Conserved elements containing NF-E2 and tandem GATA binding sites are required for erythroid-specific chromatin structure reorganization within the human beta-globin locus control region.

Proper expression of the genes of the human beta-globin gene locus requires the associated locus control region (LCR). Structurally, the LCR is defined by the presence of four domains of erythroid-specific chromatin structure. These domains, which have been characterized as DNase I hypersensitive sites (HSs), comprise the active elements of the LCR. The major focus of this research is to define the cis -acting elements which are required for the formation of these domains of unique chromatin structure. Our previous investigations on the formation of LCR HS4 demonstrated that NF-E2 and tandem, inverted GATA binding sites are required for the formation of the native HS. Similarly arranged NF-E2 and tandem GATA sites are present within the core regions of the other human LCR HSs and are evolutionarily conserved. Using site-directed mutagenesis of human HSs 2 and 3 we have tested the hypothesis that these NF-E2 and GATA sites are common requirements for the formation of all LCR HSs. We find that mutation of these elements, and particularly the GATA elements, results in a decrease or complete loss of DNase I hypersensitivity. These data imply the presence of common structural elements within the core of each LCR HS which are required for erythroid-specific chromatin structure reorganization.     

In vivo formation of a human beta-globin locus control region core element requires 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 located within domains of unique chromatin structure. These nuclease hypersensitive sites (HSs) are characterized by high DNase I sensitivity, erythroid specificity, similar nucleosomal structure, and evolutionarily conserved clusters of cis-acting elements that are required for the formation and function of the core elements. To determine the requirements for HS core formation in the setting of nuclear chromatin, we constructed a series of artificial 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 constructs were stably integrated in mouse erythroleukemia cells the binding sites 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-E2, Sp1, and tandem inverted GATA elements, result in core structure formation. Both new cis-acting elements bind Sp1, and one binds erythroid Kruppel-like factor (EKLF). We conclude that in vivo beta-globin LCR HS core formation is more complex than previously thought and that several factors are required for this process to occur.