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 identity and 17 conserved consensus cis-regulatory/transcription factor binding elements 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 Hlx 5' sequences had three segments each necessary but not sufficient for luciferase expression in NIH 3T3 cells (which express Hlx). Nine of the conserved putative regulatory elements are positioned within these segments, including two CCAAT boxes on opposite strands within a conserved 44 bp inverted repeat sequence. To test the hypothesis that these elements are required for promoter activity, we compared the reporter expression activity of segments containing mutations of these elements with activity of the parent Hlx promoter sequence. We found that mutation of either CCAAT box or a conserved AP-2 site resulted in a significant decrease in promoter activity. 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.

Structural and functional characterization of the mouse Hlx homeobox gene.

Hlx is a mesenchymally expressed homeobox transcription factor gene that is essential for normal intestinal and hepatic development in the mouse. Here we report further characterization of the mouse Hlx gene, including an additional 3.7 kb of 5' sequence as well as the sequence of the three introns. Comparison of the sequence of the mouse Hlx gene 5' to the coding region with that of the human gene revealed multiple regions of high conservation. Neither the mouse nor the human gene contained a TATA box, and ribonuclease protection studies defined heterogeneous transcription start sites for the mouse gene. A number of consensus transcription factor binding sites were conserved between the mouse and human Hlx genes both within and outside of the highly conserved regions. Reporter constructs containing 4.2 or 1.4 kb of mouse 5' sequence showed active expression in cell lines that express Hlx. Further characterization of the mouse Hlx gene will provide insight into the developmental regulation of the mouse digestive system.