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: 8970153
• 负责人: ANN J FEENEY
• 金额: $ 33.16万
• 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/8970153
• 关键词: Abelson murine leukemia virus; Address; Affect; Antibodies; Antibody Repertoire; Area; B-Lymphocytes; Biological Assay; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; 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（由适用提供）：30年前首次观察到了未脱落的V，J和C基因的非编码RNA或“生殖线转录”，这引起了公认的可访问性假设。但是，在V（d）J重排中发挥了种系转录，尤其是V基因种系转录的精确作用，但仍未得到阐明。尽管在相对基因座是积极重新排列时，IGH和Igκ基因座的紧密聚集的J和C基因的GT水平很高。还有变量 但通常在大多数功能V基因上种系转录。另外，在Ig和I座位的大型2.5-3 MB V区域的几个位置都有基因间转录。已经表明，在TCRJα基因座的一部分中阻断种系转录会减少某些直接下游Jα基因的重排，但是对于通过Ig或TCR基因座的大V基因部分的生殖线转录的作用一无所知。在这里，我们将通过在Abelson病毒转化（ABL）PER-B细胞系中使用CRISPR/CAS9删除来删除几个Vκ基因的启动子来确定解决此问题。在用ABL激酶抑制剂STI571培养后，细胞上调了抹布和种系转录本的转录，并在48小时内进行了强大的Vκ-Jκ重排。我们已经证明它们诱导了潜水员Vκ曲目，与野生型前B细胞的相似之处。这是一个健壮，快速，易于操纵，可诱导的系统，可批判地评估V基因种系转录在V（d）J重排中的作用。这将清楚地证明是否需要有效V（d）J重排的低到现代V基因种系转录。如果重排减少或消除，我们将评估生殖线转录是否影响表观遗传谱或核小体定位。我们还将确定种系转录在基因座收缩中的作用。我们提出，随着Vκ基因经历种系转录，它们与强κ启动子相同的转录工厂， 与Jκ基因相邻，因此V基因种系转录本身的作用将导致所转录区域的位点压实。为了检验这一假设，我们将 执行RNA聚合酶II芯片环，以确定是否直接远程 相互作用是pol II介导的，因此很可能发生在转录工厂中。我们提出，Ig基因座的3D结构在V基因使用中起重要作用。这里提出的研究将清楚地证明特定V基因上的种系转录是否在该V基因重排的频率中起作用。我们还将确定种系转录在V（d）J重组期间塑造Igκ基因座的远程循环结构中是否起着更全球的作用