In vivo formation of a human beta-globin locus control region core elementrequires binding sites for multiple factors including GATA-1, NF-E2, erythroid Kruppel-like factor, and Sp1

The active elements of the beta -globin locus control region (LCR) are loca ted within domains of unique chromatin structure. These nuclease hypersensi tive sites (HSs) are characterized by high DNase I sensitivity, erythroid s pecificity, similar nucleosomal structure, and evolutionarily conserved clu sters of cis-acting elements that are required for the formation and functi on of the core elements. To determine the requirements for HS core formatio n in the setting of nuclear chromatin, we constructed a series of artificia l HS cores containing binding sites for GATA-1, NF-E2, and Sp1, In contrast to the results of previous in vitro experiments, we found that when constr ucts were stably integrated in mouse erythroleukemia cells the binding site s for NF-E2, GATA-1, or Sp1 alone or in any combination were unable to form core HS structures, We subsequently identified two new cis-acting elements from the LCR HS4 core that, when combined with the NF-ES, Sp1, and tandem inverted GATA elements, result in core structure formation. Both new cis-ac ting elements bind Sp1, and one binds erythroid Kruppel-like factor (EKLF). We conclude that in vivo beta -globin LCR HS core formation is more comple x than previously thought and that several factors are required for this pr ocess to occur.