The molecular mechanism of Aire: partnering with DNA-PK

描述（由申请人提供）：自身免疫性疾病困扰着7-10%的美国人，其发病率正在上升。APECED患者具有多器官自身免疫性疾病，对其的研究已经产生了对免疫耐受和更普遍的自身免疫性疾病的重要见解。APECED的突变基因编码Aire，这是一种具有转录调节因子特征的几个结构域的大蛋白质。编码小鼠Aire的基因座的突变也导致多器官自身免疫。对该模型的研究显示，Aire主要通过诱导大量外周组织抗原（PTA）的表达在罕见的胸腺基质细胞群中起作用。因此，当分化的T细胞渗透通过胸腺时，那些能够对这种PTA做出反应的T细胞避免了克隆缺失;当它们出现在外周并遇到同源抗原时，自身免疫增强。艾尔的分子机制仍然是个谜。它控制基因表达，但似乎不作为一个传统的转录因子。相反，它似乎在其活动中高度合作，参与大型多蛋白复合物，该复合物包含不同功能的蛋白质，参与核转运，染色质结合/结构，转录调控或前mRNA加工。我们的初步研究强调了一个意想不到的Aire伙伴：DNA依赖性蛋白激酶（DNA-PK），通常与通过非同源末端连接修复DNA双链断裂（DSB）有关，但最近也参与了转录延伸的控制。这个项目的目标是阐明Aire和DNA-PK如何相互作用，以促进一个大的，但选择，基因组的一部分，特别是在罕见的胸腺基质细胞的转录。拟议的研究旨在：1）在结构上和功能上定义含有[Aire/DNA-PK]的复合物，结合顺序亲和纯化、候选伴侣共沉淀、细胞中的shRNA敲除和基因敲除小鼠。2)确定Aire是否促进DNA DSB的产生/稳定性，追求假设（从我们的初步研究中产生），即它像抗癌药物依托泊苷一样抑制DNA拓扑异构酶-2在引入DNA切割以减轻与转录相关的扭转应力后解析DNA切割。3)确定Aire是否通过组蛋白“驱逐”机制影响转录，检验假设，即它促进了负责在转录机制沿着DNA进行时拆卸和重新组装核小体的复合物（包括DNA-PK、PARP 1、TOP2、FACT、H2 AX）的募集和/或有效性。这些研究将为Aire控制免疫耐受的分子机制提供新的见解。除了APECED，这种T细胞耐受化模式被认为在常见的自身免疫性疾病中起重要作用，特别是1型糖尿病和重症肌无力。用抗癌药物依托泊苷或相关药物成功干预Aire缺陷小鼠的自身免疫性疾病将代表原理证明，这种耐受途径可以靶向治疗益处。

DESCRIPTION (provided by applicant): Autoimmune diseases afflict 7-10% of Americans, and their incidence is rising. APECED patients have a multi-organ autoimmune disorder, the study of which has yielded important insights into immunological tolerance and autoimmune diseases more generally. The mutated gene underlying APECED encodes Aire, a large protein with several structural domains characteristic of a transcriptional regulator. Mutation of the locus encoding murine Aire also resulted in multi-organ autoimmunity. Studies on this model revealed Aire to operate primarily within a rare population of thymic stromal cells by inducing the expression of a large repertoire of peripheral-tissue antigens, or PTAs. Consequently, as differentiating T cells percolate through the thymus, those capable of responding to such PTAs avoid clonal deletion; when they emerge into the periphery and encounter cognate antigen, autoimmunity ensues. Aire's molecular mechanisms remain enigmatic. It controls gene expression but does not appear to act as a conventional transcription factor. Rather, it seems to be highly cooperative in its activities, participating in large multi-protein complexes that incorporate proteins of diverse function, involved in nuclear transport, chromatin binding/structure, transcriptional regulation or pre-mRNA processing. Our preliminary studies highlighted an unexpected Aire partner: DNA-dependent protein kinase (DNA-PK), usually associated with the repair of DNA double-stranded breaks (DSBs) via non-homologous end joining, but also recently implicated in the control of transcriptional elongation. The goal of this proposed project is to elucidate how Aire and DNA-PK interact to promote transcription of a large, but select, portion of the genome specifically in rare thymic stromal cells. Proposed studies aim to: 1) Structurally and functionally define the [Aire/DNA-PK]-containing complex(es), combining sequential affinity-purifications, candidate partner co-precipitations, shRNA knockdowns in cells, and gene-knockout mice. 2) Determine whether Aire promotes the generation/stability of DNA DSBs, pursuing the hypothesis (issuing from our preliminary studies) that it operates like the anti-cancer drug etoposide to inhibit DNA topoisomerase-2 from resolving DNA cuts after introducing them to relieve the torsional stress associated with transcription. 3) Determine whether Aire impacts on transcription via the histone "eviction" machinery, testing the hypothesis, that it promotes the recruitment and/or effectiveness of a complex (including DNA-PK, PARP1, TOP2, FACT, H2AX) responsible for disassembling and re-assembling nucleosomes as the transcriptional machinery progresses along DNA. These studies should yield new insights into the molecular mechanisms by which Aire controls immunological tolerance. Besides APECED, this mode of T cell tolerization is thought to play an important role in common autoimmune diseases, notably type-1 diabetes and myasthenia gravis. Successful intervention in the autoimmune disease of Aire-deficient mice with the cancer drug, etoposide, or allied drugs would represent proof-of-principle that this tolerance pathway can be targeted for therapeutic benefit.