FUNCTIONAL DISSECTION OF THE BRAIN-SPECIFIC RAT ALDOLASE-C GENE PROMOTER IN TRANSGENIC MICE - ESSENTIAL ROLE OF 2 GC-RICH BOXES AND AN HNF3BINDING-SITE

The aldolase C gene product is a glycolytic isoenzyme specifically det ected in brain. We have previously defined a short 115-base pair promo ter fragment able to confer on a reporter chloramphenicol acetyltransf erase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A' and B boxes) were d etected by in vitro DNase1 footprinting experiments with brain, fibrob last, or liver nuclear extracts. Both A/A' and B boxes, sharing struct ural homology, are able to interact with Sp1, Krox20/Krox24 factors an d with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A, and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). In this paper, we de scribe a new ubiquitous factor termed Ub able to bind the A/A' box. We also delimit a third element (box C) binding a hepatocyte-enriched pr otein displaced by a hepatocyte nuclear factor 3-specific oligonucleot ide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mi ce carrying different mutant promoters cloned in front of the CAT gene . A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A' and B boxes. However, mu tations or deletions of either the A/A' or the B box have no significa nt effect on the CAT gene expression. We therefore hypothesize that th e A/A' and B sites may be functionally redundant. Indeed, constructs h arboring only one of these two boxes (A/A' or B) linked to the C box d isplayed a brain-specific CAT activity similar to that obtained with t he wild type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A' and B boxes, was totally inactive, sug gesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.