Myc versus USF: discrimination at the cad gene is determined by core promoter elements.
Carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase, which is encoded by the cad gene, is required for the first three rate-limiting steps of de novo pyrimidine biosynthesis. It has been previously demonstrated that cad transcription increases at the G1/S-phase boundary, as quiescent cells reenter the proliferative cell cycle. The growth-responsive element has been mapped to an E box at +65 in the hamster cad promoter. Using an in vivo UV cross-linking and immunoprecipitation assay, we show that Myc, Max, and upstream stimulatory factor (USF) bind to the chromosomal cad promoter. To determine whether binding of Myc-Max or USF is critical for cad growth regulation, we analyzed promoter constructs which contain mutations in the nucleotides flanking the E box. We demonstrate that altering nucleotides which flank the cad E box to sequences which decrease Myc-Max binding in vitro correlates with a loss of cad G1/S-phase transcriptional activation. This result supports the conclusion that binding of Myc-Max, but not USF, is essential for cad regulation. Our investigations demonstrate that the endogenous cad E box can be bound by more than one transcription factor, but growth-induced cad expression is achieved only by Myc.
Boyd
K E
KE
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA.
Farnham
P J
PJ
D016475:3T3 Cells; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D016384:Consensus Sequence; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D004080:Dihydroorotase / Q000235:genetics*; D016193:G1 Phase; D016259:Genes, myc / Q000235:genetics*; D018257:Helix-Loop-Helix Motifs; D051379:Mice; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016196:S Phase; D014157:Transcription Factors / Q000235:genetics*; D014466:Ultraviolet Rays; D051720:Upstream Stimulatory Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; C033626:CAD trifunctional enzyme; D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; C067850:Myc associated factor X; D009363:Neoplasm Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C068422:Max protein, mouse; D004247:DNA; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)