The lymphoid-specific immunoglobulin μ heavy chain gene intron enhancer (μE) contains multiple binding sites for trans-acting nuclear factors. We have used a combination of in vitro and in vivo assays to reconstruct protein-DNA interactions on a minimal B cell-specific μ enhancer that contains three motifs, μA, μB, and μE3. Using ETS-domain proteins that transactivate the minimal enhancer in non-lymphoid cells, we show that (i) PU.1 binds coordinately to both μA and μB sites in vitro and (ii) in the presence of Ets-1, this factor binds to the μA site and PU.1 to the μB site. Two factors, TFE3 and USF, bind to the μE3 element. When the ETS proteins are present together with μE3 binding proteins, a three-protein- DNA complex is generated. Furthermore, we provide evidence for protein- protein interactions between Ets-1 and PU.1 proteins that bind to μA and μB sites, and between Ets-1 and TFE3 bound to the μA and μE3 sites. We propose that this domain of the μ enhancer is assembled into a nucleoprotein complex that contains two tissue-restricted ETS domain proteins that recognize DNA from the same side of the helix and one ubiquitously expressed bHLH-leucine zipper protein that binds between them, recognizing its site from a different side of the helix.
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