IDENTIFICATION OF NOVEL GLUCOCORTICOID-RESPONSE ELEMENTS IN HUMAN ELASTIN PROMOTER AND DEMONSTRATION OF NUCLEOTIDE-SEQUENCE SPECIFICITY OF THE RECEPTOR-BINDING

Glucocorticoids exert their action on gene expression through activati on of cytoplasmic glucocorticoid receptors (GRs) that bind to glucocor ticoid response elements (GREs). The consensus GRE consists of two hal f sites (underlined), AGAACANNNTGTTCT. We have recently cloned the ent ire human elastin gene. Nucleotide sequencing of the promoter region d isclosed the presence of three putative GREs with the downstream half- site sequence TGTTCC that has homology with the consensus GRE, althoug h the upstream half site showed no homology. To examine the functional ity of these putative GREs in binding to the GRs, we performed gel mob ility shift and supershift assays with synthetic oligomers containing the putative GREs and a recombinant GR protein, expressed in a baculov irus system. All three GREs identified in the elastin promoter bound t he receptor. A chimeric oligonucleotide containing the upstream consen sus GRE half site and the downstream elastin promoter GRE half site wa s capable of binding the receptor, and this binding could be competed with the elastin promoter GRE. Nonconservative substitution of single nucleotides (positions 1-6) in the elastin GRE indicated that mutation s in the positions 1-3 and 6 had relatively little effect, but substit utions in positions 4 and 5 rendered the oligomer less effective in co mpeting for the binding. These observations suggest that the downstrea m half site of GREs in the human elastin promoter is sufficient for re ceptor binding and certain nucleotides are critical for the efficient binding. The results also imply that the three GREs within the human e lastin promoter are active and mediate the glucocorticoid-induced up-r egulation of human elastin promoter activity.