FUNCTIONAL DIFFERENCES BETWEEN HOX PROTEINS CONFERRED BY 2 RESIDUES IN THE HOMEODOMAIN N-TERMINAL ARM

Hox genes encode homeodomain-containing transcriptional regulators tha t function during development to specify positional identity along emb ryonic axes. The homeodomain is composed of a flexible N-terminal arm and three alpha helices, and it differentially binds DNA. A number of homeodomains recognize sites containing a TAAT core moth. The product of the murine Hoxd-4 (Hox-4.2) gene functions in a positive autoregula tory fashion in P19 cells that is dependent on two TAAT motifs in the Hoxd-4 promoter. This effect is specific in that murine HOXA-1 (HOX-1. 6) is unable to activate transcription through the Hoxd-4 autoregulato ry element. Here we show that this is due to an inability of the HOXA- 1 homeodomain to bind a HOXD-4 recognition site effectively. We have p roduced chimeras between HOXD-4 and HOXA-1 to map specific residues re sponsible for this functional difference. When positions 2 and 3 in th e N-terminal arm of HOXA-1 were converted to HOXD-4 identity, both str ong DNA binding and transcriptional activation were rescued. This subs titution appears to confer an increased DNA-binding ability on the HOX A-1 homeodomain, since we were unable to detect a high-affinity recogn ition sequence for HOXA-1 in a randomized pool of DNA probes. The cont ribution of position 3 to DNA binding has been implicated by structura l studies, but this is the first report of the importance of position 2 in regulating homeodomain-DNA interactions. Additionally, specific h omeodomain residues that confer major differences in DNA binding and t ranscriptional activation between Hox gene products have not been prev iously determined. Identity at these two positions is generally conser ved among paralogs but varies between Hox gene subfamilies. As a resul t, these residues may be important for the regulation of target gene e xpression by specific Hox products.