The regulation of GM-CSF is dependent on a complex interplay of multiple nuclear proteins

Abstract

GM-CSF is an important mediator of hematopoiesis and its dysregulation may play a role in neoplastic and inflammatory conditions. Previous studies have demonstrated that GM-CSF production depends upon the accumulation of specific mRNA, which occurs by transcriptional and post-transcriptional 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 analysis suggests that of the previously-described functional elements of the GM-CSF promoter, κB and a repetitive CATTT A motif, the former co-exists with an overlapping 9 nucleotide site which silences promoter activity, and the CATTT A complex binds multiple polypeptides which differentially contribute to basal and inducible promoter activity. These two sites interact to provide tissue-appropriate and stimulus-specific promoter function. Using DNA-protein cross-linking and co-transfection studies, we demonstrate that the c-rel-related proteins p65 and p50 bind to the GM-CSF promoter and that p65 binding is primarily responsible for the enhancing effects at this site. In addition, we show that the GM-CSF κB decanucleotide is inadequate to provide full binding affinity; mutation of nucleotides flanking this site affect promoter function by altering NF-κB binding affinity. Together these results suggest that the transcriptional response of GM-CSF is dependent on a complex interplay of multiple DNA binding proteins.