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Nucleic Acids Research, 2003, Vol. 31, No. 19 5483-5489
© 2003 Oxford University Press

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

Holly Maier, Rachel Ostraat, Sarah Parenti, Daniel Fitzsimmons, Lawrence J. Abraham1, Colin W. Garvie2 and James Hagman*

Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado Health Sciences Center, Denver, CO 80262, USA, 1 Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences and Western Australia Institute for Medical Research, The University of Western Australia, Crawley 6009, Australia and 2 Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250, USA

*To whom correspondence should be addressed. Tel: +1 303 398 1398; Fax: +1 303 398 1396; Email: hagmanj{at}njc.org

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 GABP{alpha} (with GABPß1), 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 ß-hairpin/ß-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 GABP{alpha} (with GABPß1). These data further define requirements for Pax-5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors.


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