EVIDENCE THAT THE TRANSCRIPTION FACTOR USF IS A COMPONENT OF THE HUMAN BETA-GLOBIN LOCUS-CONTROL REGION HETEROMERIC PROTEIN COMPLEX

The human locus control region (LCR) consists of four DNase I hypersen sitive sites upstream of the epsilon-globin gene and is intimately inv olved in globin gene transcription. We have used DNase I footprinting with K562 erythroleukemia cell extracts to identify protein components of the minimal LCR element, hypersensitive site 2. Six major regions of protection were observed, and the occupation of two regions (sites II and V) was strongly temperature-dependent. Fractionation of K562 nu clear proteins revealed a single major protein that bound tightly to s ite II. An E-box was necessary for high affinity binding to DNA. We us ed antibodies and recombinant USF protein to prove that the helix-loop -helix transcription factor USF is the only detectible component in K5 62 cells that binds to this site. Despite significant differences betw een site II and a canonical USF-binding site, the USF binding affinity was comparable for the two sites. In both cases the interaction with the E-box of either wild-type USF or a approximately 15-kDa minimal US F DNA binding polypeptide displays an unusual positive temperature dep endence, consistent with the observed footprinting behavior. The resul ts show that a relatively ubiquitous factor, not confined to erythroid cells, is an important part of the complex of proteins bound at hyper sensitive site 2 of the LCR in K562 cells.