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We show here that the Drosophila MADF/BESS domain transcription factor Dip3, which is expressed in differentiating photoreceptors, regulates neuronal differentiation in the compound eye. Loss of Dip3 activity in photoreceptors leads to an extra photoreceptor in many ommatidia, while ectopic expression of Dip3 in non-neuronal cells results in photoreceptor loss. These findings are consistent with the idea that Dip3 is required non-cell autonomously to block extra photoreceptor formation. Dip3 may mediate the spatially restricted potentiation of Notch (N) signaling since the Dip3 misexpression phenotype is suppressed by reducing N signaling and misexpression of Dip3 leads to ectopic activity of a N-responsive enhancer. Analysis of mosaic ommatidia suggests that no specific photoreceptor must be mutant to generate the mutant phenotype. Remarkably, however, mosaic pupal ommatidia with three or fewer Dip3(+) photoreceptors always differentiate an extra photoreceptor, while those with four or more Dip3(+) photoreceptors never differentiate an extra photoreceptor. These findings are consistent with the notion that Dip3 in photoreceptors activates a heretofore unsuspected diffusible ligand that may work in conjunction with the N pathway to prevent a subpopulation of undifferentiated cells from choosing a neuronal fate.

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal Rel homology domain. We show here that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters.

The Rel family transcription factor Dorsal patterns the dorsoventral axis of the Drosophila embryo by activating genes such as twist and snail and repressing genes such as decapentaplegic and zerknüllt. Dorsal represses transcription by recruiting the co-repressor Groucho. However, repression occurs only when Dorsal-binding sites are close to binding sites for other factors that also bind Groucho. The need for additional factors to assist Dorsal in repression may result from the intrinsically weak interaction between Dorsal and Groucho. To test this idea, we generated a Dorsal variant containing a high-affinity Groucho recruitment motif at its C terminus. As predicted, this variant functions as a dedicated repressor, silencing decapentaplegic and zerknüllt while failing to activate twist and snail. We also converted Dorsal into a dedicated activator by replacing its weak Groucho-recruitment motif with heterologous activation domains. Although the dedicated activator alleles fail to repress decapentaplegic and zerknüllt in the syncytial blastoderm embryo, they are able to pattern the dorsoventral axis. This indicates that dorsoventral patterning is not dependent upon Dorsal-mediated repression, reflecting the existence of redundant mechanisms to block Decapentaplegic signaling.

During Drosophila melanogaster eye development, signaling through receptor tyrosine kinases (RTKs) leads to activation of a mitogen activated protein tyrosine kinase, called Rolled. Key nuclear targets of Rolled are two antagonistic transcription factors: Yan, a repressor, and Pointed-P2 (Pnt-P2), an activator. A critical regulator of this process, Mae, can interact with both Yan and Pnt-P2 through their SAM domains. Although earlier work showed that Mae derepresses Yan-regulated transcription by depolymerizing the Yan polymer, the mechanism of Pnt-P2 regulation by Mae remained undefined. We find that efficient phosphorylation and consequent activation of Pnt-P2 requires a three-dimensional docking surface on its SAM domain for the MAP kinase, Rolled. Mae binding to Pnt-P2 occludes this docking surface, thereby acting to downregulate Pnt-P2 activity. Docking site blocking provides a new mechanism whereby the cell can precisely modulate kinase signaling at specific targets, providing another layer of regulation beyond the more global changes effected by alterations in the activity of the kinase itself.

The transcription factors Dorsal and Twist regulate dorsoventral axis formation during Drosophila embryogenesis. Dorsal and Twist bind to closely linked DNA elements in a number of promoters and synergistically activate transcription. We have identified a novel protein named Dorsal-interacting protein 3 (Dip3) that may play a role in this synergy. Dip3 functions as a coactivator to stimulate synergistic activation by Dorsal and Twist, but does not stimulate simple activation of promoters containing only Dorsal or only Twist binding sites. In addition, Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor. We assess the possibility that the MADF and BESS domains are related to the SANT domain, a well-characterized motif found in many transcriptional regulators and coregulators.

The Dorsal morphogen directs formation of the Drosophila dorsoventral axis by both activating and repressing transcription. It contains an N-terminal Rel homology domain (RHD), which is responsible for DNA binding and regulated nuclear import, and a C-terminal domain (CTD) that contains activation and repression motifs. To determine if the RHD has a direct role in transcriptional control, we analyzed a series of RHD mutations in S2 cells and embryos. Two classes of mutations (termed class I and class II mutations) that alter activation without affecting DNA binding or nuclear import were identified. The two classes appear to define distinct protein interaction surfaces on opposite faces of the RHD. Class I mutations enhance an apparently inhibitory interaction between the RHD and the CTD and eliminate both activation and repression by Dorsal. In contrast, class II mutations result in increased activation in S2 cells but severely decreased activation in embryos and have little effect on repression. Analysis of the cuticles of class II mutant embryos suggests that, in the absence of Dorsal-mediated activation, Dorsal-mediated repression is not sufficient to pattern the embryo. These results provide some of the first evidence that the RHD plays an active role in transcriptional regulation in intact multicellular organisms.

A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. We previously showed that the Drosophila melanogaster rel family factor, Dorsal, is a substrate for Smt3 conjugation. The conjugation machinery was found to enhance Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. In this report, we show that Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, we examined the role of Smt3 conjugation in the immune response. We find that the conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo.

In the Drosophila embryo, Dorsal, a maternally expressed Rel family transcription factor, regulates dorsoventral pattern formation by activating and repressing zygotically active fate-determining genes. Dorsal is distributed in a ventral-to-dorsal nuclear concentration gradient in the embryo, the formation of which depends upon the spatially regulated inhibition of Dorsal nuclear uptake by Cactus. Using maternally expressed Gal4/Dorsal fusion proteins, we have explored the mechanism of activation and repression by Dorsal. We find that a fusion protein containing the Gal4 DNA-binding domain fused to full-length Dorsal is distributed in a nuclear concentration gradient that is similar to that of endogenous Dorsal, despite the presence of a constitutively active nuclear localization signal in the Gal4 domain. Whether this fusion protein activates or represses reporter genes depends upon the context of the Gal4-binding sites in the reporter. A Gal4/Dorsal fusion protein lacking the conserved Rel homology domain of Dorsal, but containing the non-conserved C-terminal domain also mediates both activation and repression, depending upon Gal4-binding site context. A region close to the C-terminal end of the C-terminal domain has homology to a repression motif in Engrailed - the eh1 motif. Deletion analysis indicates that this region mediates transcriptional repression and binding to Groucho, a co-repressor known to be required for Dorsal-mediated repression. As has previously been shown for repression by Dorsal, we find that activation by Dorsal, in particular by the C-terminal domain, is modulated by the maternal terminal pattern-forming system.

To identify proteins that regulate the function of Dorsal, a Drosophila Rel family transcription factor, we employed a yeast two-hybrid screen to search for genes encoding Dorsal-interacting proteins. Six genes were identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus), three that encode novel proteins, and one that encodes Drosophila Ubc9 (DmUbc9), a protein thought to conjugate the ubiquitin-like polypeptide Smt3 to protein substrates. We have found that DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 was found to relieve inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity was potentiated by the overexpression of DmSmt3. We have also identified a DmSmt3-activating enzyme, DmSAE1/DmSAE2 and found that it further potentiates Dorsal-mediated activation.

The Rel family transcription factor, Dorsal, determines cell fate as a function of position along the dorsoventral axis of the Drosophila embryo. This process depends on interactions between Dorsal and a large number of additional proteins present in the early embryo. Cytoplasmic interactions regulate the nuclear uptake of Dorsal, resulting in the establishment of the Dorsal nuclear concentration gradient, which determines the dorsoventral polarity of the embryo. Nuclear protein-protein interactions then enable Dorsal to activate some target genes and to repress others, thereby promoting the division of the embryo into distinct developmental domains. Because of this broad array of regulatory interactions, Dorsal serves as an excellent paradigm for eukaryotic transcriptional regulation.

The Dorsal morphogen acts as both an activator and a repressor of transcription in the Drosophila embryo to regulate the expression of dorsal/ventral patterning genes. Circumstantial evidence has suggested that Dorsal is an intrinsic activator and that additional factors (corepressors) convert it into a repressor. These corepressors, however, have previously eluded definitive identification. We show here, via the analysis of embryos lacking the maternally encoded Groucho corepressor and via protein-binding assays, that recruitment of Groucho to the template by protein:protein interactions is required for the conversion of Dorsal from an activator to a repressor. Groucho is therefore a critical component of the dorsal/ventral patterning system.

The establishment of mesoderm and neuroectoderm in the early Drosophila embryo relies on interactions between the Dorsal morphogen and basic-helix-loop-helix (bHLH) activators. Here we show that Dorsal and the bHLH activator Twist synergistically activate transcription in cell culture and in vitro from a promoter containing binding sites for both factors. Somewhat surprisingly, a region of Twist outside the conserved bHLH domain is required for the synergy. In Dorsal, the rel homology domain appears to be sufficient for synergy. Protein-protein interaction assays show that Twist and Dorsal bind to one another in vitro. However, this interaction does not appear to be of sufficient strength to yield cooperative binding to DNA. Nonetheless, the regions of Twist and Dorsal required for the binding interaction are also required for synergistic transcriptional activation.

The Dorsal morphogen is a transcription factor that activates some genes and represses others to establish multiple domains of gene expression along the dorsal/ventral axis of the early Drosophila embryo. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). We have identified a corepression element in decapentaplegic (dpp), a zygotically active gene that is repressed by the Dorsal morphogen. This dpp repression element (DRE) is located within a previously identified VRR and close to essential Dorsal-binding sites. We have purified a factor from Drosophila embryo extracts that binds to the DRE but not to mutant forms of the DRE that fail to support efficient repression. This protein also binds to an apparently essential region in a VRR associated with the zerknüllt (zen) gene. One of the DREs in the dpp VRR overlaps the binding site for a potential activator protein suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. We have found the DRE-binding protein to be identical to NTF-1 (equivalent to Elf-1, the product of the grainyhead gene), a factor originally identified as an activator of the Ultrabithorax and Dopa decarboxylase promoters. NTF-1 mRNA is synthesized during oogenesis and deposited in the developing oocyte where it is available to contribute to ventral repression during early embryogenesis. Previous studies have shown that overexpression of NTF-1 in the postblastoderm embryo results in a phenotype that is consistent with a role for this factor in the repression of dpp later in embryogenesis.

Modulation of transcription factor activity leading to changes in cell behavior (e.g., differentiation versus proliferation) is one of the critical outcomes of receptor tyrosine kinase (RTK) stimulation. In the early Drosophila embryo, activation of the torso (tor) RTK at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of the tailless (tll) gene. Our analysis 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 present throughout the embryo. We have mapped the tor-RE to an 11-bp sequence; using this sequence as the basis for protein purification, we have determined that the proteins GAGA and NTF-1 (also known as Elf-1, product of the grainyhead gene) bind to the tor-RE. We demonstrate that NTF-1 can be phosphorylated by MAPK (mitogen-activated protein kinase), and that tll expression is expanded in embryos lacking maternal NTF-1 activity; these results make NTF-1 a likely target for modulation by the tor RTK pathway in vivo. The data presented here support a model in which 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.

The pivotal role of twist in mesoderm determination in the Drosophila embryo depends upon two processes--the transcriptional activation of twist in the ventrally located mesodermal anlage and the regulation of downstream gene expression by the twist transcription factor. To elucidate the molecular mechanisms involved in these processes, we have compared both the coding and regulatory regions of the twist genes from Drosophila melanogaster and Drosophila virilis. Within the coding region, the basic-helix-loop-helix DNA binding and dimerization motif is highly conserved, consistent with the functional importance of this domain. A comparison of the transcriptional regulatory regions reveals a high degree of conservation in the more distal of the two ventral activator regions that have been mapped in the twist 5' flanking region. On the other hand, the more proximal ventral activator region is absent at the corresponding position in the D. virilis twist gene. Instead, there is a region in the second intron of the D. virilis gene that resembles the proximal element of the D. melanogaster gene, in that it consists of little more than a series of whole and half binding sites for the dorsal morphogen. In transformation experiments, the intronic D. virilis element directs an expression pattern that is indistinguishable from that directed by the D. melanogaster proximal VAR. Thus, the twi genes from these two species appear to have evolved enhancer elements with very similar structural and functional properties. These findings suggest that apparently redundant spatially regulated enhancer elements may each play essential roles in fine tuning the level and/or pattern of gene expression.

Like many DNA binding transcription factors, the Drosophila morphogen encoded by dorsal can both stimulate and repress promoter activity. In particular, this factor activates twist and represses zerknüllt on the ventral side of the early embryo. We find that when multiple copies of a dorsal binding site from the zerknüllt ventral repressor element are fused to a heterologous basal promoter, the resulting construct is activated by dorsal to give a ventral specific expression pattern. Thus, the ability of a dorsal binding site to mediate repression rather than activation is not an intrinsic property of the site, but depends upon its context. We also show that a hybrid promoter containing both the zerknüllt ventral repressor element and the twist ventral activator region is not ventrally active in the early embryo, demonstrating that repression is dominant over activation. Thus, the default mode of action of the dorsal protein is transcriptional activation. Additional factors may modify dorsal activity to bring about repression.

We have examined the role of protein-protein interactions in modulating the activity of Sp1, a human transcription factor that utilizes glutamine-rich activation domains. These domains may represent a commonly used structural motif, since a nonhomologous glutamine-rich segment from the Drosophila Antennapedia protein is also a potent activator when fused to the Sp1 DNA binding domain. Sp1 is generally considered a proximal promoter factor that can only stimulate transcription when bound close to the initiation site. However, here we present evidence that distally and proximally bound Sp1 can stimulate transcription synergistically. In addition, a DNA binding-deficient mutant of Sp1 that retains glutamine-rich domains can interact with proximally bound Sp1 to superactivate transcription. Glutaraldehyde cross-linking provides direct evidence for an interaction between Sp1 monomers. Thus, Sp1-Sp1 interactions may play an important role in modulating promoter activity.

We have adopted Drosophila tissue culture cells as a host system for studying the structure and function of mammalian transcription factors. These cells provide an Sp1-deficient background and have been used in a complementation assay to identify functional domains of human transcription factor Sp1. The SV40 early promoter, which contains six Sp1 binding sites (GC boxes), is induced up to 500-fold in Drosophila cells by the expression of Sp1, whereas promoters with fewer sites are activated less efficiently. Analysis of Sp1 mutants reveals multiple distinct regions outside of the DNA binding domain that are responsible for mediating transcriptional activation. The two most active domains, which appear to be functionally redundant with one another, consist of an unusual structure with a very low charge density, but a strikingly high glutamine content. A number of other sequence-specific transcription factors, such as the Drosophila zeste protein and several homeodomain proteins, contain glutamine-rich stretches, and we propose that these glutamine-rich domains represent a novel structural motif for transcriptional activation.

Sp1 is a sequence-specific DNA binding protein that activates RNA polymerase II transcription from promoters that contain properly positioned GC boxes. A series of deletion mutants of Sp1 were expressed in Escherichia coli and used to identify separate regions of the protein that are important for three different biochemical activities. The sequence-specificity of DNA binding was conferred by Zn(II) fingers, whereas a different region of Sp1 appeared to regulate the affinity of DNA binding. The E. coli-synthesized Sp1 was able to stimulate initiation of RNA synthesis in vitro, and at least two distinct segments of the protein contributed to its transcriptional activity.