BIOLOGICAL OVERVIEW

Dorsal (DL) is the focal protein in the development of dorsoventral polarity in the developing fly. It is a transcription factor, activating and repressing zygotic genes responsible for differentiation along the dorsoventral axis during the early stages of development. Over the course of time measured in hours, even before cellularization, the rapidly dividing zygotic cells take up their positions at the periphery of the fertilized egg. Nuclei in the ventral portion stain positive for DL while nuclei in the dorsal region fail to stain. Nuclear staining is graded; nuclei closest to the ventral midline stain most strongly (Rushlow, 1989, Steward, 1989 and Roth, 1989).

The dorsal group comprises a whole class of genes acting in the mother and responsible for setting up the activation and nuclear transport of DL. Early in oogenesis the egg becomes polarized. Such polarization affects the follicle cells surrounding the egg. Ventral follicle cells take on a different developmental fate from dorsal follicle cells. For example, the release and activation of the Toll ligand Spätzle occurs only in the ventral region of the egg.

Once fertilization has taken place, Spätzle triggers signaling in the Toll receptor. The signals are transmitted via Tube and Pelle into the cytoplasm, resulting in the activation of Dorsal. Activation of Dorsal is held in abeyance by Cactus protein. Cactus binds Dorsal, preventing its nuclear transport. Signals from Toll result in the destruction of Cactus in ventral cells, whereupon Dorsal is released to continue its transportation into the nucleus.

The Cactus-Dorsal interactive system is preserved in vertebrates and used in activation of the immune system. The transcription factor NF kappa B is involved in the activation of immune system cells. The vertebrate homolog of Cactus, I kappa B (where I stands for inhibitor), keeps NF kappa B restrained in the cytoplasm until cell activation, at which time NF kappa B enters the nucleus. The adult fly uses the identical system in its immune system. Bacterial challenge causes the activation of Dorsal and a second Dorsal-like factor (Dif) in the fat body, an organ that serves the immune function in flies. The developing awareness of the importance of an immune response system in flies has initiated a new focus of interest in Dorsal research (Reichart, 1993 and Lemaitre, 1995).

Dorsal's nuclear function involves an interaction with transcription factor Bang senseless, here termed Dorsal switch protein 1 or DSP1 (FlyBase link: FBgn0000231) DSP1, an HMG-1/2-like protein, binds DNA in a highly cooperative manner with three members of the Rel family of transcriptional regulators (NF-kappaB, the p50 subunit of NF-kappaB, and the Rel domain of Dorsal). This cooperativity is apparent with DNA molecules bearing consensus Rel-protein-binding sites and is unaffected by the presence of a negative regulatory element, a sequence previously proposed to be important for mediating repression by these Rel proteins. The cooperativity observed in these DNA-binding assays is paralleled by interactions between protein pairs in the absence of DNA. In HeLa cells, as assayed by transient transfection, expression of DSP1 increases activation by Dorsal from the twist promoter and inhibits that activation from the zen promoter, consistent with the previously proposed idea that DSP1 can affect the action of Dorsal in a promoter-specific fashion (Brickman, 1999).

DSP1 has opposite effects on the activity of Dorsal assayed with regulatory sequences excised from the twist and zen promoters. These experiments were performed by transiently transfecting mammalian cells in culture. Thus, reporters containing either a 180-bp fragment from zen (a fragment sufficient to mediate repression in Drosophila) or the entire regulatory region of twist (from -1,438 to +38) were activated by cotransfection with DNA encoding Dorsal. Cotransfection with DNA encoding DSP1 has just the opposite effects on this Dorsal mediated activation of the two promoters: activation from the twist promoter is stimulated 4-fold, whereas that from the zen promoter is inhibited 3-fold. DSP1's stimulation of Dorsal-mediated activation from the twist promoter can be mapped to the defined enhancer elements or VARs. Thus, DSP1 also stimulates Dorsal-mediated activation if the template bears, instead of the intact twist promoter, a cassette that contains the two VARs that drive ventral-specific expression of the twist gene in the Drosophila embryo. The two VARs together constitute approximately 300 bp and contain multiple Rel-protein-binding sites (Brickman, 1999).

It is not known what DNA sequences in the zen and twist promoters determine the opposite effects of DSP1 on dorsal-mediated activation. The finding that a negative regulatory element (NRE) has no effect on cooperative binding to DNA of DSP1 and various Rel proteins prompted a reexamination of the earlier claims that DSP1 converts Dorsal, the p50 homodimer, and the NF-kappaB heterodimer into repressors and that this effect requires the NRE. In each case, DSP1 inhibits Rel-protein-dependent activation both in the presence and absence of an NRE. In no case was NRE-dependent conversion of the Rel protein to a repressor by cotransfection with DSP1 observed. It is not understood why the current results differ from those reported previously (Brickman, 1999 and references therein).

Sites of the described protein-protein interactions are found in the conserved Rel domains and in the fragment of DSP1 that bears both HMG domains. The Rel domains of p65 and of Dif differ from those of Dorsal and of p50 in that they lack the HMG-domain-interaction site. The HMG domain of DSP1 also interacts with the TATA-binding protein. Similar interactions have been reported for HMG-1 and HMG-2 with the steroid hormone receptors, for HMG-1 with p53, for HMG-1 with HOXD9, and for HMG-2 with Oct2. Thus, the HMG domain may contain a common structural motif for cooperative DNA binding and interaction with other transcription factors. The interaction between TATA-binding protein and DSP1 also seems to be influenced by the glutamine-rich amino-terminal domain in that the full-length DSP1 interacts more avidly with TATA-binding protein than does the HMG-1 domain. These experiments suggest that the amino-terminal glutamine-rich domain may also potentiate the DSP1-Rel protein interaction as well, because all DSP1-Rel interactions seem stronger with full-length DSP1, particularly the weak interactions seen between DSP1 and p65 or Dif, which are observed only with GST-DSP1 and not with GST-DSP1 (178-393) (Brickman, 1999).

PROTEIN STRUCTURE

Amino Acids - 678

Structural Domains

The Dorsal protein has a large N-terminal region of 294 amino acids, homologous to the vertebrate c-rel and its corresponding viral oncogene V-rel, the transforming gene of the reticuloendotheliosis virus strain T (Steward, 1987).

An in vivo structure-function analysis of Dorsal has been performed in order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus. All these functions are carried out by regions within the conserved Rel-homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N-terminal 40 amino acids abolishes the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region (Govind, 1996).

date revised: 22 January 2000 

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