M. Sigvardsson, D.R. Clark, D. Fitzsimmons, M. Doyle, P. Akerblad, T. Breslin, S. Bilke, R. Li, C. Yeamans, G. Zhang and J. Hagman Early B-Cell Factor, E2A, and Pax-5 Cooperate To Activate the Early B Cell-Specific mb-1 Promoter Molecular and Cellular Biology 22, 8539-51 (2002) Abstract: Previous studies have shown that early B cell-specific mb-1 promoter is regulated by EBF and Pax-5. Here, we used in vivo footprinting assays to identify promoter binding sites in cells that express mb-1 transcripts, but not in non-expressing cells. Protection of a consensus binding site for E2A proteins (E box) was detected just downstream of the protected EBF site. The E box specifically binds nuclear extracted proteins with the expected DNA recognition properties of E2A proteins, and these complexes are specifically recognized by E2A-specific antisera. Transfection assays with mb-1 promoter:reporter plasmids show that EBF, E2A, and Pax-5:Ets ternary complex binding sites are cruicial for function in pre-B cells, but are less important in pro-B and germinal center stages of differentiation. The EBF and E2A sites functionally synergize in transfection assays, and EBD and E2A (E47) proteins bind the promoter with modest cooperativity. DNA array analysis shows that of the genes tested, expression of mb-1 correlated positively with EBD, and negatively with Id1, an inhibitor of E2A protein functions. Together, ourr studies demonstrate the complexity of factors regulating tissue-specific transcription in early B cells. LU TP 02-19