Interactive Fly, Drosophila

Hox proteins are transcription factors that assign positional identities along the body axis of animal embryos. Different Hox proteins have similar DNA-binding functions in vitro and require cofactors to achieve their biological functions. Cofactors can function by enhancement of the DNA-binding specificity of Hox proteins, as has been shown for Extradenticle (Exd). Three results support a novel mechanism for Hox cofactor function. (1) The Hox protein Deformed (Dfd) can interact with simple DNA-binding sites in Drosophila embryos in the absence of Exd, but this binding is not sufficient for transcriptional activation of reporter genes. (2) Either Dfd or a Dfd-VP16 hybrid (VP16 is a transcriptional activation domain) mediate much stronger activation in embryos on a Dfd-Exd composite site than on a simple Dfd-binding site, even though the two sites possess similar Dfd-binding affinities. This suggests that Exd is required to release the transcriptional activation function of Dfd independent of Exd enhancement of Dfd-binding affinity on the composite site. (3) Transfection assays confirm that Dfd possesses an activation domain, which is suppressed in a manner dependent on the presence of the homeodomain. The regulation of Hox transcriptional activation functions may underlie the different functional specificities of proteins belonging to this developmental patterning family (Li, 1999a).

The neutral state of Dfd on simple binding sites indicates that additional regulatory steps and regulatory sequences are required for Dfd to activate gene expression. To test the hypothesis that Dfd binding per se is inherently neutral in embryos, a test was performed to see whether high levels of Dfd or Dfd-VP16 proteins could activate transcription through simple Dfd recognition sites. In vitro, a DNA sequence consisting of two tandem copies of the simple Deformed binding site (D site or 2×D), is bound by Dfd with high affinity but not detectably bound by Exd. The affinity of Dfd protein for the 2×D-site is not enhanced by the inclusion of Exd protein (Li, 1999a).

A test was performed of the embryonic function a varient of the D site reporter construct. This varient contains two tandem copies of a core sequence, to which Dfd and Exd bind together (2×ED2 sites). In vitro, the 2×ED2 site is bound weakly by Dfd protein alone, but is not bound detectably by Exd alone. Binding of Dfd to the 2×ED2 site is enhanced in the presence of Exd as shown by the formation of an abundant complex that contains Dfd, Exd and 2×ED2. The affinity of the Dfd-Exd heterodimer for the 2×ED2 site is approximately the same as the affinity of the Dfd protein alone for the 2×D site. Although the 2×D site and the 2×ED2 site have very similar in vitro affinity for Dfd in the presence of Exd, the 2×ED2 site is much more responsive than the 2×D site to either Dfd or Dfd-VP16 proteins in embryos. This strongly suggests that Exd is required to release the transcriptional activation function of Dfd in a way that is independent of the Exd enhancement of Dfd binding affinity on the 2×ED2 site. At present, the most widely accepted models propose Exd as a cofactor that has its effect on Hox specificity by acting to increase the binding affinity of different Hox proteins to different composite binding sites. The results presented here indicate that Exd has other regulatory effects on Hox proteins that may play a role in the diversification of function within the Hox family (Li, 1999a).

Dfd protein contains an autonomous activation domain that is functional in transfection assays when separated from the C-terminal half of the protein. On tandem repeats of simple Dfd-binding sites, the function of the Dfd transcription activation domain is suppressed both in cultured cells and in embryos. In embryos, this suppression can be partially relieved by the addition of Exd-binding sites to simple Dfd-binding sites. This is apparently due to the function of the Exd protein, since exd genetic function is required for the relief of the suppression of Dfd activation function on 2×ED2 sites. In cultured cells, the suppression of Dfd activation function can be conferred by the homeodomain regions from either Dfd or Ubx. Since no evidence is found that there is a direct intramolecular interaction between the Dfd homeodomain and its transcriptional activation region, a model is proposed that invokes a masking factor that suppresses the function of the activation domain by contacting the homeodomain region. In addition, it is speculated that Exd may be required to alleviate the suppressive effect of the proposed masking factor by competing for overlapping protein-protein interaction sites on the homeodomain (Li, 1999a).

DFD and UBX bind to DNA with the recognition helix in the major groove 3' to the TAAT core sequence and the N-terminal arm in the adjacent minor groove. The N-terminal arm of a homeodomain is capable of distinguishing an A.T base-pair from T.A in the minor groove. Specific orientation of the N-terminal arm within the binding site appears to vary between the homeodomains and influences the interaction of the recognition helix with the major groove (Draganescu, 1995).

The DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins were compared. One of the four, Abdominal-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity (Ekker, 1994).

Specific amino acid residues at the amino end of the Ultrabithorax homeodomain are required to specifically regulate Antennapedia transcription: in the context of a Deformed protein, these amino-end residues are sufficient to switch from Deformed- to Ultrabithorax-like targeting specificity. Although residues in the amino end of the homeodomain are also important in determining a Deformed-like targeting specificity, other regions of the Deformed homeodomain are also required for full activity (Lin, 1992).

Deformed possesses an acidic region just N-terminal to the homeodomain and a C-terminal sequence called the C-tail region, containing poly-glutamine and poly-asparagine tracts. Removal of the acidic domain and the C-tail region converts a chimeric Deformed/Abdominal-B protein, possessing the Abdominal-B homeodomain, from a strong activator to a repressor of a Distal-less promoter element, but has little effect on activation of an empty spiracles element. Constructs without a third domain, the N-terminal N domain, fail to show any regulatory activity. These results suggest transcriptional activation by the N domain can be modulated by acidic and C-tail domains (Zhu, 1996).

A heat-shock promoter/selector gene was constructed that encodes a Deformed/Abdominal-B chimera in which the Abdominal-B homeodomain is substituted for that of Deformed. Expression of this chimeric protein throughout the embryo causes morphological transformation of anterior segments toward more posterior identities. A number of other homeotic selector genes, all normally repressed by Abdominal-B, are ectopically activated by the chimeric protein. These results support the hypothesis that the target specificity of similar homeodomain proteins is largely determined by the amino acid sequence of the homeodomain (Kuziora, 1990).

The relevance of functional interactions between Prospero and homeodomain proteins is supported by the observation that Prospero, together with the homeodomain protein Deformed, is required for proper regulation of a Deformed-dependent neural-specific transcriptional enhancer. Deformed and mouse Hoxa-5 binding to this neuronal enhancer is increased more than 10 fold by Pros. Pros reduces Eve's DNA binding to this enhancer, but does not modulate the binding of Engrailed. This interaction is unidirectional and specific, since neither Dfd, Eve nor En has an effect on Pros binding. The modulation by Pros does not require Pros binding to DNA. Pros protein modifies the trypsin sensitivity of Dfd protein, suggesting that Pros binds Dfd and is able to induce a conformation change in Dfd. Nevertheless, Pros is able to bind the Dfd neuronal autoregulatory enhancer and enhances Dfd binding to this DNA sequence. The DNA-binding and homeodomain protein-interacting activities of Prospero are localized to its highly conserved C-terminal region, and the two regulatory capacities are independent (Hassan, 1997).

Hox transcription factors, in combination with cofactors such as Exd protein and its mammalian Pbx homologs (PBC proteins), provide diverse developmental fates to cells on the anteroposterior body axis of animal embryos. However, the mechanisms by which the different Hox proteins and their cofactors generate those diverse fates remain unclear. Recent findings have provided support for a model where the DNA binding sites that directly interact with Hox-PBC heterodimers determine which member of the Hox protein family occupies and thereby regulates a given target element. In the experiments reported here, the function of chimeric Hox response elements is tested, and, surprisingly, evidence is found that runs counter to this view. A 21 bp cofactor binding sequence from an embryonic Deformed Hox response element (region 6), containing no Hox or Hox-PBC binding sites, was combined with single or multimeric sites that binds heterodimers of Labial-type Hox and PBC proteins (region 3). Normally, multimerized Labial-PBC binding sites are sufficient to trigger a Labial-specific activation response in either Drosophila or mouse embryos. The 21 bp sequence element plays an important role in Deformed specificity, because it is capable of switching a Labial-PBC binding site/response element to a Deformed response element. Thus, cofactor binding sites that are separate and distinct from homeodomain binding sites can dictate the regulatory specificity of a Hox response element (Li, 1999b).

The instructive role of factors bound to non-Hox binding sites in controlling Hox responses is probably a general mechanism by which different Hox proteins acquire distinct functions. Exd is a well-characterized example that is used in a subset of Hox-activated response elements. However, the influence of Exd on Hox specificity may be superseded in complex elements that contain sequences such as region 6. How the specificity code is generated in the average Dfd or Ubx response element is likely to vary depending on the cell type, the presence or absence of Exd in the cell, the stage of development, and the extracellular signals that are received by a given response element. The putative activating cofactor binding site(s) (GGC..AAAGC) in the region 6 element are present in other naturally derived Dfd response elements, so there may be an important subset of Dfd response elements that rely on these sites for maxillary specificity. At present, none of the known complex elements that respond to other Hox proteins contain good matches to the GGC..AAAGC motifs. The region 6 cofactor(s) that are required to elicit a Dfd-dependent activation response by interacting with the GGCnn(n)AAAGC motif are not yet known. The unknown region 6 cofactors might selectively release covert activation functions of Dfd, or interact with Dfd to form new activation functions. In this view, although multiple Hox proteins (e.g., Dfd and Lab) may bind to the region 3 Dfd binding site or the Lab-Exd composite site, only Dfd would functionally interact with the cofactors bound nearby on region 6 to activate transcription, while other Hox proteins would not (Li, 1999b).

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