Regulation of Tcra/Tcrd Locus Conformation during Thymocyte Development

Abstract

<p>The chromatin architecture of antigen receptor loci has been hypothesized to facilitate the assembly of variable (V), diversity (D), and joining (J) gene segments during lymphocyte development. The 1.6 megabase Tcra/Tcrd locus is unique since it undergoes highly divergent Tcrd and Tcra recombination programs in CD4&ndash;CD8&ndash; double negative (DN) thymocytes and CD4+CD8+ double positive (DP) thymocytes, respectively. In this dissertation, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus by using three-dimensional fluorescence in situ hybridization (3D-FISH) and chromosome conformation capture (3C). </p><p>Using 3D-FISH, we found the 3' portion of the locus is contracted in both DN and DP thymocytes as compared to B cells. Remarkably, the 5' portion of the locus is contracted in DN thymocytes, but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving V&alpha; gene segments distributed over one megabase, whereas the unique 3'-contracted, 5'-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3' of the available V&alpha; gene segments. This would maintain a large pool of distal V&alpha; gene segments for subsequent rounds of recombination.</p><p>To study the conformational changes at the molecular level, we used 3C to detect interactions between different sites spanning 400kb in the contracted 3' portion of the locus. The Tcra enhancer (E&alpha;) is known to activate V&alpha; and J&alpha; segment promoters and to stimulate V&alpha;-to-J&alpha; recombination in DP thymocytes. We detected various pair-wise interactions between elements essential for initial Tcra recombination, including proximal V&alpha; segments, TEA promoter, 5' J&alpha; array and E&alpha;. Notably, these interactions occur specifically in DP thymocytes and all are E&alpha;-dependent. We proposed that in addition to regulating transcriptional activity, E&#945; promotes synapsis of RSSs by tethering proximal V&alpha; and 5'J&alpha; segments together to facilitate initial Tcra recombination. </p><p>We also asked whether a known chromatin organizer, CTCF, regulates the formation of the DP stage-specific, E&alpha;-dependent chromatin hub. Using ChIP-seq, we identified CTCF binding sites at E&alpha;, TEA promoter, and many V&alpha; promoters in DN and DP thymocytes. Loss of CTCF in DP thymocytes resulted in impaired primary V&#945;-to-J&#945; recombination, reduced V&alpha; and TEA germline transcription, and reduced interactions between E&alpha; and Tcra genes. Strikingly, we also observed aberrantly increased Tcrd gene transcription and interactions between E&alpha; and Tcrd gene segments in CTCF-deficient DP thymocytes. Our data suggest that CTCF helps E&alpha; to organize a DP stage-specific chromatin hub that sets the stage for synapsis and recombination of proximal V&alpha; and 5' J&alpha; segments in DP thymocytes.</p>