Definition of a T-Cell Receptor β Gene Core Enhancer of V(D)J Recombination by Transgenic Mapping

Abstract

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activatingcis-recombination events. A striking example is the T-cell receptor β-chain (TCRβ) gene enhancer (Eβ), which in the mouse consists of at least seven nuclear factor binding motifs (βE1 to βE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Eβ required for recombination enhancer activity. The Eβ core is comprised of a limited set of motifs (βE3 and βE4) and an additional previously uncharacterized 20-bp sequence 3′ of the βE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the βE1 through βE4 motifs but not the 3′ sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Eβ recombinational activity. Our results emphasize the multifunctionality of the TCRβ enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.

ACKNOWLEDGMENTS

We thank M. Algarté, J. Imbert, and P. Rameil for advice on in vivo genomic footprinting assays, C. Beziers La Fosse for preparing the artwork, N. Brun-Roubereau for helping in FACS sorting, and A. Loussif, M. Pontier, and G. Warcollier for maintaining the mouse colonies.

This work was supported by institutional grants from INSERM and CNRS and by specific grants from the Association pour la Recherche sur le Cancer, the Commission of the European Communities, the Fondation Princesse Grace de Monaco, the Ligue Nationale Contre le Cancer, and Rhone-Poulenc Pharmaceuticals (to P.F.). R.K.T. was a fellow of the Ministère des Affaires Etrangères and is now a fellow of the Fondation pour la Recherche Médicale. W.M.H. is the recipient of a Foreign Associate Scientist position from the CNRS.