FURTHER CHARACTERIZATION OF THE MAJOR AND MINOR RABBIT FMO1 PROMOTERSAND IDENTIFICATION OF BOTH POSITIVE AND NEGATIVE DISTAL REGULATORY ELEMENTS

Previously, two promoters were identified for the rabbit FMO1 gene: a major, upstream promoter (P-0) that initiates transcription from exon 0 and a second, minor promoter (P-1) located approximately 200 bp down stream and initiating transcription from exon 1. Transcription initiat ion from the P-0 promoter results in elimination of the exon 1 leader sequence from the mature transcript. In this report, we further define the major promoter and identify several positive and negative upstrea m regulatory domains employing deletion analysis and transient express ion in HepG2 cells. Of interest, P-0 and P-1 were equally active in th ese assays. A 49-bp fragment spanning position -41 to +8 was found ess ential for the activity of P-0 and also capable of basal transcription al activity. Interestingly, this same 49-bp region was found necessary for P-1 activity. Upstream of P-0, three positive regulatory regions (positions -348 to -176, -757 to -584, and -1196 to -829) and two nega tive regulatory regions (positions -2120 to -1724 and -829 to -757) we re identified using deletion mutants. Both P-0 and P-1 share the most proximate, positive regulatory domain but were regulated differentiall y by more distal 5' sequences. In addition to the upstream regulatory sequences, a potent negatively acting element was observed within intr on 1. Using DNA fragments representing the most potent positive (posit ion -348 to -176) and negative (position -829 to -757) regulatory sequ ences as probes, we demonstrate the formation of multiple specific DNA /protein complexes with protein factor(s) present in HepG2 nuclear ext ract. (C) 1997 Academic Press.