CHARACTERIZATION OF HUMAN INVOLUCRIN PROMOTER DISTAL REGULATORY REGION TRANSCRIPTIONAL ACTIVATOR ELEMENTS - A ROLE FOR SP1 AND AP1 BINDING-SITES

Human involucrin (hINV) is an important precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal la yers of stratifying epithelia. Previous truncation and mutagenesis exp eriments have shown that an activator protein 1 (Ap1) site, AP1-5, loc ated 2100 bp upstream of the transcription start site, is required for optimal promoter activity. These previous studies suggest that AP1-5 is part of a distal regulatory region spanning nucleotides -2473 to -2 088. In the present report, we study the distal regulatory region (DRR ), which surrounds AP1-5. Our studies show that this region contains w eak and strong activator elements spanning nucleotides -2473/-2216 and -2140/-2088, respectively. The strong activator element contains AP1- 5 and an adjacent specificity protein 1 (Spl) site. The AP1-5 site is absolutely required for DRR activity, as its mutation reduces transcri ption to basal levels. Mutagenesis studies of the AP1-5 and Sp1 sites in the presence or absence of the weak activator element indicate that the Spl site and the weak activator element synergistically activate the AP1-5 site-dependent transcription, The cooperation between the Sp l and AP1-5 sites is also observed in the context of the full-length p romoter. Gel mobility shift and supershift studies show that Spl, but not Sp2, Sp3 or Sp4 binds to the Sp1 site. When the Sp1 site is mutate d or the distance between the AP1-5 and Sp1 site is increased, the bin ding of AP1 factors to AP1-5 is markedly reduced. Surprisingly, gel sh ift studies suggest that activation does not require the formation of a stable AP1/Sp1/DNA ternary complex. These studies suggest that the A P1-5 site is absolutely required for transcriptional activation, that the weak activator element and Spl sites serve to enhance this activat ion, and that the Spl site is required for optimal AP1 factor binding at the AP1-5 site.