A CONSERVED CLUSTER OF HOMEODOMAIN BINDING-SITES IN THE MOUSE HOXA-4 INTRON FUNCTIONS IN DROSOPHILA EMBRYOS AS AN ENHANCER THAT IS DIRECTLYREGULATED BY ULTRABITHORAX

The evolutionary conservation of the homeodomains suggests that their in vivo DNA binding sites may also be conserved between vertebrates an d invertebrates. The regulatory function of the mouse Hoxa-4 and Hoxb- 4 introns were analyzed in Drosophila since they both contain a cluste r of three homeodomain binding sites, the HB1 element, which was also found in the introns of other Hox genes ranging from fish to humans as well as in the Ultrabithorax (Ubx) and decapentaplegic (dpp) genes of Drosophila. The enhancer of the Hoxa-4 intron was found to respond to several homeobox genes activating a lacZ reporter gene in particular cells of the epidermis in Drosophila embryos. The enhancer activity wa s found to be similar to previously described autoregulatory elements of Deformed (Dfd), the Drosophila homolog of Hoxa-4 but additional exp ression was observed in more posterior segments activated by Ubx and r epressed by abdominal-A (abd-A). Point mutations in the homeodomain bi nding sites in HB1 abolished the enhancer activity. A second site supp ression experiment showed that UBX interacts directly with the HB1 ele ment. When the HB1 element in the Hoxa-4 intron was replaced by that o f the mesodermal enhancer of dpp, which was previously shown to be dir ectly controlled by Ubx, Ubx-dependent activation was retained, but re pression by abd-A was lost. The same result was obtained when the thir d binding site of HB1 was altered, suggesting that this site is respon sible for abd-A-dependent repression. Finally, deletion of potential c ofactor binding sites flanking the HB1 element that are also conserved in the medaka, chicken, and mouse genes revealed that they are import ant for enhancer function in Drosophila and that the Dfd-dependent and the Ubx-dependent expression requires different sites. The evolutiona ry and functional conservation of the HB1 elements indicates that not only the homeodomains but also some of their in vivo binding sites are conserved between vertebrates and invertebrates. (C) 1997 Academic Pr ess.