Role of germline transcription in V(D)J rearrangement

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 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