ENHANCER ACTIVITY OF UPSTREAM HYPERSENSITIVE SITE-2 OF THE CHICKEN BETA-GLOBIN CLUSTER IS MEDIATED BY GATA SITES

Upstream of the chicken beta-globin gene cluster are four DNase I-hype rsensitive sites (HS1-4), Hypersensitive sites located upstream of the mammalian beta-globin clusters have enhancer activity and mediate pos ition-independent gene expression, In contrast, a region inside the ch icken cluster has enhancer activity and mediates position-independent expression, Here we investigate the function of the chicken upstream s ites, which are different from the mammalian ones in sequence, number, and distance from the genes, Each was tested for its effect on report er gene expression in transfected primary erythroid cells, HS2 and HS3 (4.4 and 6.4 kilobases upstream of rho-globin) showed significant enh ancer activity while HS1 and HS4 (1.6 and 11 kilobases upstream of rho -globin) did not, A 237-base pair region of HS2 contained the sequence s necessary for enhancer activity. Proteins from erythroid extracts bo und HS2 in seven different regions; six of these sites were characteri zed, GATA-1 bound to four of the sites, Each site contributed to the e nhancer activity of HS2, Two Other sequences bound proteins that may b e related to Spl and erythroid krup-pel-like factor. Surprisingly, mut ations in these elements, which disrupted protein binding, did not aff ect enhancer activity, Thus, the observed enhancer activity of HS2 is due to the four GATA sites, The existence of multiple GATA sites in bo th chicken HS2 and the mammalian upstream sites may be due to evolutio n from a common element with preservation of only very short sequences or to convergent evolution. These observations highlight the crucial role for GATA proteins in globin regulation.