MYC VERSUS USF - DISCRIMINATION AT THE CAD GENE IS DETERMINED BY COREPROMOTER ELEMENTS

Carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydrooro tase, which is encoded by the can gene, is required for the first thre e 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 cyc le. The growth-responsive element has been mapped to an E box at +65 i n the hamster cad promoter, Using an in vivo UV cross-linking and immu noprecipitation assay, we show that MSc, Max, and upstream stimulatory factor (USF) bind to the chromosomal cad promoter. To determine wheth er binding of Myc-Max or USF is critical for can growth regulation, we analyzed promoter constructs which contain mutations in the nucleotid es flanking the E box, We demonstrate that altering nucleotides which flank the cad E box to sequences which decrease Myc-Max binding in vit ro correlates with a loss of cad G(1)/S-phase transcriptional activati on. This result supports the conclusion that binding of Myc-Max, but n ot USF, is essential for cad regulation. Our investigations demonstrat e that the endogenous cad E box can be bound by more than one transcri ption factor, but growth-induced cad expression is achieved only by My c.