A protein complex containing Ubx, Exd and Hth is required for repression of Distalless gene expression. B.A. Gebelein ¹, J. Culi ¹, H-D. Ryoo ², R.S. Mann ¹. 1) Biochemistry and Molecular Biophysics, Columbia University, New York, NY; 2) Laboratory of Apoptosis and Cancer Biology, Rockefeller University, New York, NY.

   The Hox selector genes specify unique morphogenetic pathways along the anterior-posterior axis of metazoan organisms. They encode homeodomain-containing transcription factors that bind to DNA and regulate gene expression. It is thought that the different Hox proteins select for distinct developmental pathways by controlling the expression of unique combinations of target genes. However, many of the Hox proteins were found to interact with identical or very similar DNA sequences in vitro. The Extradenticle (Exd) and Homothorax (Hth) proteins have been shown to form complexes with the Hox transcription factors to increase their DNA-binding specificity. To characterize the role these cofactors and the Hox proteins play in the control of gene expression, we have focused on the regulation of the Distalless (Dll) target gene. Other laboratories have reported that the Dll gene contains a Hox/Exd binding site that is required for the repression of Dll expression by Ultrabithorax (Ubx) in the abdomen. In this study, we extend these observations by identifying a Hth binding site that is also required for the in vivo repression of Dll in the embryo. Furthermore, we show that Ubx and not Antennapedia (Antp) is able to repress Dll expression, and that their in vitro DNA binding affinities correlate well with their in vivo activities. In addition, another mechanism used by Ubx proteins to attain Dll target specificity is through differentially regulating transcription. Deletion analysis revealed the presence of a region in Ubx that is required for optimal Dll-lacZ repression in vivo but dispensable for Ubx/Exd/Hth binding to the Dll repressor DNA element in vitro.