Role of germline transcription in V(D)J rearrangement

种系转录在 V(D)J 重排中的作用

• 批准号: 9096069
• 负责人: ANN J FEENEY
• 金额: $ 19.25万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9096069
• 关键词: Abelson murine leukemia virus; Address; Affect; Antibodies; Antibody Repertoire; Area; B-Lymphocytes; Biological Assay; CRISPR/Cas technology; Cell Line; Cells; Complex; DNA; DNA Double Strand Break; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Deposition; Epigenetic Process; Event; Frequencies; Gene Rearrangement; Genes; Genetic Transcription; Guide RNA; Health; Hour; Immune system; Immunoglobulin Genes; Individual; Induced Mutation; Light; Location; Mediating; Modeling; Movement; Mutation; Nuclear Structure; Nucleosomes; Oncogenes; Play; Positioning Attribute; Process; Published Comment; Publishing; RNA; RNA Polymerase II; Recruitment Activity; Regulation; Role; STI571; Shapes; Structure; System; Testing; Time; Transcript; Travel; Untranslated RNA; V(D)J Recombination; base; combat; deep sequencing; kinase inhibitor; leukemia/lymphoma; mutant; pathogen; promoter; research study; three dimensional structure

 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

 描述（由申请人提供）：未重排的 V、J 和 C 基因的非编码 RNA 或“种系转录”在 30 多年前首次被观察到，从而提出了良好可及性假说。转录，特别是 V 基因种系转录，在 V(D)J 重排中的作用尚未阐明。尽管 Igh 和 Igh 的 J 和 C 基因紧密聚集，但 GT 水平非常高。当各个基因座主动重排时，Igκ基因座也存在可变性。 但大多数功能性 V 基因的种系转录通常较低。此外，Igh 和 I� 基因座的大 2.5-3 Mb V 区部分的多个位置存在基因间转录。 TCR Jα 基因座减少了一些直接下游 Jα 基因的重排，但对于通过 Ig 或 TCR 基因座的大 V 基因部分进行种系转录的作用，以及是否需要它，我们一无所知。通过在 Abelson 病毒转化的 (Abl) 前 B 细胞系中使用 CRISPR/Cas9 删除来删除多个 Vκ 基因的启动子来解决这个问题。在与 Abl 激酶抑制剂 STI571 一起培养后，细胞上调 RAG 和种系转录物的转录，在 48 小时内，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。是一个强大、快速、易于操作的诱导系统，用于严格评估 V 基因种系转录在 V(D)J 重排中的作用，这将清楚地证明低至中度 V 基因种系转录是否是有效 V(D) 所必需的。 J 重排。如果重排减少或消除，我们将评估种系转录是否影响表观遗传图谱或核小体定位。我们还将确定种系转录在基因座收缩中的作用。种系转录，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基因种系转录本身的作用将直接导致被转录区域的基因座压缩。为了检验这一假设，我们将进行验证。 执行 RNA 聚合酶 II ChIP-loop 以确定是否直接长程 相互作用是 Pol II 介导的，因此可能发生在转录工厂中。我们提出 Ig 位点的 3D 结构在 V 基因利用中发挥着重要作用，此处提出的研究将清楚地证明种系转录是否过度。特定的 V 基因在该 V 基因的重排频率中发挥作用，我们还将确定种系转录是否在 V(D)J 重组过程中塑造 Igκ 位点的长程循环结构中发挥更全面的作用。