B细胞发育过程中免疫球蛋白基因染色质构象调节研究

机体通过V(D)J重组产生高度多样的抗原受体来对抗外源病原体感染和自身细胞癌变，而异常重组会导致免疫力低下或自身免疫疾病易感。抗原受体基因的染色质空间组织对于抗原受体多样性至关重要，然而人们对于其空间组织方式和调节机制的认识还不完整。研究表明B细胞特异性转录因子Pax5通过调节免疫球蛋白重链基因（Igh）染色质构象来保证B细胞受体多样性。然而其它抗原受体基因染色质构象的发育特异性调节因子还不清楚。我们的预实验结果提示Pax5也调节轻链基因κ（Igκ）的染色质构象，同时发现重组酶Rag1可能在调节Igh和Igκ染色质构象方面存在重组酶活性不依赖的功能。为了验证这一假说，我们将采用染色质构象捕获碳拷贝（5C）技术结合表观遗传学技术研究Igh和Igκ染色质构象调节中Rag1和Pax5的新功能。本课题将有助于完整认识染色质空间组织对抗体多样性产生和等位排斥建立的作用，为免疫疾病研究提供理论基础。

Given their essential role in adaptive immunity, antigen receptor loci have been the focus of analysis for many years and are among a handful of the most well studied genes in the genome. However, we are still left with an incomplete picture of the structure of each locus and the impact of cis and trans acting factors on looping. Most of the early work focusing on understanding the mechanisms underlying locus contraction centered on the Igh locus, in part because the B cell specific transcription factor, Pax5 was the first factor to be identified as essential for altering locus conformation. In contrast to the Igh locus, no lineage and stage specific factors have been identified as essential for locus contraction of Igκ, Tcrb or Tcra. Our preliminary data showed that Pax5 play a critical role in mediating locus contraction of Igκ at the pro-B cell stage of development. Furthermore, we discovered a novel role for RAG1 in differentially altering the conformation of the Igκ and Igh loci. Here we aim to test the hypothesis that RAG1 and Pax5 mediated alterations in locus architecture impact the accessibility and rearrangement potential of Igh and Igκ in both a positive and negative manner. To determine whether RAG1- and Pax5-mediated alterations in locus conformation impact Igh and Igκ accessibility and transcriptional output we will integrate our 5C data with ChIP-seq, RNA-seq and ATAC-seq analyses. Together these studies will provide insight into the functional role of RAG1 and Pax5 in altering immunoglobulin loci conformation.