HOXB-2 TRANSCRIPTIONAL ACTIVATION IN RHOMBOMERE-3 AND RHOMBOMERE-5 REQUIRES AN EVOLUTIONARILY CONSERVED CIS-ACTING ELEMENT IN ADDITION TO THE KROX-20 BINDING-SITE

Segmentation is a key feature of the development of the vertebrate hin dbrain where it involves the generation of repetitive morphological un its termed rhombomeres (r), Hox genes are likely to play an essential role in the specification of segmental identity and me have been inves tigating their regulation. We show here that the mouse and chicken Hox b-2 genes are dependent for their expression in r3 and r5 on homologou s enhancer elements and on binding to this enhancer of the r3/r5-speci fic transcriptional activator Krox-20, Among the three Krox-20 binding sites of the mouse Hoxb-2 enhancer, only the high-affinity site is ab solutely necessary for activity, In contrast, we have identified an ad ditional cis-acting element, Box1, essential for r3/r5 enhancer activi ty. It is conserved both in sequence and in position respective to the high-affinity Krox-20 binding site within the mouse and chicken enhan cers. Furthermore, a short 44 bp sequence spanning the Box1 and Krox-2 0 sites can act as an r3/r5 enhancer when oligomerized. Box1 may there fore constitute a recognition sequence for another factor cooperating with Krox-20. Taken together, these data demonstrate the conservation of Hox gene regulation and of Krox-20 function during vertebrate evolu tion.