The ubiquitously expressed basic helix-loop-helix (bHLH) transcription factors E12 and E47, products of alternative splicing of the E2A/TCF3 gene, regulate diverse biological processes including cell growth, differentiation and development. To search for novel protein interactions for E12, we utilized the bHLH domain of E12 as a bait in yeast two-hybrid screening. Yeast two-hybrid, mammalian two-hybrid, and co-immunoprecipitation analyses demonstrate specific interaction of E12 with RANBP17, a novel member of the importin-beta superfamily; this interaction maps to the CRM1 homology region of RANBP17. Ectopic expression of RANBP17 leads to a approximately 3-fold increase in E2A/MyoD mediated transactivation of an E-box regulated luciferase reporter gene. Interaction and transactivation studies also revealed similar functions for RANBP16/XPO7. Furthermore, ectopic expression of either RANBP16 or RANBP17 resulted in increased level of endogenous transcript for the cyclin-dependent kinase inhibitor, p21(Waf1/Cip1), a well-characterized E2A target gene. Together, these biochemical and functional data reveal RANBP16 and RANBP17 as novel regulators of E2A protein action.
(c) 2010 Wiley-Liss, Inc.