Chromatin conformations and contacts that support Tcra/d locus rearrangement (62.2)

Abstract

Abstract The Tcra/d locus rearranges in DN thymocytes to assemble Tcrd genes and in DP thymocytes to assemble Tcra genes. We used 3D-FISH to show that the 3’ portion of the locus is contracted in both DN and DP thymocytes, whereas the 5’ portion is contracted in DN but decontracts in DP thymocytes. We proposed that the fully contracted conformation in DN thymocytes allows dispersed Vαs to be used in a single round of Vδ-Dδ-Jδ rearrangement, whereas the 3’-contracted, 5’-decontracted conformation in DP thymocytes allows for multiple rounds of Vα-to-Jα rearrangement initiating with 3' Vαs. For high resolution analysis, we used the 3C method to detect molecular interactions between different sites in the locus. The Tcra enhancer (Eα) activates the T early α promoter (TEA) to target 5’Jαs for initial rearrangement. Eα also activates proximal Vαs over 500 kb. We detected molecular interactions between Eα and TEA, between Eα and proximal Vαs, and between different proximal Vαs, in DP but not DN thymocytes. All pairwise interactions depended on Eα. With TEA deleted, Eα also interacted with downstream Jαs. We propose that Eα nucleates a chromatin hub that supports transcription and ordered Vα-to-Jα recombination. However, deletion of Eα does not impact Tcra/d locus 3’end contraction as measured by 3D-FISH. Thus, overall locus conformation is independent of Eα, but this conformation may facilitate Eα interactions with distant sites.