EBF is essential for B cell development. It stimulates the transcription of multiple genes of the B lineage-specific program and is essential for the expression of proteins that assemble the pre-B- and mature B cell-receptors (Hagman and Lukin, 2005
). In addition to its functional significance, EBF is a novel type of DNA binding protein. Due to the lack of structural information, very little is understood concerning how EBF binds DNA and activates transcription. Chromatin remodeling activities of EBF were described only recently (Maier et al., 2004
). A better understanding of these mechanisms is essential for determining how EBF functions in the B cell-specific regulatory network (Medina et al., 2004
; Medina and Singh, 2005
To better understand how transcription factors activate gene transcription in B cells, we developed an assay that measures the activation of endogenous mb-1
genes in plasmacytoma cells. In previous studies we used this assay to identify functionally important residues in Pax5, which activated mb-1
genes in 558LµM plasmacytoma cells (Maier et al., 2003b
). However, in a subset of these cells, Pax5 was unable to activate mb-1
transcription by itself due to the epigenetic state of mb-1
promoter chromatin (Maier et al., 2003a
). We concluded that the transcription of mb-1
genes in this context requires additional proteins. Further studies suggested that one of these proteins is EBF (Maier et al., 2004
). The assignment of this function to EBF is logical. The onset of EBF expression, which precedes that of Pax5 during early B lymphopoiesis, parallels the progressive demethylation of mb-1
promoters. Moreover, the mb-1
gene fails to become hypomethylated in B cell progenitors from EBF-deficient mice. EBF can initiate CpG demethylation of mb-1
genes in plasmacytoma cells. Furthermore, EBF enhances chromatin accessibility of mb-1
promoters, which, in turn, facilitates synergy between EBF and Pax5. Thus, mb-1
gene activation is dependent on a hierarchy of transcription factors in which EBF acts upstream of Pax5.
In our current studies we introduced mutations into EBF and measured mb-1 and Vpreb1 gene activation. Transactivation of these genes in plasmacytoma cells expressing the Pax5 DBD was robust in response to wild type EBF. Notably, endogenous Ebf1 and Pax5 genes in µM.2.21 cells were not stimulated by enforced expression of EBF and the Pax5 DBD (S.F., data not shown); therefore, the observed gene activation was due to the ectopically expressed factors. Mutations predicted to reduce zinc binding by EBF (H157A, C161S, C164S and C170S) each reduced mb-1 and Vpreb1 transcripts to nearly background levels. These mutations revealed the importance of the Zn-knuckle in functional DNA binding by EBF. R163 is also essential for DNA binding. Other residues, including R152, T156, K167/K168 and N172 are important for binding and activating the mb-1 promoter, but mutations of these residues had stronger effects on binding and activation of the Vpreb1 promoter. R173A significantly reduced activation of Vpreb1 transcription, but it had only minor effects on mb-1 transcription. Although residues including lysines 167 and 168 are not predicted to coordinate zinc, they may be essential for maintaining the Zn-knuckle in the proper orientation for DNA binding or for contacting DNA directly. Interestingly, E149A and E175A had opposing effects on mb-1 (decreased) vs. Vpreb1 (increased) transcription. We conclude that EBF utilizes different sets of amino acid side chains for binding different DNA sequences. It is likely that this flexibility of DNA recognition contributes to the degenerate DNA-binding specificity of EBF.
The Zn-knuckle and surrounding residues between prolines 148 and 177 of EBF are conserved between EBF family proteins of diverse species (Altschul et al., 1997
; Schäffer et al., 2001
). The four zinc coordination residues and flanking residues are perfectly conserved between EBF (EBF1), EBF3 and other EBF orthologs and paralogs of many vertebrates including mice and humans. Identical sequences are present in EBF proteins of more distant species including sea urchins (S. purpuratus
) and flour beetles (T. castaneum
). Nearly perfect matches are also notable between murine EBF and the ortholog Collier/Knot proteins of insects. Collier/Knot regulates cell fates during development of head structures of D. melanogaster
(Crozatier et al., 1996
). Intriguingly, Collier/Knot also regulates the innate immune system in flies (Crozatier et al., 2004
; Crozatier et al., 1996
). Vertebrate EBF2 and the Unc-3 protein of C. elegans
feature only conservative changes, including variation of an aspartic acid (D166 of EBF) to glutamic acid. Zn-knuckles within vertebrate EBF4 proteins exhibit conservative changes of a single lysine (K168 of EBF) to arginine. Interestingly, one cysteine (similar to C151 of EBF) in the Zn-knuckle region varies extensively in EBF-like proteins. Our studies demonstrated that C151 is not required for functions of murine EBF. Overall, the very high degree of conservation of the Zn-knuckle suggests its importance for functional activities of EBF family proteins in organisms as diverse as humans, fish and insects.
The configuration of metal coordinating residues in the EBF Zn-knuckle, HX3
C, is highly unusual. The most common zinc fingers are similar to repeated elements in the TFIIIA transcription factor and conform to the conserved sequence Y/FXCX2–5
H. Zinc finger motifs in nuclear hormone receptors have a different configuration of cysteines, CX2
C (C2C2 zinc fingers). Other zinc binding motifs include the C6-zinc clusters (as in Gal4 in S. cerevisiae
) and the CHCC motifs of retroviral gag
proteins. Only two other groups of proteins possess a configuration of zinc coordinating residues similar to the HCCC motif of EBF. Highly conserved proteins that regulate sex determination in metazoans possess a cyteine-rich DNA-binding domain, termed the Doublesex–MAB-3 (D–M) domain (Lints and Emmons, 2002
; Volff et al., 2003
). The D–M motif comprises intertwined zinc-binding structures featuring CCHC and HCCC configurations of metal coordinating residues (Zhu et al., 2000
). Similar to EBF, D–M proteins exhibit zinc-dependent DNA binding. HCCC motifs have also been reported in the CREB Binding Protein (CBP)/p300 co-activators (De Guzman et al., 2000
; De Guzman et al., 2005
). CBP and p300 possess three tandem HCCC motifs (TAZ1–3) that are required for interactions with other proteins (e.g. Hypoxia-Inducible transcription Factor-1α). The three-dimensional structures of these domains (TAZ1 and/or TAZ2) have been determined to include zinc ions. It is not known whether the Zn-knuckle of EBF adopts a fold similar to that of TAZ1 and TAZ2. However, it is notable that the Zn-knuckle of EBF is similar in overall length (fourteen residues) to TAZ2 (sixteen residues). It is not known whether, similar to the TAZ motifs, the Zn-knuckle of EBF mediates protein:protein interactions in addition to its DNA binding functions.
In conclusion, our data demonstrate that the Zn-knuckle of EBF is important for DNA recognition and transcriptional regulation of early B cell-specific genes. However, it is currently unknown whether Zn-knuckles strictly function as: 1) DNA recognition motifs, 2) scaffolding of the DNA-binding domain and/or 3) for recruitment of accessory proteins, such as chromatin remodeling complexes necessary for the ‘pioneer’ activity of EBF. Further studies, including determination of the three-dimensional structure of EBF, are important for resolving these questions.