Role of germline transcription in V(D)J rearrangement

 描述（由申请人提供）：30多年前首次观察到未重排的V、J和C基因的非编码RNA或“生殖系转录”，从而产生了广为接受的可及性假说。然而，生殖系转录，特别是V基因生殖系转录，在V（D）J重排中发挥的确切作用尚未阐明。当Igh和IGκ基因座活跃重排时，通过紧密聚集的J和C基因存在非常高水平的GT。也有变数 但在大多数功能性V基因上通常较低的生殖系转录。此外，在Igh和Ih基因座的2.5-3 Mb V区部分的几个位置存在基因间转录。研究表明，阻断TCR Jα基因座部分的生殖系转录可以减少一些直接下游Jα基因的重排，但对于通过IG或TCR基因座的大V基因部分进行生殖系转录的作用，以及是否需要生殖系转录，我们一无所知。在这里，我们将通过在Abelson病毒转化的（Abl）前B细胞系中使用CRISPR/Cas9缺失来删除几个Vκ基因的启动子来明确解决这个问题。在与Abl激酶抑制剂STI 571一起培养时，细胞上调RAG和生殖系转录物的转录，并且在48小时内经历稳健的Vκ-Jκ重排。我们已经证明，它们诱导了与野生型前B细胞非常相似的多样化Vκ库。因此，这是一个强大的，快速的，易于操作的，诱导系统，严格评估V基因生殖系转录在V（D）J重排的作用。这将清楚地证明有效的V（D）J重排是否需要低至中等的V基因种系转录。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谱或核小体定位。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基因进行胚系转录时，它们与强κ°启动子进入相同的转录工厂， 与Jκ基因相邻，因此V基因生殖系转录本身的行为将直接导致被转录区域的基因座压缩。为了验证这个假设，我们将 进行RNA聚合酶II ChIP环，以确定是否直接长距离 相互作用是Pol II介导的，因此可能发生在转录工厂中。我们已经提出，IG基因座的三维结构在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表明，如果生殖细胞转录特定的V基因在该V基因的重排频率中发挥作用。我们还将确定在V（D）J重组过程中，生殖系转录是否在形成IGκ基因座的长程环状结构中发挥更全面的作用

 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