Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement

The major distal regulatory sequence for the beta -globin gene locus, the l ocus control region (LCR), is composed of multiple hypersensitive sites (HS s). Different models for LCR function postulate that the HSs act either ind ependently or synergistically. To test these possibilities, we have constru cted a series of expression cassettes in which the gene encoding the enhanc ed green fluorescent protein (EGFP) is under the control of DNA fragments c ontaining single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (BS core s) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the BS cores contributed to e nhancement. Expression of these constructs was measured after targeted inte gration into three defined loci in murine erythroleukemia cells using recom binase-mediated cassette exchange. At all three marked loci, synergistic en hancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units, in contrast, HS2, HS3, and HS4 cores (without f lanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus Banking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LC R. The three targeted integration sites differ substantially in their permi ssivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three si tes.