FC0053
Homeotic protein ultrabithorax (Ubx)  -  DNA, Exd

Biological function
Development of all bilaterally symmetric animals requires reliable temporal and spatial regulation of gene expression by members of the Hox protein family. Hox proteins are expressed in contiguous domains along the anterior-posterior axis, where they regulate region-specific differentiation, patterning, and proliferation. Misexpression of a Hox protein transforms one region into another, altering tissue and appendage fates. These dramatic phenotypes underscore the absolute requirement for specific and reliable Hox function in vivo. Ultrabithorax regulates the development of halteres and the posterior-most pair of thoracic legs from analogous tissues. The disparity between the absolute requirement for distinct Hox activities in vivo and the similarity of homeodomain-DNA recognition in vitro has been termed the ’Hox paradox’. This paradox is resolved, in part, through Hox interactions with other transcription factors, increasing specificity by requiring tandem Hox and partner DNA binding sites. Since the expression and activity of many Hox partners is limited to specific tissues, protein interactions potentially provide contextual information to Hox proteins as well as contribute to target site selection.

Domain organization/sequence features
Ubx binds to DNA via its homeodomain (HD), which is flanked by three disordered regions: The I1 region of Ubx reduces affinity twofold, and the I2 region reduces affinity a further 40-fold; and R restores the loss in affinity. The I1 region embeds the YPWM Exd interaction motif), which relieves repression of DNA binding by I1. The large N-terminal disordered domain encodes most of the transcription activation domain and directly impacts DNA binding affinity by the Ubx homeodomain. The C-terminal disordered domain alters DNA binding affinity and specificity, interaction with a Hox binding protein, and strongly influences both transcription activation and repression. Phosphorylation of the N-terminal disordered domain and alternative splicing of the C-terminal disordered domain could allow the cell to both regulate and coordinate DNA binding, protein interactions, and transcription regulation.

Structural evidence
Native proteolysis experiments indicate that three disordered regulatory regions (I1,I2 and R). CD spectroscopy shows negative ellipticity ~ 200 nm, which is characteristic to disordered regions.

Biochemical evidence
The structured homeodomain (HD) of the Hox transcription factor Ubx binds its optimal DNA sequence with very high affinity (K_d=63±24 pM), yet with little ability to distinguish target DNA sequences in vivo (3-fold variation in affinity). By contrast, the full-length Ubx has lower affinity, yet higher selectivity for the optimal DNA sequence (K_d=160±33 pM, 12-fold affinity variation), which indicates that regions outside the HD are important for DNA affinity and sequence selectivity. Most of the non-HD regulatory regions have ID character. The I1 region of Ubx reduces affinity twofold, and the I2 region reduces affinity a further 40-fold; both via the competitive binding mechanism. The R region restores much of this loss in affinity in a length-dependent manner.

Structure/Mechanism
Ubx HD–DNA complex formation is strongly pH dependent. I1, but not I2, reduces the pH-dependence of DNA binding, which indicates that I1 must directly interact with the HD to shift the pK_as of residues that are crucial for DNA interactions. By contrast, I2 does not establish direct contact with the HD; instead, it rapidly fluctuates to hinder DNA access. The R region either directly interacts with I1 and I2 to perturb their conformational equilibrium, or nonspecifically blocks their access to DNA.

Mechanism category
competitive binding

Isoforms, context-dependence
Protein–protein interactions or alternative splicing enable ’context specific gene regulation’ of Ubx; that is, cognate DNA sequences depend on the cellular context. The I1 region contains a YPWM motif, which mediates communication with the Hox cofactor Extradenticle (Exd), which relieves repression of DNA binding by I1. Different Ubx splicing isoforms are produced in a stage- and tissue-specific manner by alternative splicing of three microexons that are located in the region that links the YPWM motif to the HD. Removal of the microexons (alone or in combination) affects Ubx DNA affinity and selectivity both in vitro and in vivo.

Significance
Fuzzy region based regulatory mechanisms enable Ubx to sense tissue identity and respond by implementing a context-specific gene regulatory cascade.