Lymphoid-specific cDNA clones were isolated that encode a nuclear protein with homology to the chromosomal nonhistone protein HMG-1 and to putative regulators of cell specialization, including the mammalian testis-determining factor SRY and fungal mating-type proteins. The gene represented by the isolated cDNA clones, termed LEF-I {lymphoid enhancer-binding _factor 1), is developmentally regulated and expressed in pre-B and T lymphocytes but not in later-stage B cells or nonlymphoid tissues. Both endogenous and recombinant LEF-1 were shown to bind to a functionally important site in the T-cell antigen receptor (TCR) a enhancer. Maximal TCRa enhancer activity was found to parallel the cell type-specific expression pattern of LEF-1. Moreover, forced expression of recombinant LEF-1 in late stage B cells increases TCRot enhancer function. Taken together, these data suggest that LEF-1 is a regulatory participant in lymphocyte gene expression and differentiation.

Early B-cell factor (EBF) was identified previously as a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific rob-1 gene. Partial amino acid sequences obtained from purified EBF were used to isolate eDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide formed sequence-specific complexes with the EBF-binding site in the rob-1 promoter. The eDNA hybridized to multiple transcripts in pre-B and B-cell lines, but transcripts were not detected at significant levels in plasmaeytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfeeted nonlymphoid cells strongly activated transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with eotranslated wild-type and truncated forms of EBF indicated that the protein interacts with its site as a homodimer. Deletions delineated a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression.

The mb‐1 gene, which encodes a protein associated with membrane‐bound antibody, is expressed only at the early stages of B cell differentiation. To gain insight into the mechanisms that underlie temporally regulated gene expression, we examined the mb‐1 promoter region for interactions with cell type‐specific DNA binding proteins. Here, we report the characterization of a novel nuclear factor that recognizes the mb‐1 promoter. This DNA binding activity, termed Early B cell Factor, or EBF, is expressed in early stage B cells, but not in late stage B cells, T cells or non‐lymphoid cells. EBF recognizes the nucleotide sequence 5′‐CAAGGGAAT‐3′ in the mb‐1 and major histocompatibility complex (MHC) class II A alpha d promoters. The binding of EBF to DNA was characterized by DNase I footprinting and by methylation interference analysis which indicated both major and minor groove contacts. The specificity of EBF binding is distinct from that of other nuclear factors expressed in hematopoietic cells. EBF appears to consist of at least two polypeptides of approximately 70–75 kDa and 80–85 kDa. The EBF binding site was important for maximal mb‐1 promoter activity in early stage B cells. Moreover, the EBF binding site conferred correct lineage‐ and stage‐specific transcriptional activity upon a heterologous promoter in a context‐dependent manner. Thus, EBF appears to represent an important transcriptional regulator of B cell specific gene expression.

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.