An E-box-mediated increase in cad transcription at the G1/S-phase boundary is suppressed by inhibitory c-Myc mutants

R. J. Miltenberger, K. A. Sukow, P. J. Farnham

Research output: Contribution to journalArticle

145 Scopus citations

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 +65 in the 5' untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding to the cad sequence at +55 to +75 (+55/+75) 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 +55/+75 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.

Original languageEnglish (US)
Pages (from-to)2527-2535
Number of pages9
JournalMolecular and Cellular Biology
Volume15
Issue number5
StatePublished - 1995
Externally publishedYes

    Fingerprint

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this