Sp1-mediated transcriptional activation from the dominant promoter of the rat alpha1B adrenergic receptor gene in DDT1MF-2 cells.
In the rat liver, NF1 and CP1 bind to the major P2 promoter of the alpha1B adrenergic receptor gene to generate footprint II. Here we show that, in DDT1MF-2 smooth muscle cells, the major protein bound to footprint II is not NF1 but Sp1, which binds to the 5'-portion of the footprint II sequence (footprint IIb). Mutational analyses demonstrate that the CCCGCG sequence in footprint IIb is critical for Sp1 binding and P2 promoter activity. A second GC box in the P2 promoter also binds the Sp1 protein and contributes to the P2 promoter activity. Gel shift assays indicate that footprint II can bind Sp1, NF1, and CP1, and that the binding of these 3 proteins is mutually exclusive. This is also indicated by the results of functional cotransfection experiments, where transient overexpression of NF1 and Sp1 together caused a similar increase in the activity of a P2/CAT reporter construct as overexpression of either Sp1 or NF1 alone, indicating lack of additivity. The preferential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF1 in liver appears to be due to low levels of NF1 expression in DDT1MF-2 cells and low levels of Sp1 in liver. These observations suggest that NF1 and Sp1 are the major transcription factors involved in controlling the P2 promoter in liver versus DDT1MF-2 cells, respectively, which may be one of the mechanisms responsible for the complex tissue-specific regulation of the expression of the alpha1B adrenergic receptor gene.
Chen
J
J
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Spector
M S
MS
Kunos
G
G
Gao
B
B
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D008099:Liver / Q000502:physiology; D008957:Models, Genetic; D009130:Muscle, Smooth / Q000502:physiology*; D009154:Mutation; D051839:NFI Transcription Factors; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000096:biosynthesis / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014018:Tissue Distribution; D014157:Transcription Factors / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D018340:Receptors, Adrenergic, alpha-1; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C080333:transcription factor nuclear factor 1