Interaction of Ap1, Ap2, and Sp1 with the Regulatory Regions of the Human Pro-α1(I) Collagen Gene

In the pro-α1(I) collagen gene a number of cis-regulatory elements, which interact with a variety of trans-acting factors, are present in the promoter and first intron. We have undertaken a comprehensive study of Sp1, Ap1, and Ap2 binding in the region spanning −442 to +1697 nt. DNase I footprinting analysis revealed these factors bind with varying affinities to some of the potential sites: Sp1 binds to 16 of 34 potential sites, Ap2 binds to 22 of 40 potential binding sites, and Ap1 binds to its only potential site. The Sp1 sites were mostly clustered in the intron region, while the Ap2 sites were clustered in the promoter region. Transmission electron microscopic analysis of DNA–protein complexes not only confirmed these results, but also clearly showed that heterologous and/or homologous protein–protein interactions between Sp1 and/or Ap2 bring the promoter and intron in contact with each other, with the resulting looping out of the intervening DNA. This strongly suggests that the DNA-looping model is an explanation for the orientation preference of the enhancing element in the first intron as these interactions possibly create an optimum environment for the binding of the rest of the transcriptional machinery.