Purification of early-B-cell factor and characterization of its DNA-binding specificity.

Abstract

Early-B-cell factor (EBF) is a nuclear protein that recognizes a functionally important sequence in the promoter of the mb-i gene. Like the mb-i gene, which encodes an immunoglobulin-associated protein, EBF is specifically expressed in the early stages of B-lymphocyte differentiation. 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-i promoter that were indistinguishable in electrophoretic mobility shift and DNase I footprint assays. Fractionation of affinitypurified EBF by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation of isolated polypeptides indicated that EBF DNA-binding activity could be reconstituted from polypeptides with molecular masses of 62 to 65 kDa. Gel filtration chromatography suggested 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 examined various mutant EBF-binding sites for interaction with purified EBF in an electrophoretic mobility shift assay. Second, we used oligonucleotides containing pairs of randomized bases in a binding-site selection 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-sites.

Early B-cell Factor-1 (EBF1) Is a Key Regulator of Metabolic and Inflammatory Signaling Pathways in Mature Adipocytes

“…These "surviving" genes would essentially score as false negatives in the GSEA. 3) we cannot rule out the possibility of EBF2 and/or EBF3 compensating for EBF1 at some sites in EBF1 knockdown cells, because the consensus sites for all three proteins are similar, and all EBFs are predicted to be capable of binding to the same consensus sequence (46). In support of this, the expression of Ebf2 in the knockdown cells increases slightly (Fig.…”

A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

“…First, phylogenetic footprinting analysis of the ciselement by multiple species alignment of synthenic regions (for review, see Wasserman and Sandelin 2004) will often reveal highly conserved nucleotides, which can be mutated in the control. Second, the use of the complete reverse sequence of the cis-element or part of it can be a suitable control (Travis et al 1993), because it does not change the overall nucleotide composition of the double helix and conserves the GC/AT content of each DNA strand. The fact that the vast majority of binding partners identified in our pilot screens either reproduce and confirm published data or can be placed in a functional biological context argues for a low false-positive detection rate of the technique, especially when the robust statistics of the MaxQuant software are applied (Graumann et al 2007(Graumann et al , 2008.…”

The Human V-PreB Promoter Is a Target for Coordinated Activation by Early B Cell Factor and E47

“…The mouse promoter contains one high affinity binding site for EBF important for full functional activity of the promoter (30). Investigation of the nucleotide sequence of the human promoter revealed several potential EBF binding sites with varying degrees of similarity to the defined EBF consensus site ATTCCCNNGGGAAT (31) (Fig. 1A).…”