retained

Targets of Activity

Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknullt (zen) contains Dorsal binding sites as well as an essential element termed AT2. An AT2 DNA binding activity has been identified (called ZREB) and purified in embryos. It consists of the cut (ct) and dead ringer (dri) gene products. Studies of loss-of-function mutations in ct and dri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, all of which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors (Valentine, 1998).

To determine if cut and dir are required for the activity of the AT2 site in vivo, the effects of mutations in these genes were examined on the activity of the lacZ transgene under control of the minimal zen VRR. For both cut and dir, germ line clones were generated to test the effects of eliminating maternally contributed gene products; in addition, the effects of eliminating zygotically produced gene products were examined. A null mutation in ct (which is an X-linked gene) results in strong ventral derepression of the transgene. This ventral derepression is observed in about one-half the embryos derived from a cross between females containing ct germ line clones and hemizygous males. It was never observed in a cross between heterozygous females and hemizygous males, suggesting that derepression requires simultaneous elimination of both maternal and zygotic Ct. A strong hypomorphic mutation in dri (which is an autosomal gene) also results in strong derepression. In contrast to the results observed with ct, this effect is strictly zygotic. It is observed in a cross between heterozygous dri males and females but not in a cross between females carrying dri germ line clones and wild-type males. Most strikingly, in the absence of zygotic Dri, the zen VRR directs strong ventral expression in the blastoderm embryo, reminiscent of the results observed when the AT2 element is mutagenized. These results strongly suggest that, in the context of the minimal zen VRR, Dri plays an essential role in converting Dorsal from an activator into a repressor. The dri mutation results in a significant weakening of the transverse eve stripe (generated by the minimal even skipped (eve) stripe 2 enhancer (MSE) as well as a shift in the position of the stripe toward the anterior pole of the embryo, presumably due to a role for Dri in anteroposterior pattern formation. Despite the strong effects of the cut and dir mutations on the activity of the minimal zen VRR, both genes make only minor contributions to the ventral repression of the endogenous zen gene in the stage 4 embryo. In the absence of both zygotic and maternal Ct or in the absence of zygotic Dri, zen expression in the stage 4 embryo is still largely restricted to the dorsal 40% to 50% of the embryo, although weak ventral patches of zen expression are observed with high frequency. Such patches are never observed in wild-type embryos stained in parallel with these embryos. The contrast between the strong effect observed for the minimal VRR and the weak effect observed for the endogenous zen gene suggests redundancy in the zen locus. In other words, there may be additional unidentified ventral repression regions in the zen locus that function in a Ct- and Dri-independent manner. Although neither Ct nor Dri is essential for ventral repression of the endogenous zen gene in the stage 4 embryo, both factors appear to play essential roles in the refinement of the zen pattern that normally occurs in stage 5 embryos. Normally, zen expression refines during cellularization to a stripe approximately three to five cells in width. However, in the absence of both maternal and zygotic Ct or in the absence of zygotic Dri, a severe refinement defect is observed (Valentine, 1998).

Both Dorsal and Dri bind to the corepressor Gro in vitro, suggesting a possible mechanism for repression in which Dorsal and Dri recruit Gro to the template. This model is strengthened by results showing that Dorsal and Dri, when bound to DNA, can cooperatively recruit Gro to the zen VRR in vitro. However, the magnitude of the cooperativity observed in vitro is small (twofold) and therefore does not completely account for the absolute requirement for the Dorsal and AT2 sites observed in germ line transformation assays. This suggests that factors in addition to Dorsal and Dri are required for the efficient recruitment of Gro in vivo. For example, it is possible that the addition of Ct would enhance cooperative recruitment, an idea that could not be tested due to difficulty obtaining sufficient amounts of recombinant Ct. It is also likely that elements in addition to Dorsal sites and AT2 are required for efficient Gro recruitment and therefore for efficient repression, since previous experiments indicate that, while these sites are required for repression, they are not sufficient for repression. Finally, it is possible that the cooperativity of Gro recruitment would be enhanced in the context of chromatin templates rather than naked DNA templates (Valentine, 1998).

Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFB homolog Dorsal (Dl). Dl acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), Dl is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of Dl by sequence-specific transcription factors such as Dead Ringer (Dri). The interplay between Dl, Gro and Dri on the hkb enhancer was examined and it was shown that when acting over a distance, Gro abolishes rather than converts Dl activator function. However, reducing the distance between Dl- and Dri-binding sites switches Dl into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro -- quenching and silencing of transcription -- are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with Dl. RTK signaling provides a way of modulating Dl function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer (Hader, 1999).

The cis-acting element has been identified that mediates expression of the Drosophila gene hkb, which is necessary for terminal pattern formation and to size the mesoderm anlage in the blastoderm embryo. Deletion analysis of this element reveals a 162 base pair (bp) sub-element that integrates the activities of the Tor-dependent RTK signaling cascade and the morphogen Dl. This element, termed hkb ventral element (VE), comprises a 112 bp ventral activator element (VAE) and a 50 bp ventral repressor element (VRE) (Hader, 1999).

The VAE contains a Dl-binding site, identified in vitro, and mediates gene activation along the ventral side of the embryo. VAE-mediated gene expression is absent in embryos lacking Dl activity and extends throughout Toll^10b mutants, in which Dl is present in all nuclei of the embryo. The expression pattern is not altered in embryos lacking snail and twist, the zygotic mediators of Dl. It is also not affected in embryos that lack Tor or express constitutively active Tor^Y9, which causes RTK signaling throughout the embryo. In contrast, the VE fails to activate in the absence of Tor and mediates broad ventral expression in tor^Y9 embryos not seen in the absence of Dl activity. This indicates that VAE mediates transcriptional activation by Dl, that the VRE, which by itself fails to activate transcription, is necessary to prevent Dl-dependent activation in the central region of the embryo, and that the activity of the unknown repressor, mediated by the VRE, is relieved by RTK signaling (Hader, 1999).

The evolutionarily conserved co-repressor Gro acts as a repressor of Dl activity, since both hkb expression and VE-driven gene expression expand along the ventral side of embryos lacking groucho (gro) activity. However, VAE-driven gene expression and the terminal expression domains of hkb are not significantly affected by lack of Gro. Thus, Gro functions as a repressor of VAE-directed, Dl-dependent transcriptional activation in the ventral region of the embryo and must act through the VRE (Hader, 1999).

Previous results have shown that Gro switches the transcriptional activator Dl into a potent silencer of transcription. This requires the formation of a multiprotein repressor complex of which Dl and Gro are obligatory components. Complex formation requires that Gro is recruited next to Dl by sequence-specific transcription factors such as Cut or Dri. Lack of either Gro or Dri activity results in VE-driven gene expression along the ventral axis of the embryo, indicating that both factors are necessary for repression of Dl-dependent activation. A single binding site has been identified for Dri in the VRE. Replacement of 5 bp in this site (VE-^DRI) results in loss of repression in the central region of the embryo, indicating that Dri is necessary for recruitment of Gro to the VE (Hader, 1999).

The VE differs from the cis-acting elements of the genes zerknullt (zen) and decapentaplegic (dpp), both of which mediate long-range Dl-dependent transcriptional silencing by Gro. In these elements, binding sites for Dri and Dl are directly adjacent, whereas in the VE they are some 90 bp apart. This distance suggested the possibility that Gro cannot associate with Dl on the VE, implying that Gro must prevent Dl-dependent activation by a means other than formation of a long-range silencing complex, for example, by short-range quenching. This proposal was tested by monitoring gene expression patterns directed by a cis-acting activator element of the gene knirps (kni-element) to which the VRE, the VAE, the VE or molecularly defined variants of the VE were fused (Hader, 1999).

The kni-element drives gene expression throughout the embryo except in the posterior pole region. It mediates activation in response to the transcriptional activators Bicoid (Bcd) and Caudal (Cad) and acts in a Dl-independent fashion. Addition of the VRE to the kni-element does not cause ventral repression, nor does addition of the VE or the VAE. This indicates that within the VE, Gro abolishes the activator function of Dl instead of converting Dl into a long-range repressor that interferes with transcriptional activation by Bcd and Cad (Hader, 1999).

To investigate whether this action of Gro on Dl is determined by the arrangement of Dri- and Dl-binding sites in the VE, the transcription patterns driven by a modified VE-kni-element were examined in which the normal distance of 91 bp between the binding sites was reduced to 45 bp. This reduction results in Dl-dependent repression along the ventral side of wild-type embryos. Repression is not observed in the absence of Gro or Dl or in embryos expressing the constitutively active Tor^Y9 protein. In contrast, the repression domain expands anteriorly in tor mutant embryos, which lack RTK signaling, and is found to be Dl-dependent. This suggests that the spatial arrangement of the Dl- and Dri-binding sites dictates the mechanism by which Gro and Dl act within the enhancer element. In one case, Dl is suppressed by Gro, in the other, Dl is converted into a potent silencer of transcription that can override activation by Bcd and Cad. Both modes of repression are controlled by Tor-dependent RTK signaling (Hader, 1999).

In the zen and dpp cis-acting elements, Gro causes Dl-mediated long-range silencing. Gro functions either by inhibiting the assembly and function of the core RNA polymerase II complex, by positioning nucleosomes over the core promoter and/or by recruiting the histone deacetylase Rpd3 to the template, where the enzyme can modulate local chromatin structure. However, in the VE, Gro only inhibits Dl-dependent activation without converting Dl into a repressor. The different modes of Gro function, that is, long-range silencing and short-range quenching, as shown here, are dependent on the distance between the Dl- and Dri-binding sites and/or their orientation on the enhancer, since shortening of the spacer distance converts the VE into a dpp- or zen-like element. This suggests that the way in which Gro regulates Dl activity depends on whether or not the two proteins can directly interact in vivo. Furthermore, both regulatory options of Gro on Dl are abolished by RTK signaling, a phenomenon that corresponds to the observation that Dl-dependent repression of dpp and zen is relieved by local Tor activity in the pole regions of the embryo. RTK-dependent phosphorylation may therefore interfere with the binding of Dri to the DNA template, the recruitment of Gro, or with both. Phosphorylation of the vertebrate Gro homolog TLE1 has been demonstrated, and many potential phosphorylation sites have been noted in Dri. Thus, local RTK-dependent phosphorylation may render one or both factors inactive, preventing Gro-dependent repression of Dl in the termini of the wild-type embryo (Hader, 1999).

These results establish that the cooperation between two maternal signaling systems, which determines the spatial limits of the Drosophila mesoderm anlage through hkb expression, is based on the management of the ubiquitously distributed factors Gro and Dri by local RTK signaling and that Gro can act through different modes on Dl. Lack of dead ringer (dri) activity does not result in an overt expansion of hkb expression on the ventral side of the embryo. However, as has been observed for VE-dependent gene expression, it causes only weak defects in mesoderm formation as compared with Gro-deficient embryos or embryos that express hkb under the control of the VAE. Thus, the interactions shown here represent only the Dri-dependent aspect of Gro's effect on hkb expression. The full picture of hkb control is likely to involve additional and redundantly acting factor(s) that recruit Gro to sites flanking the VE within the hkb control region (Hader, 1999).

Dead ringer interaction with DNA

The AT-rich interaction domain (ARID) is a DNA-binding module found in many eukaryotic transcription factors. Using NMR spectroscopy, the first ever three-dimensional structure has been determined of an ARID-DNA complex (mol. wt 25.7 kDa) formed in Drosophila by Dead ringer. ARIDs recognize DNA through a novel mechanism involving major groove immobilization of a large loop that connects the helices of a non-canonical helix-turn-helix motif, and through a concomitant structural rearrangement that produces stabilizing contacts from a ß-hairpin. Dead ringer's preference for AT-rich DNA originates from three positions within the ARID fold that form energetically significant contacts to an adenine-thymine base step. Amino acids that dictate binding specificity are not highly conserved, suggesting that ARIDs will bind to a range of nucleotide sequences. Extended ARIDs, found in several sequence-specific transcription factors, are distinguished by the presence of a C-terminal helix that may increase their intrinsic affinity for DNA. The prevalence of serine amino acids at all specificity determining positions suggests that ARIDs within SWI/SNF-related complexes will interact with DNA on a non-sequence specific basis (Iwahara, 2002).

ARIDs are structurally unrelated to any other protein family, but close inspection reveals the presence of a non-canonical HTH motif (helices H5 and H6) that interacts with DNA in an unusual manner. The HTH is a ubiquitous DNA-binding motif that typically inserts the second helix, the so called 'recognition helix', into the major groove for base-specific hydrogen bonding. In contrast, in the DRI-DBDF355L-DNA complex, no direct hydrogen bonds are made from its recognition helix (helix H6); rather, the nine-residue preceding 'turn' adapts its structure upon immobilization to form several base-specific hydrogen bonds to the Ade10-Thy11 base step. Other protein-DNA complexes contain lengthy turns separating the helices of their HTH motif, but their turns either protrude into the solvent or contact the sugar-phosphate backbone exclusively. The prominent role of the loop in the Dead ringer complex is a direct result of the anomalous positioning of the recognition helix (helix H6), which is placed at a steep angle extending away from the duplex, compared with other HTH-containing protein- DNA complexes. This unusual arrangement largely precludes extensive interactions with the duplex, allowing only van der Waals contacts from the side chains of Leu355 and Thr356, and a potential water-mediated hydrogen bond from the hydroxyl of Thr356. Interestingly, Dead ringer and the Engrailed homeodomain have the same sequence specificity, but the DNA contacts from their respective HTH units are not conserved (Iwahara, 2002 and references therein).

During dorsal-ventral axis formation, Dorsal and Dead ringer bind to adjacent sites within the VRR to repress the transcription of the zen gene. In order to understand this process a model was constructed of the ternary Dorsal-DRI-DBD-DNA complex based on the complex structures of Dead ringer and the close dorsal homolog Gambif1 (an Anopheles gambiae protein). Interestingly, the model predicts that the Dorsal and Dead ringer DNA-binding domains will interact with one another at the AT2-dl2 site, and that this interaction will be mediated by helix H8 of Dead ringer. This protein- protein interaction may partially explain Dorsal and Dead ringer's co-operative recruitment of Groucho to the VRR, as well as the observation that the spacing between the dl2 and AT2 sites is important for repression. It also suggests that in addition to DNA binding, the C-terminal helix within extended ARIDs may mediate protein- protein interactions (Iwahara, 2002 and references therein).

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