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患者患有多器官自身免疫性疾病，对其的研究为更普遍的免疫耐受和自身免疫性疾病提供了重要见解。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。小鼠Aire基因位点的突变也会导致多器官自身免疫。对该模型的研究表明，Aire主要通过诱导大量外周组织抗原（PTAs）的表达，在罕见的胸腺基质细胞群体中起作用。因此，当分化的T细胞通过胸腺渗透时，那些能够对这些pta作出反应的T细胞避免了克隆缺失；当它们进入外周并遇到同源抗原时，就会产生自身免疫。Aire的分子机制仍然是个谜。它控制基因表达，但似乎不像传统的转录因子那样起作用。相反，它似乎在其活动中高度合作，参与包含多种功能蛋白质的大型多蛋白复合物，参与核运输，染色质结合/结构，转录调节或mrna前加工。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），通常通过非同源末端连接与DNA双链断裂（DSBs）的修复有关，但最近也涉及转录延伸的控制。本项目的目标是阐明Aire和DNA-PK如何相互作用以促进罕见胸腺基质细胞中大量但有选择性的基因组部分的转录。拟开展的研究旨在：1)结合序列亲和纯化、候选伴侣共沉淀、细胞shRNA敲低和基因敲除小鼠，从结构和功能上定义含[Aire/DNA-PK]复合物（es）。2)确定Aire是否促进DNA dsb的生成/稳定性，根据我们的初步研究假设，它的作用类似于抗癌药物依托oposide，在引入DNA拓扑异构酶-2 （topoisomerase-2）后抑制DNA切割，以缓解转录相关的扭转应力。3)确定Aire是否通过组蛋白“逐出”机制影响转录，验证假设，即随着转录机制沿着DNA进行，它促进了负责核小体拆卸和重组的复合体（包括DNA- pk， PARP1, TOP2， FACT, H2AX）的招募和/或有效性。这些研究应该对Aire控制免疫耐受的分子机制产生新的见解。除了APECED，这种T细胞耐受模式被认为在常见的自身免疫性疾病中发挥重要作用，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空气缺陷小鼠的自身免疫性疾病，将证明这种耐受途径可以靶向治疗。

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.