Single cell visualization of the V(D)J recombinase complex

不同的抗原受体库是在淋巴细胞增殖过程中启动的。 通过V（D）J重组的发展。这种遗传机制重新排列生殖细胞 抗原受体基因座通过连接组成基因片段，共同编码 受体的抗原特异性。最初的位点特异性DNA切割步骤是 由重组激活基因蛋白RAG 1和RAG 2催化。一起 RAG蛋白在DNA内序列特异性位点引入DNA双链断裂， 抗原受体位点。随后，非同源末端连接因子加入到 合适的DNA末端在一起产生功能性抗原受体基因。而 RAG蛋白可以在脱靶位点错误地切割，它们的活性通常是 仅限于抗原受体位点。然而，RAG蛋白也已知结合至 修饰的组蛋白在开放的染色质位点富集，将它们置于 非常接近许多非抗原受体DNA位点。这些违反直觉的 研究结果强调，关于RAG蛋白如何在 在发育中的淋巴细胞中受到调节。了解RAG蛋白的调节将 通过对RAG蛋白-染色质相互作用的单细胞分析来促进， 相对于它们在特定DNA识别位点的相互作用， 抗原受体位点。然而，用于可视化单个复合物的工具 结合RAG的组蛋白，特别是结合RAG蛋白的特异性DNA位点， 缺乏我们在这个项目中的目标是开发新的方法来测量RAG染色质 细胞核中的相互作用，并在特定的基因组中可视化局部RAG相互作用 单细胞中的DNA位点。这些方法的开发和优化将是 在解决与V（D）J重组的调节相关的问题方面很重要， 这个过程中的缺陷如何导致疾病。

The diverse repertoire of antigen receptors is initiated during lymphocyte development by V(D)J recombination. This genetic mechanism rearranges germline antigen receptor loci by joining component gene segments, which together encode for the antigen specificity of the receptor. The initial site-specific DNA cleavage steps are catalyzed by the recombination activating gene proteins, RAG1 and RAG2. Together the RAG proteins introduce DNA double strand breaks at sequence-specific sites within the antigen receptor loci. Subsequently, non-homologous end joining factors join the appropriate DNA ends together to yield functional antigen receptor genes. While the RAG proteins can erroneously cleave at off-target sites, their activity is typically restricted to the antigen receptor loci. Yet, the RAG proteins are also known to bind to modified histone proteins that are enriched at open chromatin sites, placing them in close proximity to numerous non-antigen receptor DNA sites. These counterintuitive results highlight that much is still unknown regarding how the RAG proteins are regulated in developing lymphocytes. Understanding regulation of the RAG proteins will be facilitated by single cell analysis of RAG protein-chromatin interactions that occur throughout the nucleus relative to their interactions at specific DNA recognition sites in the antigen receptor loci. However, the tools to visualize single complexes containing RAG-bound histones and, in particular, specific DNA sites with bound RAG proteins are lacking. Our goal in this project is to develop new methods to measure RAG-chromatin interactions in cell nuclei and to visualize localized RAG interactions at specific genomic DNA sites in single cells. Development and optimization of these methods will be important in addressing questions related to the regulation of V(D)J recombination, and how defects in this process can lead to disease.