An E-Box-Mediated Increase in cad Transcription at the G₁/S-Phase Boundary Is Suppressed by Inhibitory c-Myc Mutants

Abstract

To better understand the signaling pathways which lead to DNA synthesis in mammalian cells, we have studied the transcriptional activation of genes needed during the S phase of the cell cycle. Transcription of the gene encoding a pyrimidine biosynthetic enzyme, carbamoyl-phosphate synthase (glutamine-hydrolyzing)/ aspartate carbamoyltransferase/dihydroorotase (cad), increases at the G₁/S-phase boundary. We have mapped the growth-dependent response element in the hamster cad gene to the extended palindromic E-box sequence, CCACGTGG, which is centered at 165 in the 5′ untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding to the cad sequence at 155 to 175 (155/175) restored growth-dependent regulation to nonresponsive cad promoter mutants when placed downstream of the transcription start site. The same oligonucleotide conferred less G₁/S-phase induction when placed upstream of basal promoter elements. An analogous oligonucleotide containing the mutant E box had no effect in either location. Nuclear proteins bound the cad 155/175 element 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 function of c-Myc and Max significantly inhibited cad transcription during S phase but had no effect on transcription from another G₁/S-phase-activated promoter, dhfr. These data support a model whereby E-box-binding proteins activate serum-induced transcription from the cad promoter at the G₁/S-phase boundary and suggest that a Max-associated protein complex contributes to the serum response.