Two CCAAT boxes in a novel inverted repeat motif are required for Hlx homeobox gene expression

Hlx is a homeobox transcription factor gene required for normal intestinal and hepatic growth in development. We previously found high sequence identi ty and 17 conserved consensus cis-regulatory/transcription factor binding e lements in the mouse and human Hlx 5' regions. A 594 bp sequence in the Hlx 5' region possessing the same activity in driving luciferase expression as larger His 5' sequences had three segments each necessary but not sufficie nt for luciferase expression in NM: 3T3 cells (which express Hlx). Nine of the conserved putative regulatory elements are positioned within these segm ents, including two CCAAT boxes on opposite strands within a conserved 44 b p inverted repeat sequence. To test the hypothesis that these elements are required for promoter activity, we compared the reporter expression activit y pf segments containing mutations of these elements with activity of the p arent His promoter sequence. We found that mutation of either CCAAT box or a conserved AP-2 site resulted in a significant decrease in promoter activi ty. Restoration of the inverted repeat with complementary mutations of both CCAAT boxes did not restore activity. Further, mutation of other portions of the inverted repeat did not affect promoter activity. Mutation of other elements had no effect on promoter activity. (C) 2001 Elsevier Science B.V. All rights reserved.