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 journalArticlepeer-review

148 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

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

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