Ultrabithorax

How do the HOM proteins achieve their developmental specificity despite the very similar DNA binding specificities of isolated HOM proteins in vitro? Specificity could be achieved by differential interactions with protein cofactors. The extradenticle gene might encode such a cofactor since it interacts genetically in parallel with Ultrabithorax and abdominal-A. There is a selective interaction of the Extradenticle with certain UBX and ABD-A proteins. Strong interaction with Ultrabithorax proteins requires only the Ultrabithorax homeodomain and a 15-residue N-terminal extension that includes Tyr-Pro-Trp-Met (YPWM), a tetrapeptide motif found near the homeodomain in most HOM proteins and their mammalian Hox counterparts. The size and sequence of the region between the YPWM element and the homeodomain differ among Ultrabithorax isoforms (Johnson, 1995).

During the development of multicellular organisms, gene expression must be tightly regulated, both spatially and temporally. One set of transcription factors that are important in animal development is encoded by the homeotic (Hox) genes, which govern the choice between alternative developmental pathways along the anterior-posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbxl in mammals). To understand the structural basis of Hox-Extradenticle pairing, the crystal structure of an Ultrabithorax-Extradenticle-DNA complex at 2.4 A resolution was determined, using the minimal polypeptides that form a cooperative heterodimer. The Ultrabithorax and Extradenticle homeodomains bind opposite faces of the DNA, with their DNA-recognition helices almost touching each other. However, most of the cooperative interactions arise from the YPWM amino-acid motif of Ultrabithorax, located amino-terminally to its homeodomain, which forms a reverse turn and inserts into a hydrophobic pocket on the Extradenticle homeodomain surface. Together, these protein-DNA and protein-protein interactions define the general principles by which homeotic proteins interact with Extradenticle (or Pbx1) to affect development along the anterior-posterior axis of animals (Passner, 1999).

The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide identified in the 5' region of the mouse Hoxb-1 (the mammalian homolog of Drosophila labial) gene that is sufficient to direct a labial-like autoregulated expression pattern in Drosophila embryos. Labial and Extradenticle, their binding sites separated by only 4 bp, bind DNA as a heterodimer in a head-to-tail orientation. Mutations in base pairs predicted to contact the HOX N-terminal arm results in a change in HOX preference in the heterodimer, from Labial to Ultrabithorax. These results demonstrate that Extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site (Chan, 1996).

The homeobox gene extradenticle (exd) acts as a cofactor of Hox function in both Drosophila and vertebrates. It has been shown that the distribution of the Exd protein is developmentally regulated at the post-translational level; in the regions where exd is not functional, Exd is present only in the cell cytoplasm, whereas it accumulates in the nuclei of cells requiring exd function. Maternal EXD mRNA lasts for a few hours and is undetectable by stage 9 of embryogenesis. Protein produced by maternal RNA is stable. Both maternally and zygotically derived protein translocates into nuclei, suggesting that the proteins translocated from both mRNAs are functional and also suggesting that regulation of protein distribution is not dependent on transcriptional control (Azpiazu, 1998).

The subcellular localization of Exd is regulated by the BX-C genes; to varying degrees, BX-C genes can prevent or reduce nuclear translocation of Exd. Embryos homozygous for the deficiency Df(3R)P9, lacking the entire Bithorax complex, contain Exd at high levels in the nuclei of epidermal cells of both thoracic and abdominal segments. Only in the Keilin's organs of each segment does Exd remain cytoplasmic. This rise in the level of nuclear Exd in the abdominal segments (in comparison with the wild-type distribution) already indicates an involvement of the BX-C genes with the subcellular distribution of the product. In contrast, the thorax-determining genes Sex combs reduced (Scr) and Antp do not appear to affect Exd localization, as Scr Antp homozygous embryos exhibit normal distribution of Exd. To discriminate the roles of individual BX-C genes in the nuclear translocation of Exd, the distribution of Exd protein was examined in two more BX-C mutant combinations: Ubx- abd-A+ Abd-B+ and Ubx- abd-A- Abd-B+. Embryos homozygous for the first combination, defective only for Ubx function, show an increased level of nuclear Exd in the first abdominal segment as compared to wild type. In more posterior abdominal segments the levels and distributions of Exd are normal. In the second combination, lacking Ubx and abd-A functions, Exd is detected at high levels in the nuclei of the abdominal segments A1 to A4. In Df(3R)P9 (Ubx abd-A Abd-B) embryos, Exd nuclear localization extends to A8: all these results indicate that each BX-C gene is capable of preventing or reducing the nuclear translocation of Exd (Azpiazu, 1998).

The inhibition of Exd nuclear transport by overexpression of BX-C genes causes exd-like phenotypes. This was shown by inducing ectopic Ubx expression with several Gal4 lines during embryonic development and examining the segmental transformations produced. In the presence of normal exd function, Ubx specifies the pattern of the first abdominal (Al) segment. In contrast, in embryos lacking exd function, Ubx specifies a pattern resembling a more posterior segment, of A3-A5 type. Under conditions in which levels of Gal4 activity are high, larvae develop all segments anterior to A2 with an A3-A5 pattern. This segment pattern closely resembles that found in the A1 segment of zygotic exd larvae and is the same overall pattern observed after heat shock-inducing Ubx expression in zygotic exd embryos. These observations indicate that high levels of Ubx protein are able to produce an exd-like phenotype, in good agreement with the observed negative effect of BX-C genes on the nuclear translocation of Exd (Azpiazu, 1998).

The Ultrabithorax and Antennapedia genes of Drosophila encode homeodomain proteins that have very similar DNA binding specificities in vitro but specify the development of different segmental patterns in vivo. Cooperative interactions occur between UBX protein and Extradenticle, that selectively increase the affinity of UBX for a particular DNA target. UBX and EXD bind to neighboring sites on this DNA and interact directly to stabilize the DNA-bound form of UBX (Chan, S. K., 1994).

Specific amino acid residues at the amino end of the Ultrabithorax homeodomain are required to specifically regulate Antennapedia transcription; and 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).

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. However, there are striking differences between the two homeodomains in their specific interactions with DNA. Sequence differences within the selected binding sites have differential effects on protein binding, depending on the identity of the homeodomain. Differences at the 3' end of the binding site on the top strand indicate that the N-terminal arm of a homeodomain is capable of distinguishing an AT base-pair from TA in the minor groove (Draganescu, 1995).

A comparison was made among the DNA sequence preferences of homeodomains encoded by four Drosophila HOM proteins. One of the four, ABD-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 UBX and ANTP homeodomains display indistinguishable preferences outside the core while DFD 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).

The Ultrabithorax, abdominal-A, and Antennapedia homeoproteins differentially regulate the Antennapedia P1 promoter in a cell culture cotransfection assay: UBX and ABD-A repress P1, whereas ANTP activates it. Homeoproteins can use the same regulatory element but in very different ways. Chimeric UBX-ANTP proteins and UBX deletion derivatives demonstrate that functional specificity in P1 regulation is dictated mainly by sequences outside the homeodomain, with important determinants in the N-terminal region of the proteins (Saffman, 1994).

Naturally occurring binding sites for UBX contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more UBX molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation. Cooperative binding is dependent on sequences outside the homeodomain (Beachy, 1993).

Specific mutations in the gene encoding the largest subunit of RNA polymerase II (RpII215) cause a partial transformation of the capitellum, a structure on the third thoracic segment, into the wing, the analogous structure on the second thoracic segment. This mutant phenotype is also caused by genetically reducing the cellular concentration of UBX. Three RpII140 alleles cause a transformation of capitellum to wing but unlike RpII215 alleles, only when the concentration of UBX protein is reduced by mutations in Ubx (Mortin, 1992).

Ultrabithorax (Ubx) and Deformed (Dfd) proteins of Drosophila melanogaster contain homeodomains (HD) that are structurally similar and recognize similar DNA sequences, despite functionally distinct genetic regulatory roles for Ubx and Dfd. The Ubx-HD binding to a single optimal target site displays significantly increased affinity and higher salt concentration dependence at lower pH, while Dfd-HD binding to DNA is unaffected by pH. Results from studies of chimeric Ubx-Dfd homeodomains show that the N- and C-terminal regions of the Ubx-HD are required for this pH dependence. The increase in binding affinity at lower pH is greater for the Ubx optimal binding site than for other DNA binding sites, indicating that subtle sequence alterations in DNA binding sites may influence pH-dependent behavior. These data demonstrate enhanced DNA binding affinity at lower pH for the Ubx-HD in vitro and suggest the potential for significant discrimination of DNA binding sites in vivo (Li, 1996).

The presence of the LIM domain of mammalian Isl-1 (Drosophila homolog: Islet) inhibits binding of the homeodomain to its DNA target. This in vitro inhibition can be released either by denaturation/renaturation of the protein or by truncation of the LIM domains. A similar inhibition is observed in vivo using reporter constructs. LIM domains in a chimeric protein can inhibit binding of the Ultrabithorax homeodomain to its target. The ability of LIM domains to inhibit DNA binding by the homeodomain provides a possible basis for negative regulation of LIM-homeodomain proteins in vivo (Sanchez-Garcia, 1993).

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