Using the recognition code to swap homeodomain target specificity in cell culture

The homeodomain (HD) is a 60 amino acid-long DNA-binding domain. A large fraction of HDs binds with high affinity sequences containing the 5′-TAAT-3′ core motif. However, NK-2 class HDs recognizes sequences containing the 5′-CAAG-3′ core motif. By using a cell transfection approach, here we show that modification of residues located in the N-terminal arm (at positions 6, 7 and 8) and in the recognition helix (at position 54) is enough to swap the “in vivo” binding specificity of TTF-1 HD (which is a member of the NK-2 class HD) from 5′-CAAG-3′ to 5′-TAAT-3′-containing targets. The role of residue at position 54 is also supported by data obtained with the HD of the Drosophila engrailed protein. These data support the notion that DNA-binding specificity “in vivo” is dictated by few critical residues.     

Homeotic Complex (Hox) gene regulation and homeosis in the mesoderm of the Drosophila melanogaster embryo: the roles of signal transduction and cell autonomous regulation

In this paper we evaluate homeosis and Homeotic Complex (Hox) regulatory hierarchies in the somatic and visceral mesoderm. We demonstrate that both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology. We present data identifying novel regulatory interactions associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm. A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided. Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction. We find that extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and propose the term autonomic dominance for this phenomenon.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

Identification of presomitic mesoderm (PSM)-specific Mesp1 enhancer and generation of a PSM-specific Mesp1/Mesp2-null mouse using BAC-based rescue technology

Bacterial artificial chromosome (BAC) modification technology is a powerful method for the identification of enhancer sequences and genetic modifications. Using this method, we have analyzed the Mesp1 and/or Mesp2 enhancers and identified P1-PSME, a PSM-specific enhancer of Mesp1, which contains a T-box binding site similar to the previously identified P2-PSME. Hence, Mesp1 and Mesp2 use different enhancers for their PSM-specific expression. In addition, we find that these two genes also use distinct enhancers for their early mesodermal expression. Based on these results, we generated a PSM-specific Mesp1/Mesp2-null mouse by introducing a BAC clone, from which only early mesodermal Mesp1 expression is possible, into the Mesp1/Mesp2 double knockout (dKO) genetic background. This successfully rescued gastrulation defects due to the lack of the early mesoderm in the dKO mouse and we thereby obtained a PSM-specific Mesp1/Mesp2-null mouse showing a lack of segmented somites.