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 delta enhancer in vivo.
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 TCRalphadelta locus, the TCR alpha enhancer and the TCR delta enhancer (Edelta), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR alpha and delta genes, respectively. Edelta 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 Edelta within wild-type and mutant versions of a human TCR delta 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 Edelta enhanceosome in the context of chromatin in vivo.
Hernández-Munain
Cristina
C
Basel Institute for Immunology, Basel, Switzerland. chmunain@cbm.uam.es
Krangel
Michael S
MS
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D050659:Core Binding Factor Alpha 1 Subunit; D050658:Core Binding Factor beta Subunit; D050660:Core Binding Factors; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004742:Enhancer Elements, Genetic / Q000276:immunology*; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D011487:Protein Conformation; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism / Q000502:physiology*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured
C493692:CBFB protein, human; D050659:Core Binding Factor Alpha 1 Subunit; D050658:Core Binding Factor beta Subunit; D050660:Core Binding Factors; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D020598:Proto-Oncogene Proteins c-myb; C493709:RUNX2 protein, human; D016692:Receptors, Antigen, T-Cell, gamma-delta; D050656:Transcription Factor AP-2; D014157:Transcription Factors