A novel DNA-binding protein, HS2NF5, interacts with a functionally important sequence of the human beta-globin locus control region.
We have identified a previously unreported DNA-binding protein, HS2NF5, which interacts with a conserved sequence within hypersensitive site II (HS2) of the human beta-globin locus control region. A minimal DNA recognition sequence of TGTTCTCA was defined. The binding site for HS2NF5 overlaps an E box, which is a preferred recognition site in vitro for the erythroid-specific transcription factor TAL1 (SCL). No evidence for TAL1 (SCL) binding was found using nuclear extracts from K562 and MEL erythroleukemia cells. Mutations that prevent HS2NF5 binding reduce the enhancer activity of HS2 by 40 and 38% in transient and stable transfection assays, respectively. Analytical gel filtration and velocity centrifugation studies revealed a Stokes' radius of 23.0 A and an s20,w of 3.45 for HS2NF5. Based on these parameters, a native molecular mass of 34,679 Da was calculated. An ultraviolet light cross-linking assay was used to cross-link HS2NF5 to a minimal oligonucleotide. The cross-linking results are consistent with a protein of 33,396-38,309 Da. We propose that HS2NF5 is a novel DNA-binding protein that modulates the transcriptional activation property of the beta-globin locus control region.
Lam
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Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA. ehbresni@facstaff.wisc.edu
Bresnick
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EH
D001483:Base Sequence; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; C102840:HS2NF5 protein, human; D015534:Trans-Activators; D005914:Globins