Requirements for selective recruitment of Ets proteins and activation of mb-1/lg-a gene transcription by Pax-5 (BSAP)

Pax-5, a member of the paired domain family oftranscription factors, is a key regulator of Blymphocyte-speci®c transcription and differentiation.A major target of Pax-5-mediated activation isthe mb-1 gene, which encodes the essential transmembranesignaling protein Ig-a. Pax-5 recruitsthree members of the Ets family of transcriptionfactors: Ets-1, Fli-1 and GABPa (with GABPb1), toassemble ternary complexes on the mb-1 promoterin vitro. Using the Pax-5:Ets-1:DNA crystal structureas a guide, we de®ned amino acid requirements fortranscriptional activation of endogenous mb-1genes using a novel cell-based assay. Mutations inthe b-hairpin/b-turn of the DNA-binding domain ofPax-5 demonstrated its importance for DNAsequence recognition and activation of mb-1 transcription.Mutations of amino acids contacting Ets-1in the crystal structure reduced or blocked mb-1promoter activation. One of these mutations, Q22A,resulted in greatly reduced mb-1 gene transcriptlevels, concurrent with the loss of its ability torecruit Fli-1 to bind the promoter in vitro. In contrast,the mutation had no effect on recruitment ofthe related Ets protein GABPa (with GABPb1).These data further de®ne requirements for Pax-5function in vivo and reveal the complexity of interactionsrequired for cooperative partnershipsbetween transcription factors.