Our dissection of the Vnd homeodomain protein in cell culture suggests that this transcription factor is a complex combination of repression and activation domains. Both intra- and inter-molecular interactions are apparently involved in the DNA binding, activation and repression activity of Vnd. Chimeric analyses of Vnd subdomains indicate that there are at least three repression domains and two activation domains that can contribute to the regulatory capacity of this protein. These activation and repression domains likely interact in the intact protein to generate different patterns of regulation. Our analyses indicate that the co-repressor, Groucho, promotes the repressional activity of Vnd, whereas interaction with the transcriptional activator and DNA bending protein, Dichaete, leads to target activation. summarizes our current model for Vnd regulation of target gene expression that is based on the data presented.
Figure 7 Summary of the regulatory domains in Vnd, and the mechanism of Vnd-mediated target gene regulation. (A) Schematic representation of the Vnd ORF showing the NK decapeptide (black box), the homeodomain (open box) and the NK-2 box (solid gray box). Below, (more ...)
The Vnd homeodomain plays a complex role in the function of this protein. Although capable of binding its target in vitro
), the homeodomain apparently does so very inefficiently within the context of the native protein in transient transfection assays. Watada et al
) showed that removal of the C-terminal of NKx2.2 downstream of the homeodomain leads to NKx2.2-activating reporter expression in tissue culture, although the intact protein was unable to do so. Co-factors are potentially required to stabilize the binding of the homeodomain to its target in the context of the complete protein. NMR analyses indicate that the conformation of the Vnd homeodomain is altered upon binding to target DNA (30
). This potentially results in the protein surfaces available for contact with the transcriptional machinery, and with other regulatory proteins being altered. These intra-molecular interactions would then provide a mechanism for transducing information from the DBD to the other regulatory domains within the protein. Our deletion analyses of Vnd–Gal4 chimeras in transient transfection assays suggest that the homeodomain interacts with repression domains at the N-terminal of the protein. In keeping with this speculation, we found that Groucho did not interact with a Vnd deletion, lacking the N-terminal 200 amino acids including the homeodomain and the NK-2 box, despite the presence of the EH domain (). Paradoxically, the homeodomain and sequences on its C-terminal side are also required for the interaction of Vnd with the transcription activator and DNA bending protein, Dichaete. Likewise, Bicoid and Oct 2A have been shown to interact with HMG proteins through their homeodomains (24
). Taken together, these results suggest that conformational changes in the native protein, including local protein folding, play important roles in the capacity of Vnd to regulate gene expression.
Extending the findings of Xhao and Skeath (11
), we show that Vnd's interaction with the high-mobility Sox protein, Dichaete, confers weak activation capacity on this homeodomain protein in transient transfections. Although Dichaete co-operated with Vnd to activate reporter expression driven by a vnd
enhancer containing both Dichaete and Vnd binding sites, no effects were seen when both proteins were expressed in cells with a reporter driven by the ind
enhancer (data not shown). Thus, Vnd's capacity to activate is dependent on both the availability of co-factors and the DNA target. In the context of the vnd
enhancer, Vnd's interaction with Dichaete changes both the context of these proteins and also that of the target DNA. As an HMG protein, Dichaete's transcriptional co-activator ability is associated with its capacity to bend DNA to align non-contiguous sites for interaction with other transcription regulators [(20
) and ]. The capacity of Dichaete to modulate target DNA architecture so that the contact DNA is altered potentially generates a new dimension to NKx2.2/Vnd regulation. To address the possibility that NKx2.2 regulates neural tube patterning by activating transcription, Muhr et al
) over-expressed the NKx2.2 homeodomain in frame with the VP16 activation domain in the chick neural tube. This substitution did not affect patterning of the neural tube, whereas over-expression of the NKx2.2 homeodomain fused to the Engrailed repression domain did. Consequently, Muhr et al
) concluded that NKx2.2's capacity to activate gene expression was not important in ventral neural tube patterning. However, these experiments modified the NKx2.2 protein but not its target DNA. Sox 1, 2 and 3, the vertebrate homologues of Dichaete, are expressed in overlapping patterns in the developing neural tube and Sox 2 function is necessary, but not sufficient, to direct cells to a neural fate (31
). If Nkx2.2 interacts with one, or all, of these proteins in the neural tube, this interaction could potentially confer transcriptional activation capacity on NKx2.2, by both reorganizing the target DNA and directly interacting with this transcription factor. This possibility has not yet been addressed. Interestingly, Watada et al
) also identified an activation domain at the C-terminal end of NKx2.2 that could be used in the presence of the appropriate transcriptional co-activators to activate target gene expression.
The intrinsic activity of Vnd's activation domains is suppressed in transient transfection assays in the context of the full-length protein, or when full-length Vnd is fused to Gal4. Both activation domains that we identified are predicted to have subdomains that are helical and enriched in homopolymeric alanine residues interspersed with bulky amino acids. The structure of the activation domain is as important as the amino acid sequence to its function (33
). Further work will be required to define the limits of these activation domains and the critical amino acids involved.
We were surprised to find that a domain with no homology to the published regulatory domains at the N-terminus of Vnd had the strongest repression activity in our domain swap experiments. This region is moderately conserved between Drosophila virilis, Drosophila pseudoobscura and Drosophila melanogaster (Z. Yu and D.M. Mellerick, unpublished data). However, the amino acid composition is not striking. One possibility is that the N-terminal extension of Vnd, lacking in vertebrate Vnd-type proteins, reflects greater functional complexity relative to the vertebrate counterparts. The alternative possibility that this domain is not functionally significant in the developing Drosophila embryo cannot be excluded at this time. Although, full-length Vnd co-precipitates with Groucho, a Vnd deletion lacking the N-terminal 200 amino acids, including the homeodomain and the Nk-2 box, failed to do so ( and ). Potentially, this explains why we were unable to assign repressor activity to the EH domain in the Gal4–Vnd chimera dissection analyses ( and ).
It should be kept in mind that how a transcription factor functions in a transient transfection assay does not always fully reflect its function in the context of the developing embryo, where the availability of co-activators, repressors and targets may be different. Tolkunova et al
) previously found that Engrailed uses two mechanisms to repress transcription—one that is predominant under normal transient transfection assay conditions, the other, which is predominant in an in vivo
repression assay. The EH domain had only weak activity in transient transfection assays, despite the fact that this Engrailed domain mediates the in vivo
repression activity and interacts specifically with Groucho, whereas two additional repression domains were more potent in transient transfections than in vivo
. Koizumi et al
) recently found that over-expression of mutant Vnd lacking either the NK-2 box or the NK domain in transgenic embryos did not affect the early repression activity of the vnd
transgene. Since both these domains are highly conserved, and associated with the repressor activity of the protein, these results were unexpected. One possible explanation for Koizumi's result is that deleting repression domains does not alter the activation capacity of Vnd. The mutant transgenes may be able to autoactivate expression of the endogenous vnd
gene, resulting in its over-expression. Consequently, the effects of the mutations would be hidden by the over-expression of the endogenous gene. Determining whether the mutant vnd
transgenes can rescue loss-of-function, vnd
mutants will address this possibility. Thus, both tissue culture and transgenic vnd
over-expression experiments have limitations in terms of their capacity to assign a specific function to an individual domain. Both these approaches highlight the context-dependence of Vnd activity.