MULTIPLE POSITIVE AND NEGATIVE REGULATORY ELEMENTS IN THE PROMOTER OFTHE MOUSE HOMEOBOX GENE HOXB-4

Mouse Hoxb-4 (Hox-2.6) is a homeobox gene that belongs to a family whi ch also includes Hoxa-4, Hoxc-4, and Hoxd-4 and that is related to the Deformed gene in Drosophila melanogaster. We have determined the sequ ence of 1.2 kb of 5' flanking DNA of mouse Hoxb-4 and by nuclease S1 a nd primer extension experiments identified two transcription start sit es, P1 and P2, 285 and 207 nucleotides upstream of the ATG initiator c odon, respectively. We have shown that this region harbors two indepen dent promoters which drive CAT expression in several different cell li nes with various efficiencies, suggesting that they are subject to cel l-type-specific regulation. Through detailed mutational analysis, we h ave identified several cis-regulatory elements, located upstream and d ownstream of the transcription start sites. They include two cell-type -specific negative regulatory elements, which are more active in F9 em bryonal carcinoma cells than in neuroblastoma cells (regions a and d a t -226 to -186 and +169 to +205, respectively). An additional negative regulatory element has been delimited (region b between +22 and +113) . Positive regulation is achieved by binding of HoxTF, a previously un known factor, to the sequence GCCATTGG (+148 to +155) that is essentia l for efficient Hoxb-4 expression. We have also defined the minimal pr omoter sequences and found that they include two 12-bp initiator eleme nts centered around each transcription start site. The complex archite cture of the Hoxb-4 promoter provides the framework for fine-tuned tra nscriptional regulation during embryonic development.