THE REGULATION OF GM-CSF IS DEPENDENT ON A COMPLEX INTERPLAY OF MULTIPLE NUCLEAR PROTEINS

GM-CSF is an important mediator of hematopoiesis and its dysregulation may play a role in neoplastic and inflammatory conditions. Previous s tudies have demonstrated that GM-CSF production depends upon the accum ulation of specific mRNA, which occurs by transcriptional and post-tra nscriptional mechanisms. In order to dissect the cis-acting sequences responsible for its regulation, we performed an extensive mutagenesis study spanning 54 nucleotides 5' of the GM-CSF coding region. Our anal ysis suggests that of the previously-described functional elements of the GM-CSF promoter, kappa B and a repetitive CATTT/A motif, the forme r co-exists with an overlapping 9 nucleotide site which silences promo ter activity, and the CATTT/A complex binds multiple polypeptides whic h differentially contribute to basal and inducible promoter activity. These two sites interact to provide tissue-appropriate and stimulus-sp ecific promoter function. Using DNA-protein cross-linking and co-trans fection studies, we demonstrate that the c-rel-related proteins p65 an d p50 bind to the GM-CSF promoter and that p65 binding is primarily re sponsible for the enhancing effects at this site. In addition, we show that the GM-CSF kappa B decanucleotide is inadequate to provide full binding affinity; mutation of nucleotides flanking this site affect pr omoter function by altering NF-kappa B binding affinity. Together thes e results suggest that the transcriptional response of GM-CSF is depen dent on a complex interplay of multiple DNA binding proteins. Copyrigh t (C) 1996 Elsevier Science Ltd.