THE V(D)J RECOMBINATION REACTION AND ITS IMPACT ON LYMPHOCYTE DEVELOPMENT

描述(由申请人提供)：淋巴细胞的发育受多种细胞信号的调节，其中许多信号来自细胞表面受体。所有发育中的淋巴细胞必须组装编码不同链的B细胞受体(BCR)或T细胞受体(TCR)的基因，并表达一种功能性的、非自身反应的抗原受体，以离开胸腺(T细胞)或骨髓(B细胞)进入外周。抗原受体基因的组装是通过V(D)J重组过程进行的，当RAG蛋白在抗原受体基因的V、D和J基因片段上引入DNA双链断裂(DSB)时，V(D)J重组就开始了。这些DSB被加工并连接到DSB修复的NHEJ途径的蛋白上，附加V、D和J基因片段，完成抗原受体基因的第二外显子。遗传毒性物质(如电离辐射)产生的双链断裂激活细胞DNA损伤反应，包括修复途径和促进细胞周期停滞和细胞死亡的途径，如果双链断裂持续未修复的话。目前尚不清楚生理性DNA断裂(如RAG DSB)是否也激活了广泛的DNA损伤反应，或者是否阻止了它们这样做，除非它们持续未修复，从而对细胞构成危险。在这里，我们证明了在发育中的淋巴细胞中，RAG DSB激活ATM，ATM是DNA损伤反应信号的主要转导者，这通过参与包括STAT3、Foxo1和NF：B在内的几个转录途径来促进广泛的遗传程序。然而，令人惊讶的是，很大一部分在DNA损伤反应中没有已知的功能。相反，这些基因编码的蛋白质在正常的淋巴细胞发育过程中发挥重要作用。由此，我们假设RAG DSB提供了调节发育过程的信号，这些信号至少对组装抗原受体基因的淋巴细胞的发育是重要的。在这里，我们将检验这一假说，阐明RAG DSB激活发育中淋巴细胞转录途径的机制，并确定这些途径的功能如何与其他淋巴细胞发育信号整合。

DESCRIPTION (provided by applicant): Lymphocyte development is regulated by a variety of cellular cues, many of which emanate from cell surface receptors. All developing lymphocytes must assemble genes encoding the different chains of the B cell receptor (BCR), or T cell receptor (TCR) and express a functional, non-auto-reactive, antigen receptor to exit the thymus (T cells) or bone marrow (B cells) and enter the periphery. Antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG proteins introduce DNA double strand breaks (DSBs) at V, D and J gene segments in antigen receptor loci. These DSBs are processed and joined by the proteins of the NHEJ pathway of DSB repair, appending the V, D and J gene segments to complete the second exon of antigen receptor genes. DSBs generated by genotoxic agents (such as ionizing radiation) activate cellular DNA damage responses including repair pathways and pathways that promote cell cycle arrest and cell death if the DSBs persist unrepaired. It has been unclear whether physiologic DNA breaks such as RAG DSBs also activate broad DNA damage responses or whether they are prevented from doing so unless they persist un-repaired, thus posing a hazard to the cell. Here, we show that in developing lymphocytes, RAG DSBs activate Atm, a kinase that is a major transducer of DNA damage response signals, and that this promotes a broad genetic program by engaging several transcriptional pathways including STAT3, Foxo1 and NF:B. Several of the genes regulated by RAG DSBs encode proteins that function in canonical DNA damage responses. Surprisingly however, a large fraction has no known function in DNA damage responses. Rather, the genes encode proteins that function in processes that are important for normal lymphocyte development. From this, we hypothesize that RAG DSBs provide signals that regulate developmental processes that are at a minimum, important for developing lymphocytes assembling antigen receptor genes. Here, we will test this hypothesis, elucidate the mechanisms by which RAG DSBs activate transcriptional pathways in developing lymphocytes and determine how the function of these pathways is integrated with other lymphocyte developmental signals.