TATA box and Sp1 sites mediate the activation of c-myc promoter P1 by immunoglobulin kappa enhancers.

In Burkitt's lymphoma (BL) cells the proto-oncogene c-myc is transcriptionally activated by chromosomal translocation to the immunoglobulin (Ig) gene loci. This activation is characterized by preferential transcription from the c-myc promoter P1 and accomplished by juxtaposed Ig enhancer elements. To identify promoter elements required for enhancer-activated P1 transcription, we studied the activation of c-myc reporter gene constructs by the Ig kappa intron and 3' enhancers. Deletion analysis defined the core promoter with a TATA box and two adjacent GC/GT boxes upstream sufficient for basal and enhancer-activated transcription. Gel retardation assays revealed Sp1's binding affinity to the GC/GT box proximal to the TATA box to be higher than to the distal one. This difference correlated well with the resulting levels of transcription mediated by Sp1 in contransfection experiments in BL and Sp1-deficient SL2 cells. Sp3 also bound to the core promoter in vitro, but failed to transactivate in vivo. Mutation of the distal Sp1 site moderately affected basal transcription concomitant with a modest decrease in enhancer stimulation. Mutation of the proximal Sp1 site almost entirely abolished basal as well as enhanced transcription. A considerable level of basal transcription was maintained upon mutation of the TATA box, whereas enhancer-activated transcription largely was abolished. Stable transfection of the BL cell line Raji with constructs containing core promoter mutations confirmed that the proximal Sp1 site and the TATA box are essential for the activation of promoter P1 by the Ig kappa enhancers.