Three zinc finger nuclear proteins, Sp1, Sp3, and a ZBP-89 homologue, bindto the cyclic adenosine monophosphate-responsive sequence of the bovine adrenodoxin gene and regulate transcription

Adrenocorticotropin acting through cyclic adenosine monophosphate (cAMP) re gulates transcription of the bovine adrenodoxin (Adx) gene in the adrenal c ortex. The bovine Adx cAMP-responsive transcription sequence (CRS) has prev iously been found to contain two consensus GC boxes. By use of nuclear extr acts from adrenocortical cells, Spl and Sp3 are shown here to bind to CRS. Mutations designed to enhance the identification of additional CRS binding proteins by reducing Sp protein binding showed the presence of an additiona l DNA-binding protein (Adx factor). Adx factor binding is inhibited by the zinc-chelating agent, 1,10-o-phenanthroline, suggesting it might be a zinc finger protein. By a fractionation/ renaturation technique the Adx factor i n mouse Y1 adrenocortical cells was found to be in the size range of 106-11 5 kDa by gel mobility shift assay. On the basis of size, the CRS sequence t o which it binds, and its tentative identification as a zinc finger protein , Adx factor has been identified as a Kruppel-like zinc finger protein (a m ouse ZBP-89 homologue). Further mutagenesis of CRS demonstrates that it can further be divided into two similar cAMP-responsive elements, and eliminat ion of ZBP-89 binding does not affect cAMP responsiveness of either. Expres sion of these three nuclear proteins in Drosophila SL2 cells has been used to decipher the role of Adx CRS binding proteins in regulating transcriptio n. Spl and Sp3 confer basal transcriptional activities, yet only Spl confer s cAMP-responsive activity. ZBP-89 represses basal transcriptional activity .