An E-box-mediated increase in cad transcription at the G1/S-phase boundary is suppressed by inhibitory c-Myc mutants.
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 down-stream 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.
Miltenberger
R J
RJ
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School 53706, USA.
Sukow
K A
KA
Farnham
P J
PJ
D016475:3T3 Cells; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004080:Dihydroorotase / Q000235:genetics*; D016193:G1 Phase / Q000235:genetics; D016259:Genes, myc; D008647:Mesocricetus; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D009154:Mutation; D011401:Promoter Regions, Genetic; D016196:S Phase / Q000235:genetics; D013489:Suppression, Genetic; D014158:Transcription, Genetic
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D004247:DNA; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)