Tcragene recombination is supported by aTcraenhancer- and CTCF-dependent chromatin hub

Abstract

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4 − CD8 − double-negative thymocytes and Tcra gene segments in CD4 + CD8 + double-positive thymocytes. Initial V α -to-J α recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E α ) interacts directly with V α and J α gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E α promotes interactions between these V α and J α segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E α and to many locus promoters, biases E α to interact with these promoters, and is required for efficient V α -J α recombination. Our data indicate that E α and CTCF cooperate to create a developmentally regulated chromatin hub that supports V α -J α synapsis and recombination.T-cell development | T-cell receptor | thymus T and B cells produce diverse antigen receptors through the recombination of variable (V), diversity (D), and joining (J) gene segments at the T-cell receptor (Tcra, Tcrb, Tcrg, and Tcrd) and Ig (Igh, Igκ, and Igλ) loci. This V(D)J recombination is initiated by the lymphoid-specific recombination-activating gene-1 (RAG-1) and RAG-2 proteins, which recognize the recombination signal sequences (RSSs) that flank all V, D, and J gene segments and then cleave the DNA between the RSSs and the adjacent coding gene segments (1). A critical feature of the reaction is the assembly of a synaptic complex composed of two RSSs before the generation of RAG-dependent DNA doublestrand breaks (DSBs). As such, lineage-and developmental stagespecific V(D)J recombination events can be regulated not only by changes in RAG protein expression and RSS accessibility to RAG proteins but also by the ability of those RSSs to undergo synapsis (2).Conformational changes of antigen receptor loci are believed to support V(D)J recombination events because they can bring distant RSSs into proximity and therefore increase the probability of RSS synapsis (2, 3). Studies using 3D-FISH have demonstrated that lineage-and development stage-specific locus contraction marks the recombination windows at antigen receptor loci (3). For example, the 3-Mb Igh locus contracts specifically in pro-B cells to support V H -to-D H J H recombination (4-7). This contracted conformation brings distal and proximal V H segments, which are separated by megabases in the linear DNA sequence, to the vicinity of the D H J H cluster, presumably allowing all V H segments a similar opportunity for recombination (8). In addition, the mapping of Igh locus DNA-DNA...

Unifying model for molecular determinants of the preselection Vβ repertoire

“…At most genes, many levels of control are integrated to achieve the requisite gene expression state. For example, transcriptional promoters interact with their cognate enhancers over considerable distances in the linear genome to generate "hubs" where the two cis elements are in spatial proximity (1,2).…”

Insights into the Transcriptional Regulation of the Unrearranged and Rearranged Tcra and Tcrd Genes

“…The 1.7-Mb 5´-locus region includes 132 Tcra and Tcrd V (Trav and Trdv) gene segments, while the remaining 0.1-Mb 3´-locus region contains the Tcrd D and J (Trdd and Trdj) gene segments, the Tcrd constant region (Cδ), the Trdv5 gene segment, the Tcra J (Traj) gene segments, and the Tcra constant region (Cα) (Figure 2 [27,[30][31][32][33]. Eα is responsible for activating the endogenous locus germline transcription and the Tcra VJ recombination, as well as the generation of αβ T lymphocytes [34].…”

“…Interestingly, the formation of this chromatin loop favors the use of the diverse Trdv gene segments for Tcrd VDJ recombination in DN2/3a thymocytes and indirectly increases the diverse use of the Trav/Trdv gene segments for Tcra VJ recombination in DP thymocytes [28]. In Rag -/-eDP thymocytes, binding of the pre-TCR inducible TFs to Eα triggers the formation of a chromatin hub through the physical interactions of the Eα-bound TFs, TFs bound to the promoters associated with the most 3´-Trav/Trdv gene segments and TEAp, and the CTCF bound to HS-1´ and each Eα-dependent promoter (Figure 3) [27,31]. This chromatin hub creates an additional recombination center at the 3´-Trav/Trdv and 5´-Traj gene segments to activate the primary Tcra VJ recombination in DP thymocytes [5].…”

“…TEAp orchestrates different chromatin loops at the 3´-end of the locus during T-cell development [5,27,28] (see below). Eα is part of a previously described locus control region (LCR) located between Cα and the ubiquitously expressed Dad1 gene [29].…”

“…The fully contracted locus configuration in DN3a thymocytes is thought to facilitate the Tcrd VDJ recombination using the disperse Trdv gene segments across the entire locus, whereas the Tcra/Tcrd configuration in DP thymocytes is believed to facilitate the sequentially ordered primary and secondary Tcra VJ recombination (Figure 2) [5,61]. The molecular mechanism involved in the regulation of the different configurations adopted by the Tcra/Tcrd locus during thymocyte development is currently unknown.Although the 3´-end of the locus remains similarly contracted in DN3a and DP thymocytes, chromosome conformation capture experiments (3C and 4C) have distinguished two distinct functional chromatin interactions within this 0.5-Mb region of DN3a and DP thymocytes using recombinase activating gene-deficient (Rag -/-) mice (Figure 3) [5,27,28]. In Rag -/-DN3a thymocytes, a functionally relevant discrete chromatin loop mediated by CTCF-bound INT1/2 and CTCF-bound TEAp has been recently identified [28].…”