Role of germline transcription in V(D)J rearrangement

 描述(由申请人提供)：未重排的V、J和C基因的非编码RNA或“生殖系转录”在30多年前首次被观察到，从而产生了广为接受的可及性假说。然而，胚系转录，尤其是V基因胚系转录在V(D)J重排中的确切作用尚不清楚。在IgH和Igκ基因座的J和C基因紧密聚集的同时，GT水平非常高，这两个基因在各自的座位上都处于活跃的重排状态。还有一个变量 但通常情况下，大多数功能V基因的生殖系转录水平较低。此外，在IgH和I�基因座的2.5Mb-3MbV区的几个位置还存在基因间转录。已有研究表明，阻断部分TCRJα基因座的生殖系转录会减少一些直接下游的Jα基因的重排，但关于生殖系转录通过Ig或TCR基因座的大V基因部分所起的作用，以及它是否是必需的，目前尚不清楚。在这里，我们将明确地解决这个问题，通过删除Abelson病毒转化(Abl)前B细胞系中的CRISPR/Cas9缺失来删除几个Vκ基因的启动子。在Abl激酶抑制剂STI571的培养下，细胞上调RAG和生殖系转录，并在48小时内经历强劲的Vκ-Jκ重排。我们已经证明，它们诱导出一种与野生型前B细胞非常相似的多样化的V-κ谱系。因此，这是一个健壮、快速、易于操作、可诱导的系统，可用于关键评估V基因种系转录在V(D)J重排中的作用。这将清楚地表明是否需要低到中等的V基因种系转录来进行有效的V(D)J重排。如果重排减少或消除，我们将评估生殖系转录是否影响表观遗传学特征或核小体定位。我们还将确定生殖系转录在基因座收缩中的作用。我们认为，当Vκ基因经历胚系转录时，它们与强κ°启动子进入相同的转录工厂，即 与Jκ基因相邻，因此V基因种系转录本身的行为将直接导致被转录区域的座位紧凑。为了检验这一假设，我们将 进行RNA聚合酶II芯片环以确定是否直接远程 相互作用是由POL II介导的，因此很可能发生在转录工厂中。我们认为，免疫球蛋白基因的3D结构在V基因利用中起着重要作用。总之，这里提出的研究将清楚地证明，特定V基因上的生殖系转录是否在该V基因重排的频率中发挥作用。我们还将确定种系转录在V(D)J重组过程中是否在塑造Igκ基因座的长程环状结构方面发挥更全局的作用

 DESCRIPTION (provided by applicant): Non-coding RNA, or "germline transcription", of unrearranged V, J and C genes was first observed over 30 years ago, giving rise to the well accepted accessibility hypothesis. However, the precise role that germline transcription, and in particular V gene germline transcription, plays in V(D)J rearrangement has still not been elucidated. There is a very high level of GT though the tightly clustered J and C genes of the Igh and Igκ loci at the time when the respective locus is actively rearranging. There is also variable but generally low germline transcription over most functional V genes. In addition there is intergenic transcription at several locations in the large 2.5-3 Mb V region part of the Igh and I� loci. It has been shown that blocking germline transcription in part of the TCR Jα locus reduces rearrangement of some of the immediate downstream Jα genes, but nothing is known about the role of germline transcription through the large V gene portion of Ig or TCR loci, and whether it is even required. Here we will definitively address this issue by deleting the promoter of several Vκ genes using CRISPR/Cas9 deletion in an Abelson virus-transformed (Abl) pre-B cell line. Upon culture with the Abl kinase inhibitor STI571, the cells upregulate transcription of RAG and germline transcripts, and within 48 hours undergo robust Vκ-Jκ rearrangement. We have demonstrated that they induce a diverse Vκ repertoire that closely resembles that of wild type pre-B cells. Thus, this is a robust, rapid, easily maniputable, inducible system to critically evaluate the role of V gene germline transcription in V(D)J rearrangement. This will clearly demonstrate if the low to moderate V gene germline transcription is required for efficient V(D)J rearrangement. If rearrangement is reduced or eliminated, we will assess whether germline transcription affects the epigenetic profile or nucleosome positioning. We will also determine the role of germline transcription in locus contraction. We propose that as Vκ genes undergo germline transcription, they go to the same transcription factory as the strong κ° promoter which is adjacent to the Jκ genes, and thus the act of V gene germline transcription itself will directl result in locus compaction of the area being transcribed. In order to test this hypothesis, we will perform RNA Polymerase II ChIP-loop to determine if direct long-range interactions are Pol II-mediated, and hence likely to be occurring in a transcription factory. We have proposed that the 3D structure of the Ig locus plays an important role in V gene utilization. Together the studies proposed here will clearly demonstrate if germline transcription over a specific V gene plays a role in the frequency of rearrangement of that V gene. We will also determine if germline transcription plays a more global role in shaping the long-range looping structure of the Igκ locus during V(D)J recombination