PURIFICATION OF EARLY-B-CELL FACTOR AND CHARACTERIZATION OF ITS DNA-BINDING SPECIFICITY

Early-B-cell factor (EBF) is a nuclear protein that recognizes a funct ionally important sequence in the promoter of the mb-1 gene. Like the mb-1 gene, which encodes an immunoglobulin-associated protein, EBF is specifically expressed in the early stages of B-lymphocyte differentia tion. We purified EBF by sequence-specific DNA affinity chromatography and examined its biochemical properties and DNA-binding specificity. Crude nuclear extract and affinity-purified EBF generated protein-DNA complexes with the mb-1 promoter that were indistinguishable in electr ophoretic mobility shift and DNase I footprint assays. Fractionation o f affinity-purified EBF by sodium dodecyl sulfate-polyacrylamide gel e lectrophoresis and renaturation of isolated polypeptides indicated tha t EBF DNA-binding activity could be reconstituted from polypeptides wi th molecular masses of 62 to 65 kDa. Gel filtration chromatography sug gested that native EBF has a molecular mass of 140 kDa, if a globular shape of the protein is assumed. Thus, EBF appears to be a dimer with subunits of 62 to 65 kDa. To characterize the DNA-binding specificity of purified EBF, we performed two sets of experiments. First, we exami ned various mutant EBF-binding sites for interaction with purified EBF in an electrophoretic mobility shift assay. Second, we used oligonucl eotides containing pairs of randomized bases in a binding-site selecti on and amplification experiments to determine a preferred sequence for DNA binding by EBF. Taken together, the results of these experiments indicated that EBF recognizes variations on the palindromic sequence 5 '-ATTCCCNNGGGAAT, with an optimal spacer of 2 bp between the half-site s.