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 hypersensitive sites upstream of the epsilon-globin gene and is intimately involved 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 nuclear proteins revealed a single major protein that bound tightly to site II. An E-box was necessary for high affinity binding to DNA. We used antibodies and recombinant USF protein to prove that the helix-loop-helix transcription factor USF is the only detectable component in K562 cells that binds to this site. Despite significant differences between 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 USF DNA binding polypeptide displays an unusual positive temperature dependence, consistent with the observed footprinting behavior. The results show that a relatively ubiquitous factor, not confined to erythroid cells, is an important part of the complex of proteins bound at hypersensitive site 2 of the LCR in K562 cells.
Bresnick
E H
EH
Laboratory of Molecular Biology, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Felsenfeld
G
G
D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D005914:Globins / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D015342:DNA Probes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D005914:Globins; D004247:DNA; D003850:Deoxyribonuclease I