NF-E2 DISRUPTS CHROMATIN STRUCTURE AT HUMAN BETA-GLOBIN LOCUS-CONTROLREGION HYPERSENSITIVE SITE 2 IN-VITRO

The human beta-globin locus control region (LCR) is responsible for fa rming an active chromatin structure extending over the 100-kb locus, a llowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 to -4) , DNase I hypersensitive sites are thought to represent nucleosome-fre e regions of DNA which are bound by a ans-acting factors, Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In t his study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro, NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and t he ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-lik e sites in HS2 which form tile core of its enhancer activity. In this study. we show that when bound to in vitro-reconstituted chromatin, NF -E2 forms a DNase I hypersensitive site at HS2 similar to the site obs erved in vivo. Moreover, NF-E2 binding in vitro results in a disruptio n of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the d isruption is more pronounced when NF-E2 is added to DNA Drier to chrom atin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but not to naked DNA. Inte raction of NF-E2 with ifs site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites, Lastly, nu cleosome disruption by NF-E2 is an ATP-dependent process, suggesting t he involvement of energy-dependent nucleosome remodeling factors.