The somatic assembly of antigen receptor (AgR) genes by V(D)J recombination creates the diverse antigen receptor repertoires of T and B lymphocytes. The three-dimensional architecture of AgR loci has emerged as an important facilitator of the long-distance recombination events on which AgR repertoire assembly depends. Among the various AgR loci, the Tcra-Tcrd locus is unrivaled in its complexity, with Tcrd, primary Tcra, and secondary Tcra rearrangements occurring sequentially in developing thymocytes. We have described changes in Tcra-Tcrd locus conformation and in the fine structure of the Tcra-Tcrd interactome in developing thymocytes, and have shown chromatin organizer CTCF to be a critical mediator of long-distance interactions and repertoire. Here, we will address unexplored aspects of Tcra-Tcrd locus conformation and interactome, focusing on how these parameters are configured and then reconfigured during the recombination program, and how each round of recombination and locus restructuring sets the stage for and dictates the next. In doing so, we will reveal how the structure of the Tcra-Tcrd locus supports the development of diverse Tcrd and Tcra repertoires. We will test the hypothesis that the Tcra-Tcrd interactome is a composite of stable structural loops and dynamic tethering loops, that loop dynamics are dictated by the orientation of CTCF binding elements, and that stable and dynamic loops are both essential to generate robust TCR repertoires. We have described the organization of the 3' end of the unrearranged Tcra-Tcrd locus in DN and DP thymocytes. We will identify local and long-distance interactions that organize the V gene array and may allow communication with the 3' end of the locus. We will also compare interactions on V?J? rearranged alleles to unrearranged alleles to reveal how the interactome adjusts to primary Tcra rearrangement and thus sets the stage for secondary rearrangement. We will evaluate combinatorial diversity in the pre-selection Tcra repertoire, focusing on whether secondary Tcra recombination consumes V gene segments in an ordered or stochastic fashion. We will enhance this analysis using genetic models to assess how Tcrd rearrangement influences primary Tcra rearrangement, and how primary Tcra rearrangement influences secondary Tcra rearrangement, to understand how the Tcra repertoire evolves in developing thymocytes. Finally, we will evaluate a three-step model for Tcra-Tcrd locus conformation, in which the 5' portion of the locus contracts for Tcrd recombination, extends for primary Tcra recombination, and contracts again for secondary Tcra recombination. We will relate the resulting physical model of Tcra-Tcrd locus organization to our data on repertoire development through primary and secondary Tcra recombination. Successful completion of this work is expected to provide an integrated view of locus conformation, interactome, and repertoire through Tcrd, primary Tcra, and secondary Tcra recombination. It should also provide fundamental insights into organizational features of CTCF-mediated loopscapes that will impact understanding of how genome architecture dictates genome function at AgR loci and beyond.