Myc versus USF: Discrimination at the cad gene is determined by core promoter elements

Carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase, which is encoded by the can gene, is required for the first three rate-limiting steps of de novo pyrimidine biosynthesis. It has been previously demonstrated that cad transcription increases at the G(1)/S-phase boundary, as quiescent cells reenter the proliferative cell cycle. The growth-responsive element has been mapped to an E box at +65 in the hamster cad promoter, Using an in vivo UV cross-linking and immunoprecipitation assay, we show that MSc, Max, and upstream stimulatory factor (USF) bind to the chromosomal cad promoter. To determine whether binding of Myc-Max or USF is critical for can growth regulation, we analyzed promoter constructs which contain mutations in the nucleotides flanking the E box, We demonstrate that altering nucleotides which flank the cad E box to sequences which decrease Myc-Max binding in vitro correlates with a loss of cad G(1)/S-phase transcriptional activation. This result supports the conclusion that binding of Myc-Max, but not USF, is essential for cad regulation. Our investigations demonstrate that the endogenous cad E box can be bound by more than one transcription factor, but growth-induced cad expression is achieved only by Myc.