title: Functional elements of the steroid hormone-responsive promoter of mouse mammary tumor virus.
abstract: Transcription from the promoter of mouse mammary tumor virus is subject to induction by several classes of steroid hormones as well as to repression by a negative regulatory element present in the long terminal repeats of proviral DNA. In order to characterize the functional elements of the promoter that in some way must respond to these regulatory signals, a number of promoter mutations were constructed, including a set of linker-scanning mutations across the entire promoter region. Analysis of these mutated promoters with a transient-transfection assay defined at least three mutation-sensitive promoter elements that are required for both basal and hormone-induced transcription. One mutation-sensitive region contains a TATA element located at approximately position -30 with respect to the start of transcription. A second mutation-sensitive region contains two 10-base-pair direct repeats located between positions -60 and -38, within which are embedded three copies of octamer-related sequences; complete disruption of this region of the promoter leads to a more severe decrease in transcription than do any of the linker-scanning mutations, suggesting that the repeated sequences may be at least partially functionally redundant. Gel electrophoresis mobility shift assays were used to demonstrate specific binding of a nuclear protein to this region of the promoter. A third mutation-sensitive region contains a binding site for nuclear factor 1 (NF-1) located between positions -77 and -63. Site-directed mutations in the NF-1-binding site which increase the apparent affinity of NF-1 for the promoter in vitro do not decrease the hormone dependence of transcription, suggesting that transcriptional activation mediated by steroid hormone-receptor complexes cannot be explained by facilitation or stabilization of the interaction of promoter sequences with NF-1 and consistent with the idea that binding of NF-1 is not rate determining in transcription from the mouse mammary tumor virus promoter. None of the promoter mutations functionally separates basal from glucocorticoid-induced transcription, suggesting that hormone induction does not make the promoter independent of any of the DNA-binding factors required for its basal activity.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
authors.forename: M G
authors.initials: MG
authors.lastname: Toohey
authors.forename: J W
authors.initials: JW
authors.lastname: Lee
authors.forename: M
authors.initials: M
authors.lastname: Huang
authors.forename: D O
authors.initials: DO
authors.lastname: Peterson
mesh_terms: D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004278:DNA, Superhelical / Q000235:genetics / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006728:Hormones / Q000494:pharmacology; D007739:L Cells / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011533:Proviruses / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D013256:Steroids / Q000494:pharmacology; D014157:Transcription Factors; D014158:Transcription, Genetic; D051840:Y-Box-Binding Protein 1
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004278:DNA, Superhelical; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D006728:Hormones; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D013256:Steroids; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D015500:Chloramphenicol O-Acetyltransferase