Transcriptional activation of the minimal human Proalpha1(I) collagen promoter: obligatory requirement for Sp1.
A construct containing human Proalpha1(I) collagen gene promoter/enhancer-driven chloramphenicol acetyltransferase (CAT), pCOL-KT, failed to be expressed significantly in Sp1-deficient Schneider Drosophila line 2 (SL2) cells. However, CAT expression was induced 200-fold in SL2 cells co-transfected with pCOL-KT and pPACSp1, an Sp1-expression vector driven by the Drosophila actin 5C promoter. Elimination of the four potential Sp1-binding sites from pCOL-KT (pCOL-KTDeltaI), by removal of the first intron, did not abrogate Sp1-mediated induction of CAT. Even more significantly, a minimal Proalpha1(I) collagen promoter (-100 to +117 bp), containing a TATA box (-28 to -25 bp) and one putative Sp1-binding site (-87 to -82 bp), elicited strong Sp1-induced transactivation. Furthermore, mutation of the Sp1 motif in the minimal Proalpha1(I) collagen promoter-CAT construct abolished Sp1-induced expression of the reporter gene. Purified Sp1 protein bound specifically to DNA fragments of the Proalpha1(I) minimal promoter encompassing the putative Sp1-binding site; Sp1 binding could be competed out by a double-stranded oligonucleotide containing the wild-type Sp1 sequence, while an oligonucleotide containing a mutated Sp1 site failed to compete. Based on these results, we postulate that Sp1 plays an obligatory role in the transcriptional activation of the human Proalpha1(I) collagen gene. Additionally, we propose that a bona fide Sp1 motif, located most proximal to the TATA box, is necessary and sufficient for Sp1-mediated activation of the minimal Proalpha1(I) collagen promoter.
Poppleton
H M
HM
Department of Pharmacology, College of Medicine, The University of Tennessee, Memphis, TN 38104, USA.
Raghow
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D000818:Animals; D001665:Binding Sites; D004330:Drosophila; D006801:Humans; D007438:Introns; D016297:Mutagenesis, Site-Directed; D011347:Procollagen / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D011347:Procollagen; D016329:Sp1 Transcription Factor