Synergistic activation of a human promoter in vivo by transcription factor Sp1.
Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.
Anderson
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Molecular Biology Research Program, Henry Ford Hospital, Detroit, Michigan 48202.
Freytag
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D001124:Argininosuccinate Synthase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D016329:Sp1 Transcription Factor; D003850:Deoxyribonuclease I; D001124:Argininosuccinate Synthase