene expression. This idea is consistent with the finding that Zelda-binding sites are enriched in both promoter and promoter-distal enhancers regions, whereas STAT-binding sites are enriched in promoter regions only. It has been reported that pausing of RNA polymerase II is prominently detected at promoters of highly regulated genes, but not in those of housekeeping genes. In light of these results that STAT and Zelda sites are highly enriched in the early zygotic gene promoters, it is suggested that these transcription factors might contribute to chromatin remodeling that favors RNA polymerase II pausing at these promoters (Tsurumi, 2011).

Finally, the MZT marks the transition from a totipotent state to that of differentiation of the early embryo. As a general transcription factor at this transition, STAT, together with additional factors (such as Zelda), is important for embryonic stem cell differentiation. Further investigation is required to understand the molecular mechanism by which STAT and Zelda cooperate in controlling zygotic transcription in the early Drosophila embryo. Moreover, it would be interesting to investigate whether STAT plays similar roles in embryonic stem cell differentiation in other animals (Tsurumi, 2011).

Protein Interactions

Histone deacetylase-associating Atrophin proteins are nuclear receptor corepressors: Repression of knirps by Tll involves Atrophin

Drosophila Tailless (Tll) is an orphan nuclear receptor involved in embryonic segmentation and neurogenesis. Although Tll exerts potent transcriptional repressive effects, the underlying molecular mechanisms have not been determined. Using the established regulation of knirps by tll as a paradigm, it is reported that repression of knirps by Tll involves Atrophin, which is related to vertebrate Atrophin-1 and Atrophin-2. Atrophin interacts with Tll physically and genetically, and both proteins localize to the same knirps promoter region. Because Atrophin proteins interact with additional nuclear receptors and Atrophin-2 selectively binds histone deacetylase 1/2 (HDAC1/2) through its ELM2 (EGL-27 and MTA1 homology 2)/SANT (SWI3/ADA2/N-CoR/TFIII-B) domains, this study establishes that Atrophin proteins represent a novel class of nuclear receptor corepressors (Wang, 2006).

Since SMRT is a transcriptional corepressor for many NRs and SMRTER is the Drosophila cognate of SMRT, the first step in identifying Tll/Tlx-interacting corepressors was to test whether Tll and Tlx interact with SMRT and SMRTER. Using yeast two-hybrid assays, it was found that, whereas EcR, TR, and RAR interact with both SMRTER and SMRT, both Tll and Tlx fail to interact with SMRTER or SMRT (Wang, 2006).

To find potential corepressors of Tll/Tlx, a yeast two-hybrid screen was used, in which a Tll-expressing bait construct was deployed against a Drosophila embryonic library. A positive clone was identified, whose insert codes for the (1301-1966) region of Atro. This clone was selected for further investigation for several reasons: (1) In yeast, this clone also interacts strongly with chick and human Tlx, but not with RAR or TR; (2) Atro encodes a SANT domain, a RERE stretch, and an ELM2 domain; (3) Atro is a transcriptional corepressor of the Drosophila segmentation gene even-skipped; (4) two Atro-related proteins, Atr1 and Atr2, exist in vertebrates; and (5) Atr2 interacts with HDAC1 in mouse embryos. These properties of Atro proteins highlight the possibility that they are corepressors for Tll and Tlx (Wang, 2006).

To determine which region in Tll is required for Atro association, a series of truncated Tll expression constructs was prepared and their interactions with Atro were tested in yeast. The (192-452) region of Tll was found to be sufficient to mediate its interaction with Atro. Since this region of Tll harbors its ligand-binding domain (LBD), it suggested that an intact LBD is required for Tll to bind Atro. Indeed, no association between Tll variants lacking an intact LBD [e.g., Tll(33-161) or Tll(132-352)] and Atro could be detected (Wang, 2006).

A LBD-dependent interaction between Tll and Atro was further confirmed in human cells by using an immunostaining approach. CFP-tagged Atro (CFP-Atro) localizes to subnuclear regions when expressed in cells. This nuclear focal pattern of Atro resembles the nuclear pattern known for Atr2. Expressing Atro with Tll or Tlx in the same cells alters the nuclear distribution of Tll and Tlx: Both Tll and Tlx shift from their evenly distributed nuclear patterns to punctate nuclear patterns virtually identical to that displayed by CFP-Atro. Deleting the LBD from Tll and Tlx abrogates their localization to Atro-positive nuclear foci, confirming that Atro-Tll/Tlx interactions are mediated through the LBD of Tll and Tlx (Wang, 2006).

The regions in Atro responsible for Tll or Tlx interaction were mapped, using serial deletion Atro constructs. Two regions in Atro were found to mediate its interaction with Tll: Atro(965-1511) interacts weakly with Tll, whereas Atro(1711-1966) interacts strongly with both Tll and Tlx. The latter finding is of great interest, since the 1711-1966 region of Atro contains sequences conserved in the C-terminal regions of vertebrate Atr1 and Atr2. This correlation prompted an investigation of whether Atr1 and Atr2 interact with Tll or Tlx (Wang, 2006). Accordingly, two constructs expressing the C-terminal regions of Atr1 and Atr2 were generated and tested individually against Tll- or Tlx-expressing plasmids. As expected, both Atr1(846-1191) and Atr2(1224-1566), like Atro(1711-1966), interact strongly with Tll and Tlx in yeast, confirming that all Atro proteins are commonly targeted by Tll/Tlx (Wang, 2006).

Because the mapped Tll/Tlx-interacting regions in Atro, Atr1, and Atr2 share a stretch of highly conserved residues, whether mutations created within this region, which is called in this study the Atrobox, affect Tll/Tlx interaction was examined. Indeed, substitution of two leucine residues with alanine abolishes the interaction between Atro(1711-1966) and Tll or Tlx in yeast. Atro-Tll/Tlx interactions are, therefore, in part mediated through the Atro-box (Wang, 2006).

Tll/Tlx belong to the NR2 subfamily of the NR superfamily. The similarity shared by members of the NR2 subfamily suggests that additional NR2 proteins may interact with Atro proteins as well. This possibility was tested first with GST pull-down assays, in which several 35S-methionine-labeled NR2 and NR1 proteins were tested for their interactions with GST or GST-Atro fusion proteins. Atro proteins specifically bind Tll, Tlx, human chicken ovalbumin upstream promoter-transcription factor (COUP-TF), and Seven-Up1 (SVP1) (the Drosophila COUP-TF homolog), but not TRß and Ultraspiracle (USP). A similar interacting profile was observed between Atro proteins and COUP-TF or SVP1 in yeast. Therefore, Atro proteins do not interact with all NRs; rather, they preferentially bind a subset of NR2, including Tll, Tlx, SVP1, and COUP-TF (Wang, 2006).

Having demonstrated that Atro physically interacts with various NRs, the biological relevance of these interactions was examined. In this study, focus was placed on the in vivo relationship between Atro and Tll in flies by exploiting the known role of Tll in the segmentation process during Drosophila early embryogenesis. At this stage, Atro is expressed as a nuclear protein throughout the embryos. Consistent with previous observations that tll represses kni expression at the posterior end of the embryo, in situ hybridization for tll1 embryos shows a posterior expansion of kni stripe, especially in the ventral region of the embryos. Removal of zygotic Atro alone, as in the P-element excision line Atro35, does not cause such expansion, due to the presence of maternally deposited Atro. When maternal alone or both maternal and zygotic Atro are depleted using the dominant female sterile-FLP method, however, kni expression expands posteriorly in embryos. Because mutations of Atro and tll alter kni expression similarly, these in vivo observations suggest that Atro and tll are involved in overlapping transcriptional pathways (Wang, 2006).

To address the genetic interaction between tll and Atro further, advantage was taken of the hypomorphic nature of the tll1 allele, and it was asked whether the tll1-mediated phenotype is aggravated by additional Atro mutation. Specifically, whether the observed posterior expansion of kni stripe in tll1 embryos becomes more prominent when zygotic Atro is removed was investigated. Accordingly, a tll1, Atro35 double-mutant fly line was generated, in which both tll1 and Atro35 alleles were recombined to the same chromosome, and kni expression was tested in the resulting homozygous mutant embryos. Indeed, a further posterior expansion of kni stripe was observed in tll1, Atro35 double-mutant embryos, mimicking that found in tlle embryos. Therefore it is concluded that Atro is required for Tll to repress kni (Wang, 2006).

Since Atro is a binding factor of another terminal gap gene product, Huckebein (Hkb), the expression of kni was examined in hkb2 mutant embryos. No significant posterior expansion of kni was observed, therefore indicating that the repression of kni in the posterior-terminal region primarily results from the combined effect of Tll and Atro (Wang, 2006).

The genetic interaction between tll and Atro was further assessed by monitoring the expression of the pair-rule gene fushi tarazu (ftz) in the posterior region of the mutant embryos described above. In wild-type and in Atro35 zygotic mutant embryos, ftz is expressed as seven stripes in the central region. In tll1 embryos, however, the posterior stripes of ftz (mostly the fifth, sixth, and seventh stripes) shift toward the posterior end. In the most severely affected tll1 embryos, the seventh stripe of ftz is lost. This altered ftz pattern is known to be the consequence of cell fate changes, partly owing to the posterior expansion of kni, when tll is mutated. In tll1, Atro35 double-mutant embryos and in tlle embryos, additional loss of the sixth stripe of ftz was observed. Because the cell fate change is more pronounced in tll1, Atro35 double mutants than in tll1 or Atro35 embryos, it is concluded that Atro participates with Tll in determining posterior-terminal cell fates in early Drosophila embryos (Wang, 2006).

To verify the involvement of Atro in the regulation of kni by Tll at the chromatin level, chromatin immunoprecipitation (ChIP) assays were carried out for 0- to 4-h-old Drosophila embryos using Atro antibody, Tll antibody, and control IgG, respectively. The immunoprecipitated (IP) chromatin was subjected to PCR using primers corresponding to two separate regions, P1 and P2, in the kni gene, and a region in a randomly selected control (CG11562) promoter. In the kni promoter, P1 resides 2.5 kb upstream of the transcription initiation site and has a defined Tll-binding site. P2 corresponds to the 3' untranslated region of the kni gene, where no Tll-binding site is found (Wang, 2006).

In vivo ChIP assays revealed that both Atro and Tll antibodies, but not the control IgG, specifically precipitated chromatin that harbors the P1 site, but not chromatin containing P2 or the CG11562 promoter. These results establish that Atro, by forming protein complexes with Tll, is present naturally on the kni promoter (Wang, 2006).

Many transcriptional corepressors, including SMRT and N-CoR, are associated with HDAC activity. Because the results indicate that Atro proteins are corepressors of Tll/Tlx, the following was further investigated: (1) whether Atro proteins also show HDAC activity; (2) whether Atro proteins bind selected HDACs; and, if so, (3) which regions/domains in Atro proteins mediate their HDAC binding. To address these interconnected questions, fluorometric HDAC assays and Western blot analysis were performed on protein complexes immunoprecipitated by Flag-tagged Atro, Atr1, Atr2, or truncated Atr2 variants expressed in HEK293 cells. In parallel experiments, Flag and Flag-SMRT were used as a negative and a positive control, respectively. The expression of tested Flag fusion proteins was first examined using Western blot analysis (Wang, 2006).

As expected, Flag-SMRT is associated with potent HDAC activity that is sensitive to trichostatin A (TSA), an HDAC inhibitor. Robust levels of TSA-sensitive HDAC activity were also observed for both Atro and Atr2, confirming that both proteins' properties involve HDACs. surprisingly, Atr1 displays no prominent HDAC activity. Since Atr1 lacks the conserved ELM2 and SANT domains found in the N-terminal regions of Atr2 and Atro, it is suspected that the missing N-terminal region in Atr1 might be important for the HDAC activity of Atro proteins (Wang, 2006).

To determine whether the HDAC activity of Atro or Atr2 depends on its N-terminal region, the BAH (Bromo adjacent homology), the ELM2, and the SANT domains in this region of Atr2 were deleted sequentially. Note that the BAH domain is absent in Atro. Whereas Atr2DeltaBAH still exerts a robust level of HDAC activity, a dramatic reduction of HDAC activity was observed with Atr2DeltaBAH-ELM2. A further deletion of the SANT domain, Atr2DeltaBAH-ELM2-SANT, causes a complete loss of HDAC activity, indicating that both the ELM2 and SANT domains are central to Atr2's HDAC activity (Wang, 2006).

Next, which HDACs Atro proteins interact with was investigated, and whether Atr2's association with HDACs involves its ELM2/SANT domains. Protein complexes immunoprecipitated by Flag-tagged Atro proteins and Atr2 variants were examined by Western blot for a panel of potential associating proteins, including HDAC1, HDAC2, HDAC3, and Sin3A. Sin3A was not detected in any of the IP complexes. In contrast, a significant level of HDAC3 was precipitated along with SMRT. Although SMRT also interacts with HDAC1 or HDAC2, these interactions are considerably weaker. Conversely, abundant HDAC1 and HDAC2 (but only minimal HDAC3) are present in the protein complexes associated with Atr2. Similarly, Atro, but not Atr1, also precipitates HDAC1/2 specifically, indicating that Atro-family (except Atr1) and SMRT-family proteins display distinct preferences for different HDACs (Wang, 2006).

Consistent with HDAC assay results, removing the ELM2 domain or both the ELM2 and SANT domains from Atr2DeltaBAH impairs or disrupts its ability to associate with HDAC1/2. Given the fact that similar results were also obtained when the distribution of endogenous HDAC1 was examined in cells expressing different CFP-Atr2 variants, it is therefore concluded that the ability of Atr2 to exert HDAC activity and to recruit HDAC1/2 depends on its ELM2 and SANT domains (Wang, 2006).

In many respects, the transcriptional properties that discover in this study for Atro proteins parallel those found for SMRT, N-CoR, and SMRTER. (1) These two classes of corepressors share a SANT domain and RERE stretch; (2) they are conserved in vertebrates and in flies; (3) they bind NRs, albeit selectively, and (4) they associate with HDACs, also selectively. Additionally, Atr1, like SMRT and N-CoR, also interacts with ETO/MTG8, which is known to be a transcriptional repressor involved in acute myeloid leukemia. Considering that SMRT and N-CoR interact with a large number of NRs, with a wide variety of transcriptional factors, and also with type II HDACs, it is predicted that Atro proteins may have similar qualities as well. Therefore, more Atro-interacting factors still await discovery (Wang, 2006).

In the context of human diseases, it is known that polyglutamine expansion in human Atr1 causes DRPLA. It has been shown that Atr1 lacks HDAC activity, yet it binds Atr2 through their RERE stretches, and it associates with both Tlx and COUP-TF, two known NRs with key roles in CNS development and functioning. It is therefore proposed that mutant Atr1 may cause its pathological effects by interfering with the normal transcriptional properties of Atr2 and its associated nuclear receptors (Wang, 2006).

Drosophila Brakeless interacts with Atrophin and is required for Tailless-mediated transcriptional repression in early embryos

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. Mutations were isolated in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, an expanded expression pattern was noted of the Krüppel (Kr) and knirps (kni) genes. Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. Both Drosophila and human Brakeless and Atrophin interact in vitro, and it is proposed that they act together as a co-repressor complex in many developmental contexts. The possibility is discussed that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA) (Haecker, 2007).

Repression plays a pivotal role in establishing correct gene expression patterns that is necessary for cell fate specification during embryo development. For example, in the early Drosophila embryo, repression by gap and pair-rule proteins is essential for specifying the positions of the 14 segments of the animal. The mechanisms by which transcriptional repressors delimit gene expression borders are not well understood. However, many repressors require co-repressors for function. In the Drosophila embryo, the CtBP and Groucho co-repressors are required for activity of many repressors. Atrophin has been identified as a co-repressor for Even-skipped and Tll. Still, co-regulators for several important transcription factors in the early embryo have not yet been identified. Therefore a screen was performed for novel maternal factors that are required for establishing correct gene expression patterns in the early embryo (Haecker, 2007).

From this screen, mutations were identified in the bks gene that cause severe phenotypes on gap gene expression and embryo segmentation. The Bks protein is evolutionarily conserved between insects and deuterostomes, but has not been characterized in any species except Drosophila, in which it has been shown to repress runt expression in photoreceptor cells and thickveins expression in wing imaginal discs. However, the molecular function of Bks has been unknown. This study shows that Bks interacts with the transcriptional repressor Tll, is recruited to target gene CRMs, and will repress transcription when targeted to DNA (Haecker, 2007).

Tll has been shown to utilize Atrophin as a co-repressor. Atrophin genetically interacts with Tll and physically interacts with its ligand binding domain. Atrophin binding is conserved in nuclear receptors within the same subfamily, such as Seven-Up in Drosophila as well as Tlx and COUP-TF in mammals. When expressed in mammalian cells, Drosophila Atrophin and mouse Atrophin-2 interact with the histone deacetylases HDAC1 and HDAC2. Histone deacetylation may therefore be part of the mechanism by which Atrophin functions as a co-repressor. Another recent report described genetic interactions among bks and atrophin mutants in the formation of interocellar bristles in adult flies. Furthermore, it was shown that atrophin mutants have virtually identical phenotypes as bks mutants, including de-repression of runt expression in the eye, thickveins expression in the wing, and Kr and kni expression in the embryo (Haecker, 2007).

Both proteins are recruited to the kni CRM, a Tll-regulated target gene, in the embryo. Importantly, Atrophin and Bks interact in vitro and that they can be co-immunoprecipitated from S2 cells. It is proposed that Bks and Atrophin function together as a co-repressor complex, and based on the similar bks and atrophin mutant phenotypes at several developmental stages, the complex may function throughout development. These results are compatible with the existence of a tripartite complex consisting of Tll, Bks, and Atrophin. Bks binding to Tll is enhanced by the Tll DNA binding domain, whereas the interaction of Tll with Atrophin is mediated through the C-terminal ligand binding domain. Tll may therefore simultaneously interact with Bks and Atrophin. Alternatively, Tll interacts separately with Bks and Atrophin on the kni CRM. In either case, both Bks and Atrophin are required for full Tll activity. However, at high enough Tll concentration, Bks activity is dispensable. Some bks embryos misexpressing Tll still repress kni expression, and overexpressing Tll from a heat-shock promoter can repress the posterior kni stripe in both wt and bks mutant embryos. For this reason, it is believed that Bks and Atrophin are cooperating as Tll co-repressors, so that Tll function is only partially impaired by the absence of either one. It was found that Tet-Bks-mediated repression in cells is insensitive to the deacetylase inhibitor trichostatin A (TSA). It is possible, therefore, that whereas Atrophin-mediated repression may involve histone deacetylation, Bks could repress transcription through a separate mechanism (Haecker, 2007).

These results have not revealed any differences between the molecular functions of the two Bks isoforms. Both Bks-A and Bks-B repress transcription when tethered to DNA, and the sequences that mediated binding to Tll and Atrophin are shared between the two isoforms. However, the bks339 allele that selectively affects the Bks-B isoform causes a weaker, but comparable phenotype to the stronger bks alleles that disrupt both isoforms. Therefore, the C-terminus of Bks-B provides a function that is indispensable for embryo development and regulation of kni expression. This part of Bks-B contains two regions (D3 and D4) that are highly conserved in insects and loosely conserved in deuterostome Bks sequences, but does not resemble any sequence with known function. The only sequence similarity to domains found in other proteins is a single zinc-finger motif in Bks-B. Preliminary results indicate that the zinc finger in isolation or together with the conserved D2 domain does not exhibit sequence-specific DNA binding activity. Indeed, multiple zinc fingers are generally required to achieve DNA binding specificity. Instead, Bks is likely brought to DNA through interactions with Tll and other transcription factors (Haecker, 2007).

Atrophins are required for embryo development in C. elegans, Drosophila, zebrafish, and mice. In vertebrates, two atrophin genes are present. Atrophin-1 is dispensable for embryonic development in mice, and lacks the N-terminal MTA-2 homologous domain that interacts with histone deacetylases . However, the homologous C-termini of Atrophin-1 and Atrophin-2 can interact, and it was found that this domain can also bind to the human Bks homolog ZNF608. Atrophin-1 interacts with another co-repressor-associated protein as well, ETO/MTG8, and can repress transcription when tethered to DNA. These data are consistent with the emerging view that deregulated transcription may be an important mechanism for the pathogenesis of polyglutamine diseases. Recent evidence indicates that interactions with the normal binding partners may cause toxicity of polyglutamine-expanded proteins such as Ataxin-1 . It will be interesting to investigate whether the interaction between human Bks homologs and Atrophin-1 is important for the neuronal toxicity of polyglutamine-expanded Atrophin-1 (Haecker, 2007).

tailless: Biological Overview | Evolutionary Homologs | Targets of Activity | Developmental Biology | Effects of Mutation | References

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