Early B-Cell Factor, E2A, and Pax-5 Cooperate To Activate the Early B Cell-Specific mb-1 Promoter

this report, we show that mb-1 transcription correlates with occupancy of multiple promoter binding sites by factors in vivo. Using in vivo footprinting, we identified at least six factor binding sites and showed their importance for transcription in early B cells. Each of the sequences protected from DMS modification in intact cells contributed to promoter function in a cell type-specific manner, as evidenced by decreased activity of mutated promoters in short-term transfection assays. These data are supported by Western and EMSA analysis of proteins in representative cell lines, which showed that levels of EBF and Pax-5 proteins in cells correlate with protection of their binding sites in footprinting assays and requirements for these sites in mb-1 promoter function. DNA microarray experiments identified a very close correlation between mb-1 and EBF expression. Thus, the data suggest that mb-1 promoter function is most directly regulated by levels of EBF, but all three factors are important for high level mb-1 transcription in early B cells. At later stages of development, decreased levels of expression are mediated by Pax-5:Ets ternary complexes in the absence of EBF and E2A activit