The 'zinc knuckle' motif of Early B cell Factor is required for transcriptional activation of B cell-specific genes.

Early B cell factor (EBF) is a critical regulator of B lymphocyte-specific gene transcription. EBF functions, in part, by binding to regulatory sites of genes required for the pre-B- and mature B cell receptors. These DNA targets include the promoters of the mb-1 and Vpreb1 genes that encode Ig-alpha and one of the components of surrogate light chain, respectively. The biochemical basis of DNA binding and gene activation by EBF is poorly understood. The DNA-binding domain (DBD) of EBF includes a putative zinc-binding motif (HX(3)CX(2)CX(5)C), which we have designated the 'Zn-knuckle'. The Zn-knuckle is required for binding of the mb-1 promoter site in EMSA, but it has not been demonstrated to be important for functional activities of EBF in B cells. Therefore, we expressed EBF with mutations in the Zn-knuckle motif or flanking sequences in plasmacytoma cells in which activation of endogenous mb-1 and Vpreb1 genes is dependent on EBF. EBF with mutations that prevent zinc coordination by the Zn-knuckle did not activate transcription of either target gene. Other mutations affected the sequence preference of DNA binding and differentially inhibited activation of these genes. Our results demonstrate the importance of the Zn-knuckle motif in EBF. These experiments also confirm that EBF can re-activate multiple genes of the early B cell program in plasmacytoma cells, which provide a useful cell-based assay for dissecting mechanisms involving EBF.

Requirements for selective recruitment of Ets proteins and activation of mb-1/Ig-alpha gene transcription by Pax-5 (BSAP).

Pax-5, a member of the paired domain family of transcription factors, is a key regulator of B lymphocyte-specific transcription and differentiation. A major target of Pax-5-mediated activation is the mb-1 gene, which encodes the essential transmembrane signaling protein Ig-alpha. Pax-5 recruits three members of the Ets family of transcription factors: Ets-1, Fli-1 and GABPalpha (with GABPbeta1), to assemble ternary complexes on the mb-1 promoter in vitro. Using the Pax-5:Ets-1:DNA crystal structure as a guide, we defined amino acid requirements for transcriptional activation of endogenous mb-1 genes using a novel cell-based assay. Mutations in the beta-hairpin/beta-turn of the DNA-binding domain of Pax-5 demonstrated its importance for DNA sequence recognition and activation of mb-1 transcription. Mutations of amino acids contacting Ets-1 in the crystal structure reduced or blocked mb-1 promoter activation. One of these mutations, Q22A, resulted in greatly reduced mb-1 gene transcript levels, concurrent with the loss of its ability to recruit Fli-1 to bind the promoter in vitro. In contrast, the mutation had no effect on recruitment of the related Ets protein GABPalpha (with GABPbeta1). These data further define requirements for Pax-5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors.

Identification of an Sp factor-dependent promoter in GCET, a gene expressed at high levels in germinal center B cells.

Antigen-stimulated B lymphocytes undergo genetic and phenotypic changes in germinal centers (GCs), including affinity maturation of immunoglobulin (Ig) genes and Ig heavy chain isotype switching. Expression of the Germinal Center Expressed Transcript (GCET) gene is up-regulated in murine GC B cells. The human homolog of GCET, HGAL/GCET2, is an important prognostic marker for staging lymphomas derived from GCs. To identify mechanisms that control cell type-specific transcription of GCET, we localized promoter sequences using S1 nuclease protection and functional assays. Sequences comprising a TATA-less promoter were localized to a short region upstream of multiple mRNA start sites. In functional assays, the promoter is active in cells irrespectively of endogenous GCET gene expression. In vitro binding assays identified a non-consensus binding site for Sp factors near sites of transcriptional initiation. The site binds Spl and Sp3 in nuclear extracts and recombinant Spl in vitro, and is required for full promoter function in transient promoter assays. Activation of the promoter by Spl or Sp3 in Spl/3-deficient cells was largely dependent on the Sp site. Together, these data provide the first analysis of regulatory modules necessary for GCET expression, a model for GC B cell-specific transcription.

Early B cell factor cooperates with Runx1 and mediates epigenetic changes associated with mb-1 transcription.

Cd79a (called mb-1 here) encodes the Ig-alpha signaling component of the B cell receptor. The early B cell-specific mb-1 promoter was hypermethylated at CpG dinucleotides in hematopoietic stem cells but became progressively unmethylated as B cell development proceeded. The transcription factor Pax5 activated endogenous mb-1 transcription in a plasmacytoma cell line, but could not when the promoter was methylated. In this context, early B cell factor (EBF), a transcription factor required for B lymphopoiesis, potentiated activation of mb-1 by Pax5. EBF and the basic helix-loop-helix transcription factor E47 each contributed to epigenetic modifications of the mb-1 promoter, including CpG demethylation and nucleosomal remodeling. EBF function was enhanced by interaction with the transcription factor Runx1. These data suggest a molecular basis for the hierarchical dependence of Pax5 function on EBF and E2A in B lymphocyte development.