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 HS 2NF5 overlaps an E box, which is a preferred recognition site in vitro for the erythroid-specific transcription factor TAL1 (SCL). No eviden ce for TAL1 (SCL) binding was found using nuclear extracts from K562 a nd MEL erythroleukemia cells. Mutations that prevent HS2NF5 binding re duce the enhancer activity of HS2 by 40 and 38% in transient and stabl e transfection assays, respectively. Analytical gel filtration and vel ocity centrifugation studies revealed a Stokes' radius of 23.0 Angstro m and an s(20,w) of 3.45 for HS2NF5. Based on these parameters, a nati ve molecular mass of 34,679 Da was calculated. An ultraviolet light cr oss-linking assay was used to cross-link HS2NF5 to a minimal oligonucl eotide. 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 t hat modulates the transcriptional activation property of the beta-glob in locus control region.