AN E-BOX-MEDIATED INCREASE IN CAD TRANSCRIPTION AT THE G(1) S-PHASE BOUNDARY IS SUPPRESSED BY INHIBITORY C-MYC MUTANTS/

To better understand the signaling pathways which lead to DNA synthesi s in mammalian cells, we have studied the transcriptional activation o f genes needed during the S phase of the cell cycle. Transcription of the gene encoding a pyrimidine biosynthetic enzyme, carbamoyl-phosphat e synthase (glutamine-hydrolyzing)/ aspartate carbamoyltransferase/dih ydroorotase (cad), increases at the G(1)/S-phase boundary. We have map ped the growth-dependent response element in the hamster cad gene to t he extended palindromic E-box sequence, CCACGTGG, which is centered at +65 in the 5' untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding t o the cad sequence at +55 to +75 (+55/+75) restored growth-dependent r egulation to nonresponsive cad promoter mutants when placed downstream of the transcription start site. The same oligonucleotide conferred l ess G(1)/S-phase induction when placed upstream of basal promoter elem ents. An analogous oligonucleotide containing the mutant E box had no effect in either location. Nuclear proteins bound the cad +55/+75 elem ent in a cell cycle-dependent manner in electromobility shift assays; antibodies specific to USF and Max blocked the DNA-binding activity of different growth-regulated protein-DNA complexes. Expression of c-Myc mutants which have been shown to dominantly interfere with the functi on of c-Myc and Max significantly inhibited cad transcription during S phase but had no effect on transcription from another G(1)/S-phase-ac tivated promoter, dhfr. These data support a model whereby E-box-bindi ng proteins activate serum-induced transcription from the cad promoter at the G(1)/S-phase boundary and suggest that a Max-associated protei n complex contributes to the serum response.