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Adrenocorticotropin acting through cyclic adenosine monophosphate (cAMP) regulates transcription of the bovine adrenodoxin (Adx) gene in the adrenal cortex. The bovine Adx cAMP-responsive transcription sequence (CRS) has previously been found to contain two consensus GC boxes. By use of nuclear extracts from adrenocortical cells, Sp1 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 additional 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 in mouse Y1 adrenocortical cells was found to be in the size range of 106-115 kDa by gel mobility shift assay. On the basis of size, the CRS sequence to which it binds, and its tentative identification as a zinc finger protein, Adx factor has been identified as a Krüppel-like zinc finger protein (a mouse ZBP-89 homologue). Further mutagenesis of CRS demonstrates that it can further be divided into two similar cAMP-responsive elements, and elimination of ZBP-89 binding does not affect cAMP responsiveness of either. Expression of these three nuclear proteins in Drosophila SL2 cells has been used to decipher the role of Adx CRS binding proteins in regulating transcription. Sp1 and Sp3 confer basal transcriptional activities, yet only Sp1 confers cAMP-responsive activity. ZBP-89 represses basal transcriptional activity.