ASSOCIATION OF USF AND C-MYC WITH A HELIX-LOOP-HELIX-CONSENSUS MOTIF IN THE CORE PROMOTER OF THE MURINE TYPE II-BETA REGULATORY SUBUNIT GENE OF CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE

Previous studies showed that the core promoter of the mouse cAMP-depen dent protein kinase regulatory subunit type II beta (RII beta) gene wa s composed of two functional elements. One element was GC rich and bou nd the Sp1 transcription factor. The second element contained a helix- loop-helix (HLH)-motif. Each element conferred transcriptional activit y when inserted upstream of a reporter gene, chloramphenicol acetyltra nsferase and transfected into mouse NB2a neuroblastoma cells and Chine se hamster ovary (CHO) cells. The core promoter was further characteri zed by mutational analysis using electrophoretic mobility shift assays and by transfection into CHO and NB2a cells. Electrophoretic mobility shift assays showed that the HLH-consensus motif, CACGTG, present in the RII beta gene bound nuclear factors present in NB2a and CHO cells. Mutations in the HLH-core motif decreased the binding of these factor s and reduced the transcriptional activity of constructs containing th e chloramphenicol acetyltransferase reporter when transfected into the se cells. The results showed that the central nucleotides as well as t he adjacent bases were important for the interaction with the nuclear binding factors. UV cross-linking, Southwestern blot analysis, and int erference of the mobility shift patterns by specific antisera directed against USF and c-Myc indicated that both of these transcription fact ors were forming complexes with the HLH-consensus motif. The results s uggest that RII beta transcription may be regulated, in part, by USF a nd c-Myc in NB2a CHO cells.