Functional dissection of the brain-specific rat aldolase C gene promoter in transgenic mice. Essential role of two GC-rich boxes and an HNF3 binding site.
The aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A' and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A' and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and 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 describe 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 protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice 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, mutations or deletions of either the A/A' or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A' and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A' or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A' and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.
Thomas
M
M
Institut Cochin de Génétique Moléculaire, Génétique et Pathologie Moléculaires, INSERM U129, Université René Descartes, Paris, France.
Skala
H
H
Kahn
A
A
Tuy
F P
FP
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D003596:Cytosine; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D006147:Guanine; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D007527:Isoenzymes; D014157:Transcription Factors; D006147:Guanine; D003596:Cytosine; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D005634:Fructose-Bisphosphate Aldolase