Orientation-specific RAG activity in chromosomal loop domain contribute to Tcrd V(D)J recombination during T cell development. Mus musculus

T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination. Here, we employ a high throughput method to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in developing thymocytes. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, but Trdd1 joining is ordered with joining to Trdd2 a prerequisite for further rearrangement. We also find frequent, previously unappreciated Trdd1 and Trdd2 rearrangements that inactivate Tcrd. Moreover, we find numerous RAG off-targets that are generated via unidirectional RAG tracking across the loop-domain containing Trdd1, Trdd2 and Trdj. Correspondingly, disruption of the upstream domain boundary causes spreading of on- and off-target RAG activity to the proximal Trdv domain. Overall design: RAG-initiatd Tcrd D segment rearrangements in developing thymocytes were generated by deep sequencing using illumine Miseq

T细胞抗原受体δ链（T cell antigen receptor δ, Tcrd）的可变区外显子由重组激活基因（Recombination Activating Gene, RAG）起始的V(D)J重组组装而成。本研究采用高通量方法，对发育中胸腺细胞内数十万条由RAG起始的Tcrd D片段（Trdd1与Trdd2）重排事件进行图谱绘制。研究发现，Trdd2可直接与Trdv、Trdd1及Trdj片段发生连接重排；而Trdd1的重排连接具有严格顺序性，即需先与Trdd2完成连接，方可进行后续重排。此外，本研究还发现大量此前未被报道的Trdd1与Trdd2重排事件，此类重排会导致Tcrd功能失活。同时，我们还观察到众多RAG脱靶位点，此类位点通过单向RAG扫描包含Trdd1、Trdd2与Trdj的环结构域而产生。相应地，上游结构域边界的破坏会使RAG的靶标与脱靶活性扩散至邻近的Trdv结构域。

实验设计概述：通过Illumina MiSeq进行深度测序，获取发育中胸腺细胞内由RAG起始的Tcrd D片段重排信息。