OF THE GATA-BINDING PROTEINS, ONLY GATA-4 SELECTIVELY REGULATES THE HUMAN INTERLEUKIN-5 GENE PROMOTER IN INTERLEUKIN-5-PRODUCING CELLS WHICH EXPRESS MULTIPLE GATA-BINDING PROTEINS

Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cell s. Using ATL-16T cells which express IL-5 mRNA, we have identified a r egion within the human IL-5 gene promoter that regulates IL-5 gene tra nscription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the c loning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively int eracts with the -70 GATA site within the IL-5 proximal promoter region . By promoter deletion and mutation analyses, we established this regi on as a positive regulatory element, Cotransfection experiments reveal ed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requiremen t for another regulatory element called CLEO, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and ea ch of them has a potential to bind to the consensus (A/T)GATA(G/A) mot h. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein co mplex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed tha t GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities we re compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcription al regulation of the IL-5 gene in a circumstance where multiple member s of the GATA-binding proteins are expressed.