IDENTIFICATION OF 2 ACTIVATING ELEMENTS IN THE PROXIMAL PROMOTER REGION OF THE HUMAN GLUTATHIONE TRANSFERASE-A1 AND TRANFERASE-A2 GENES

Promoter regions derived from the human glutathione S-transferase (GST ) alpha gene cluster located on chromosome 6p12 were studied: the iden tical proximal promoters of the GST A1 and GST A2 genes and a proximal promoter of a pseudogene of this class. The sequence of the pseudogen e promoter differs in four single nucleotides at positions -86, -66, - 41, and -13, and a noncritical TTT insertion at positions -71 to -69 f rom the GST A1/A2 promoter. Here, it was shown that the GST A1/A2 prox imal promoters differed by a factor of 3.4 in their activity from the proximal pseudogene promoter. Therefore, the functional significance o f single base exchanges was examined by introducing individual point m utations at the four positions within the proximal GST A1/A2 promoter. In functional tests in transiently transfected human hepatoblastoma H epG2 cells the base exchange at position -13 showed no effect, whereas mutations at position -41 or -86 diminished the promoter activity to a level comparable to the pseudogene promoter. Promoter fragments of b oth genes spanning over these four sites were analyzed in a heterologo us promoter context for their functionality in HepG2 cells. Moreover, gel shift experiments showed specific binding of nuclear proteins to t hese promoter fragments. The results show that in the proximal GST A1/ A2 promoter the sites at position -41 or -86 are essential for the bin ding of activating transcription factor complexes. (C) 1998 Academic P ress.