THE 6-KILOBASE C-ERBB2 PROMOTER CONTAINS POSITIVE AND NEGATIVE REGULATORY ELEMENTS FUNCTIONAL IN HUMAN MAMMARY CELL-LINES

A 6-kilobase fragment extending at the 5'-end of the c-erbB2 protoonco gene was isolated from a normal human lymphocyte genomic DNA library. The full-length fragment and five subfragments with identical 3'-ends were obtained by progressive unidirectional deletion from the 5'-end a nd were cloned in front of the luciferase reporter gene. The hybrid ge nes were analyzed for transcriptional activity in human mammary cell l ines synthesizing low (HBL-100 and T-47D), moderate (MDA-MB-453), or h igh (BT-474) amounts of the c-erbB2 mRNA and were also analyzed in HeL a cells. Gene-specific expression was observed, indicating the presenc e of multiple cis-acting sequences in the c-erbB2 promoter. A major ne gative element is located in the -2- to -4-kilobase region. It is flan ked on both sides by positive elements that display enhanced transcrip tional activity in the BT-474 tumor cells only. While predominant in t he low-expressing cells, the effect of the repressor appears to be ove rcome by the distal transactivator in the high-expressing BT-474 cells , resulting in a 15 to 50 times increase in luciferase activity relati ve to the HBL-100 and T-47D cells, respectively. Cell-specific express ion relies on the trans-acting factors present in the different cell l ines. The formation of cell-specific protein-DNA complexes was demonst rated by gel retardation assay.