TRANSCRIPTIONAL ACTIVATION OF THE MINIMAL HUMAN PRO-ALPHA-1(I) COLLAGEN PROMOTER - OBLIGATORY REQUIREMENT FOR SP1

A construct containing human Pro alpha 1(I) collagen gene promoter/enh ancer-driven chloramphenicol acetyltransferase (CAT), pCOL-KT, failed to be expressed significantly in Sp1-deficient Schneider Drosophila li ne 2 (SL2) cells. However, CAT expression was induced 200-fold in SL2 cells co-transfected with pCOL-KT and pPACSp1, an Sp1-expression vecto r driven by the Drosophila actin 5C promoter. Elimination of the four potential Sp1-binding sites from pCOL-KT (pCOL-KT Delta I), by removal of the first intron, did not abrogate Sp1-mediated induction of CAT. Even more significantly, a minimal Pro alpha 1(I) collagen promoter(-1 00 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 transac tivation. Furthermore, mutation of the Sp1 motif in the minimal Pro al pha 1(I) collagen promoter-CAT construct abolished Sp1-induced express ion of the reporter gene. Purified Sp1 protein bound specifically to D NA fragments of the Pro alpha 1(I) minimal promoter encompassing the p utative Sp1-binding site; Sp1 binding could be competed out by a doubl e-stranded oligonucleotide containing the wild-type Sp1 sequence, whil e 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 Pro alpha 1(I) collage n gene. Additionally, we propose that a bona fide Sp1 motif, located m ost proximal to the TATA box, is necessary and sufficient for Sp1-medi ated activation of the minimal Pro alpha 1(I) collagen promoter.