Internal regulatory interactions determine DNA binding specificity by a Hox transcription factor

Abstract

In developing bilaterans, the Hox transcription factor family regulates batteries of downstream genes to diversify serially repeated units. Given Hox homeodomains bind a wider array of DNA binding sites in vitro than are regulated by the full-length protein in vivo, regions outside the homeodomain must aid DNA site selection. Indeed, we find affinity for disparate DNA sequences varies less than 3-fold for the homeodomain isolated from the Drosophila Hox protein Ultrabithorax Ia (UbxHD), whereas for the full-length protein (UbxIa) affinity differs by more than 10-fold. The rank order of preferred DNA sequences also differs, further demonstrating distinct DNA binding preferences. The increased specificity of UbxIa can be partially attributed to the I1 region, which lies adjacent to the homeodomain and directly impacts binding energetics. Each of three segments within I1-the Extradenticle-binding YPWM motif, the six amino acids immediately N-terminal to this motif, and the eight amino acids abutting the YPWM C-terminus-uniquely contribute to DNA specificity. Combination of these regions synergistically modifies DNA binding to further enhance specificity. Intriguingly, the presence of the YPWM motif in UbxIa inhibits DNA binding only to Ubx-Extradenticle heterodimer binding sites, potentially functioning in vivo to prevent Ubx monomers from binding and misregulating heterodimer target genes. However, removal of the surrounding region allows the YPWM motif to also inhibit binding to Hox-only recognition sequences. Despite a modular domain design for Hox proteins, these results suggest that multiple Hox protein regions form a network of regulatory interactions that coordinate context- and gene-specific responses. Since most nonhomeodomain regions are not conserved between Hox family members, these regulatory interactions have the potential to diversify binding by the highly homologous Hox homeodomains.

Fig. 1

Homeodomain structure and Ubx sequence motifs. a. A schematic of the Ubx sequence showing mRNA splicing isoforms with the location of the homeodomain (HD, blue), the regions encoded by the b, mI, and mII microexons (shades of yellow/orange), and the YPWM motif (Y, blue), which may be used to bind the general Hox cofactor Exd., The sequences corresponding to the alternatively spliced microexons are intrinsically disordered, as demonstrated by disorder prediction algorithms and extreme susceptibility to native state proteolysis. The DNA binding experiments in this work focus on UbxIa, the most abundant isoform in the Drosophila embryo. b,c. Two views of a double truncation mutant of UbxIVa, which contains none of the microexon-encoded sequences, spanning the YPWM motif through the homeodomain (blue, side chains included for the YPWM motif) bound to a composite DNA binding site (black) and the Exd homeodomain (red). The region linking the YPWM motif to the homeodomain is too disordered to be visible in the crystal structure. To demonstrate the size of the small UbaIVa linker relative to the distance between the YPWM motif and the homeodomain, a model of this loop is shown in yellow, in which each dot represents the alpha carbon of one amino acid in the UbxIVa isoform (the shortest of the isoforms). d. Alignment of a portion of Ubx orthologues, including the 18 amino acid sequence that enhances DNA binding specificity (marked by a heavy black line) and the region surrounding this crucial area. The sequences for butterfly Juonia coenia Ubx (JcUbx), beetle Tribolium castaneum Ubx (TcUbx), Artemia franciscana Ubx (AfUbx) and velvet worm Akanthokara kaputensis Ubx (AkUbx) are derived from references., Sequence homology to Drosophila UbxIa (Dm UbxIa, gray) decreases with distance from the YPWM motif. The variation of some sequences within this 18 amino acid region potentially reflects evolution of DNA binding specificity in Ubx orthologues. e. We focus on DNA binding specificity determinants within an 18 amino acid section located largely within the I1 domain. The sequence of this region is expanded to illustrate the Ubx1a mutants used in this study. The bars above the UbxIa schematic indicate the positions of the DNA binding inhibitory domains (I1 and I2) and the domain that counters this inhibition to restore near-wild type high affinity DNA interaction (R).

Fig. 2

Schematics of enhancers containing the Ubx DNA binding sites. The numbers indicate the position of the DNA sequence used relative to the transcription start site for its associated gene. Ubx binding sites are marked by red right facing arrows (5´-TAAT-3´ sites) and red left facing arrows (5´-ATTA-3´ sites)., – Atypical binding sites recognized by UbxIa are indicated by orange left facing arrows (5´-ATTT-3´) and purple left facing arrows (5´-ATGT-3´). Note that while UbxIa binds this Dpp sequence in vitro, it is not known whether it is regulated by Ubx in vivo. The scale of the schematic is indicated by the 100 bp (black) and 1 kb (blue) scale bars. Because the large size of these enhancers precludes use of the entire enhancer in DNA binding experiments, only the sequences labeled and marked by a green box were utilized.

Fig. 3

DNA binding by UbxHD and UbxIa. a,b. Gel shift of UbxHD (a) and UbxIa (b) binding UA DNA. Although multiple sites are bound by UbxHD and UbxIa, single protein binding dominates at the concentration of Ubx equal to the apparent dissociation constant, indicated by an arrow. Multiple binding by UbxHD at higher protein concentrations is consistent with previous reports. Gels for the remaining DNA sequences can be found in Supplementary Materials Fig. 1. c. All multiple site DNA sequences are bound many times by UbxHD and UbxIa. For each DNA examined, the sample in the left lane contains DNA only, whereas the sample in the right lane contains DNA and UbxIa at concentrations greater than K_d or K_app. Therefore, in addition to DNA affinity, UbxIa•UbxIa interactions and the availability of multiple sites also impact the apparent affinity reported. d. The dissociation constant, K_d, is reported for the 40AB and Dll single site DNAs. The apparent dissociation constant, K_app, is reported for DNA sequences with multiple binding sites (Dpp, UA, A1, and Sal). e–j. DNA binding curves for UbxIa (grey) and UbxHD (black). To permit consistent analysis of binding to single and multiple site DNAs, the disappearance of free DNA was monitored. Note the differences in affinity are generally more pronounced for the natural sites than for the optimal binding sequence, 40AB. Note also that the UbxIa curves are not significantly cooperative, indicating a relatively low level of protein•protein interaction.

Fig. 4

The impact of the Ubx-18 and GPGG mutations on the function and structure of Ubx. a. Comparison of sequence-dependent DNA binding by Ubx-18 and the GPGG variant with UbxIa and UbxHD. Although both of these variations cause DNA sequence-specific effects on affinity, the major impact of the GPGG mutant is limited to enhancing affinity (lowering K_d) for Dll DNA, which contains a single Hox•Exd composite sequence. The broader impact of the internal deletion in Ubx-18 suggests that sequences flanking the YPWM motif also modulate DNA binding specificity. b. UbxIa binds poorly to all Hox•Exd composite sequences examined; these sequences differ in DNA binding site sequences and spacing between binding sites. Removal of the YPWM motif, either by deletion or point mutations, improves binding to a similar degree, suggesting inhibition of binding by the YPWM motif is a common feature of monomer binding to heterodimer sites. c. Circular dichroism of UbxIa (open circles) and Ubx-18 (gray circles). The similarity of these spectra reveals that the 18 amino acid internal deletion does not cause any large-scale changes in Ubx structure.

Fig. 5

Both UbxIa and Ubx-18 cooperatively bind the Dll DNA sequence with an Exd•Hth heterodimer. Although minimal DNA binding is observed by isolated UbxIa / Ubx-18 at these low concentrations, combination of these proteins permits facile formation of a quaternary complex (Ubx, Exd, Hth, and DNA), as revealed by the presence of a strong supershifted band (marked by U•E•H•D).

Fig. 6

The impact of the Ubx-6, Ubx-8, and Ubx-14 mutations on DNA binding by Ubx. a. Comparison of the DNA specificity of the Ubx-6 and Ubx-8 internal deletion mutants with UbxIa and UbxHD. The Ubx-6 data recapitulate the DNA binding profile of the GPGG mutant, although with weaker effects. The-8 internal deletion enhances binding to Dll yet inhibits 40AB binding relative to UbxIa. b. Deletion (Ubx-8) and rearrangement (Ubx8scramble) of the 8 amino acids C-terminal to the YPWM motif similarly impact DNA binding, indicating deletion mutants can be effectively used to probe the function of this region. c. PONDR VX-LT intrinsic disorder predictions are similar for UbxIa and Ubx8scramble, suggesting the dynamics of this disordered region are not greatly altered. d. The hydrophobicity profile of this region is also similar for UbxIa and Ubx8scramble. Consequently, the differences in binding observed in panel b are likely due to changes in side chain interactions between this region and the rest of the protein. e. Comparison of DNA affinities for Ubx-14 relative to Ubx-18, Ubx1a, and UbxHD. Relative to UbxIa, the -14 amino acid deletion largely recapitulates the effects of the -8 amino acid deletion. Comparison of Ubx-14 and Ubx-18 reveals that, in the absence of the 14 surrounding amino acids, the YPWM motif can inhibit binding to all DNA sequences tested. In contrast, the YPWM motif in the full-length protein can only inhibit binding to Hox•Exd composite sequences (Fig. 4 a,b).