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Distinct Roles for c-Myb and Core Binding Factor/Polyoma Enhancer-Binding Protein 2 in the Assembly and Function of a Multiprotein Complex on the TCR Enhancer In Vivo¹

Cristina Hernández-Munain^2,* and Michael S. Krangel

^* Basel Institute for Immunology, Basel, Switzerland; and Department of Immunology, Duke University Medical Center, Durham, NC 27710

Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCR locus, the TCR enhancer and the TCR enhancer (E), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR and genes, respectively. E function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin-integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, we used in vivo footprinting to study the requirements for protein binding to E within wild-type and mutant versions of a human TCR minilocus in stably transfected Jurkat cells. Our data indicate that CBF/PEBP2 plays primarily a structural role as it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, our data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an E enhanceosome in the context of chromatin in vivo.

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