Date of Award
Doctor of Philosophy (PhD)
The adaptive immune system endows mammals with a sophisticated mechanism to recognize foreign proteins via surface antigen receptors that are expressed on the surface of all lymphocytes. This defense network is generated by V(D)J recombination, a set of sequentially controlled DNA cleavage and repair events that assembles functional antigen receptor genes from distally located Variable (V), Diversity (D) and Joining (J) gene segments. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is controlled at the levels of tissue, stage and allele specificity by a collection of architectural and regulatory elements that are distributed throughout each antigen receptor locus. Our laboratory has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (Tcrb) locus. These elements include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex, facilitating Dβ to Jβ recombination. Simultaneously, spatial apposition of the Vβ cluster to the DβJβ region (a phenomenon called locus contraction) increases the efficiency of long-range Vβ recombination. Using extensive chromatin profiling of the Tcrb locus, we have discovered that selection of Vβ genes depend upon their association with transcriptionally active chromatin and high quality Recombination Signal Sequences, which serve as substrates for the V(D)J recombinase proteins RAG1/2. We further identify a bi-functional barrier-tethering region upstream of the DβJβ cluster that is essential for stabilizing its long-range interactions with distal Vβ gene segments in progenitor CD4-CD8- double negative (DN) thymocytes. Following Tcrb rearrangement, progenitor thymocytes proliferate and differentiate into CD4+CD8+ Double Positive (DP) cells, where the Vβ genes are epigenetically silenced and the distal ends of Tcrb are spatially segregated (presumably to inhibit further rearrangements). However, we have found that the transcriptionally inactive proximal Vβ genes continue to interact with the DβJβ cluster in a proliferation independent manner. These findings divide the Tcrb locus into two architectural domains, of which only the distal part is spatially segregated in DP cells. The loss of distal Vβ interaction is also observed in DP thymocytes containing a rearranged Tcrb allele, suggesting this conformation is DP-intrinsic. Our results have unraveled new mechanisms that stabilize the long-range Tcrb conformation in DN cells, how the Vβ segments are selected to recombine and how Tcrb topology is retained by DP-intrinsic mechanisms. These studies pave the way for future investigations into the role of boundary elements and tissue specific transcription factors in sculpting AgR gene assembly and regulating genome topology.
Chair and Committee
Eugene M Oltz
Paul Allen, Maxim Artyomov, Takeshi Egawa, Kenneth Murphy, Barry Sleckman,
Majumder, Kinjal, "Spatial and Epigenetic Regulation of T-Cell Receptor Beta Gene Assembly" (2015). Arts & Sciences Electronic Theses and Dissertations. 566.