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 δ Enhancer In Vivo

摘要

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 TCRαδ locus, the TCR α enhancer and the TCR δ enhancer (Eδ), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR α and δ genes, respectively. Eδ 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 Eδ within wild-type and mutant versions of a human TCR δ 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 Eδ enhanceosome in the context of chromatin in vivo.