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 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 the previously-described functional elements of the GM-CSF promoter, kappa B 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 kappa B decanucleotide is inadequate to provide full binding affinity; mutation of nucleotides flanking this site affect promoter function by altering NF-kappa B binding affinity. Together these results suggest that the transcriptional response of GM-CSF is dependent on a complex interplay of multiple DNA binding proteins.
Kaushansky
K
K
Division of Hematology, University of Washington, Seattle 98195, USA.
O'Rork
C
C
Shoemaker
S G
SG
McCarty
J
J
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D016178:Granulocyte-Macrophage Colony-Stimulating Factor