A serum response element and a binding site for NF-Y mediate the serum response of the human thrombospondin 1 gene.
The expression of thrombospondin 1 (TSP 1), a member of the TSP gene family, is rapidly induced by growth factors. We tested the ability of human TSP 1-chloramphenicol acetyltransferase constructs to respond to serum in stably transfected NIH-3T3 cells. Two transcriptional elements in the TSP 1 promoter, a distal element at -1280 and a proximal element at -65, were required for the response of the human TSP 1 gene to serum. The distal element contains the 5'-CC(A + T)6GG-3' consensus sequence characteristic of a serum-response element (SRE). Deletions or mutations in this element reduced the serum response of the TSP 1 gene by 80-90%. In gel-shift assays, the -1280 element and the c-fos SRE cross-competed, whereas their functional and binding mutants did not. The proximal element contains the sequence 5'-GGCCAATGGG-3', which closely resembles the consensus binding motif for the CCAAT-binding factor NF-Y (CBF, CP1, alpha CP1). Deletions or mutations in this element also reduced the serum response by 80-90%. Methylation interference analysis of the -65 region identified a pattern of contacts with nuclear factors resembling that for NF-Y, and an NF-Y-binding site and the proximal TSP 1 element cross-competed in gel-shift assays, whereas their binding mutants did not. Finally, an abbreviated TSP 1 promoter/5'-flank, containing the SRE- and NF-Y-binding sites, mediated a serum response that was close in magnitude to that of the parent promoter. We conclude that the serum response of the human TSP 1 gene requires the coordinated function of an SRE- and NF-Y-binding site.
Framson
P
P
Department of Biochemistry, University of Washington, Seattle 98195.
Bornstein
P
P
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001769:Blood; D001792:Blood Platelets / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009841:Oligonucleotides; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D026362:Serum Response Factor; D019699:Thrombospondins; D014157:Transcription Factors / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D010980:Platelet Membrane Glycoproteins; D026362:Serum Response Factor; D019699:Thrombospondins; D014157:Transcription Factors