Regulation of V(D)J Recombination by Arginine Methylation

描述(申请人提供)：V(D)J重组是获得性免疫的标志。在T和B细胞中，一系列高度调控的体细胞基因重排事件从基因片段产生功能基因。这一过程导致功能性抗原受体的表达。免疫球蛋白(Ig)基因编码B细胞受体，而T细胞受体由TCR基因编码。这些基因组装的中心机制是可变区(V)的切割和连接、多样性(D，位于抗原受体基因的一个子集)以及通过V(D)J重组连接(J)片段。V(D)J重组酶复合体包含多种蛋白质，包括重组激活基因1和2(Rag1和Rag2)，当重组信号序列处于可接近状态时，它们识别和切割重组信号序列(RSS)。在这里，我们将确定蛋白质精氨酸甲基转移酶5(PRMT5)、甲基化精氨酸以及表观遗传‘橡皮擦’在这一过程中的作用。RSS进行重组的可及性在很大程度上受表观遗传机制的控制，包括DNA甲基化和组蛋白的翻译后修饰。在V(D)J重组中记录了这些机制中的每一种的重要性。然而，关于精氨酸甲基化的重要性以及在淋巴细胞发育过程中添加或去除精氨酸甲基的酶的特性，人们了解的要少得多。我们假设T细胞受体(TCRA)基因座的V(D)J重组受PRMT5的调控，PRMT5在组蛋白H3和H4的多个残基以及一些非组蛋白蛋白上对称地甲基化精氨酸。具有挑衅性的是，PRMT5转录本在发育中的胸腺细胞(和B细胞)中表达，但不在表达Rag1基因和进行V(D)J重组的阶段。这些观察结果表明，PRMT5的精氨酸甲基化抑制V(D)J重组。事实上，前T细胞系中PRMT5mRNA的缺失极大地增加了质膜上成熟的TCRmRNA的表达。我们预测，这一机制调节T和B细胞中的抗原受体组装。因此，我们将讨论精氨酸甲基化和PRMT5在体外进行有效V(D)J重组的T和B细胞系中的功能。我们的证据表明，精氨酸甲基化的丢失是一个涉及一个或多个表观遗传擦除器的活跃过程，这些表观遗传擦除器可能是Jumonji蛋白质二加氧酶家族的成员。我们将使用生化方法来鉴定淋巴细胞前体细胞中的候选精氨酸去甲基酶。我们的实验将共同解决新的表观遗传机制，这些机制在淋巴细胞发育过程中控制抗原受体组装，但在包括癌症在内的各种环境中也对基因调控很重要。

DESCRIPTION (provided by applicant): V(D)J recombination is the hallmark of adaptive immunity. In T and B cells, a series of highly regulated somatic gene rearrangement events produce functional genes from gene segments. This process results in the expression of functional antigen receptors. Immunoglobulin (Ig) genes encode B cell receptors, while T cell receptors are encoded by Tcr loci. The central mechanisms necessary for the assembly of these genes are cleavage and ligation of variable (V), diversity (D, at a subset of antigen receptor loci), and Joining (J) segments by V(D)J recombination. V(D)J recombinase complexes comprise multiple proteins including Recombination activating genes 1 and 2 (Rag1 and Rag2), which recognize and cleave recombination signal sequences (RSS) when RSS are in an 'accessible' state. Here, we will determine the roles of Protein Arginine Methyltransferase 5 (PRMT5), methylated arginine, and its removal by epigenetic 'erasers' in this process. Accessibility of RSS for recombination is largely controlled by epigenetic mechanisms including DNA methylation and post-translational modifications of histones. The importance of each of these mechanisms has been documented in V(D)J recombination. However, far less is understood concerning the importance of arginine methylation and the identities of enzymes that add or remove methyl groups from arginine during lymphocyte development. We hypothesize that V(D)J recombination of T cell receptor (Tcra) loci is regulated by PRMT5, which dimethylates arginine symmetrically at multiple residues of histones H3 and H4, as well as some non-histone proteins. Provocatively, Prmt5 transcripts are expressed in developing thymocytes (and B cells), but not at stages that express the Rag1 gene and feature ongoing V(D)J recombination. These observations suggest that arginine methylation by PRMT5 inhibits V(D)J recombination. Indeed, depletion of Prmt5 mRNA in a pre-T cell line greatly increases mature TCR� expression on the plasma membrane. We predict that this mechanism regulates antigen receptor assembly in both T and B cells. Therefore, we will address functions of arginine methylation and PRMT5 in T and B cell lines that undergo efficient V(D)J recombination in vitro. Our evidence suggests that the loss of arginine methylation is an active process involving one or more epigenetic erasers, which are likely members of the Jumonji family of protein dioxigenases. We will use biochemical methods to identify candidate arginine demethylases in lymphocyte progenitors. Together, our experiments will address novel epigenetic mechanisms that control antigen receptor assembly during lymphocyte development, but are also important for gene regulation in a wide variety of contexts including cancer.