SP1-MEDIATED TRANSCRIPTIONAL ACTIVATION OF THE HUMAN-PI-CLASS GLUTATHIONE-S-TRANSFERASE PROMOTER

Previous studies in this laboratory have identified an essential AP-1 recognition sequence (C1 region; -69 to -63) in the human Pi class glu tathione S-transferase (GSTP1) promoter and a negatively acting regula tory element (-105 to -86) that acts to suppress GSTP1 transcription i n the human mammary carcinoma cell line, MCF7 (1). The data presented here further delineate the functional characteristics of the GSTP1 pro moter by examining the significance of two potential binding sites for the transcription factor, Sp1 (-57 to -49 and -47 to -39). The introd uction of mutations within these Sp1-like elements and the use of Sp1 antisera in electrophoretic mobility shift assays demonstrated that Sp 1 was bound to this region of the GSTP1 promoter in three different ce ll lines, MCF7, VCREMS, and EJ. Moreover, these in vitro studies indic ated that only one of the two putative Sp1 response elements was utili zed. Transient transfection assays using GSTP1 promoter constructs tha t incorporated mutations of the Sp1 elements clearly demonstrated that binding of Sp1 to the GSTP1 promoter was absolutely required for opti mal levels of GSTP1 transcription. In particular, disruption of the di stal Sp1 recognition motif (-57 to -49) markedly reduced GSTP1 promote r activity in each cell line, thus indicating preferential binding of Sp1 to the distal site. However, insertion of the repressor binding si te (-105 to -86) into these constructs suggested that Sp1 was not invo lved in mediating the suppressive effects of the GSTP1 transcriptional repressor in MCF7 cells, because inhibition of Sp1 binding did not al leviate repressor activity. Therefore, these studies provide strong ev idence that Sp1 plays a central role in regulating basal levels of GST P1 transcription.