THE ROLE OF NUCLEAR WASP IN THE TRANSCRIPTIONAL REGULATION OF TH1 DIFFERENTIATION

描述(由申请人提供)：Wiskott-Aldrich综合征是一种临床严重的X连锁免疫缺陷，由WASP基因突变引起，导致Was蛋白(黄蜂)完全丧失表达或突变的黄蜂表达。WASP是EVH-1结构域蛋白家族中的一员，仅在造血系统的细胞中表达，是细胞肌动蛋白聚合的已知调节因子。然而，黄蜂对肌动蛋白细胞骨架的影响与Th功能障碍的关系尚不清楚，因为在某些情况下，尽管肌动蛋白聚合正常，但仍会发生淋巴细胞缺陷。我们的数据让我们推测黄蜂的一个新角色--作为一种适配器蛋白，将T helper-1细胞特异性信号分子聚集在一起，并将它们分割到细胞核，在那里它们成为Th1特异性转录复合体的一部分。经典的Th1信号通路在Th：DC相互作用过程中被正常激活，导致STAT1和T-bet下游的磷酸化，从而导致干扰素-β的转录。同时，依赖ITK的T-bet的磷酸化是Th2转录因子GATA-3失活所必需的。Notch信号通路似乎不同的激活对Th1和Th2的分化也很重要。在这项提案中，我们将检验黄蜂是一种核适配子蛋白的假设，该假说对于正确组装Th1特异性转录复合体至关重要，该复合体由依赖TCR和Notch的Th1激活途径(NICD、STAT1和T-bet)的成员组成，并协调Th1的分化。我们进一步假设，AS临床表型的严重程度反映了破坏这些相互作用的黄蜂突变所带来的Th功能障碍的程度。在目标1中，我们将测试在包含T-bet、NICD、STAT1和RNA Pol II的核大分子转录复合体的形成中是否需要黄蜂，以及在Th1极化条件下对Tbx21基因转录的影响。在目标2中，我们将测试致病黄蜂错义突变是否扰乱了这种Th1-核转录复合体的形成和功能。最后，在目标3中，我们将使用活体T细胞中黄蜂-DNA相互作用的全基因组图谱来确定黄蜂结合的T细胞特异性基因。这些研究应该会为黄蜂在适应性免疫生成中的作用提供新的见解，并可能揭示出过去和其他免疫缺陷的新治疗靶点。公共卫生相关性：Wiskott-Aldrich综合征是一种遗传性全身性免疫缺陷，由一种名为WASP的蛋白质缺陷引起，临床表现为严重反复感染、易擦伤和出血、自身免疫和癌症。这项研究将在分子水平上研究WASP的功能，以确定WASP上述临床并发症的原因。这些研究应该为未来设计新的治疗方法奠定基础，以治疗这种威胁生命、使人虚弱的童年疾病。

DESCRIPTION (provided by applicant): Wiskott-Aldrich syndrome is an X-linked immune deficiency of broad clinical severity arising from mutations in the WASP gene, which results in either a total loss of expression of WAS protein (WASp) or expression of a mutated WASp. WASp, a member of the EVH-1 domain family of proteins, is expressed exclusively in the cells of the hematopoeitic system, and is a known regulator of cellular actin polymerization. The relationship of WASp's effect on actin cytoskeleton and Th dysfunction, however, remains unclear, as in some instances lymphocyte defects in WAS occur despite normal actin polymerization. Our data leads us to speculate on a novel role for WASp - as an adaptor protein bringing together T helper-1 cell-specific signaling molecules and partitioning them to the nucleus where they become part of a Th1-specific transcription complex. Normal activation of the classical Th1 signaling pathway occurs during the Th:DC interaction, resulting in downstream phosphorylation of STAT1 and T-bet, and consequent transcription of IFN-?. In parallel, ITK-dependent phosphorylation of T-bet is required for deactivation of GATA-3, the Th2 transcription factor. A seemingly disparate activation of the Notch signaling pathway also is important for both Th1 and Th2 differentiation. In this proposal, we will test the hypothesis that WASp is a nuclear adaptor protein critical for the correct assembly of a Th1-specific transcriptional complex comprised of members of both the TCR- and Notch-dependent pathways of Th1 activation (NICD, STAT1, and T-bet), and orchestration of Th1 differentiation. We further posit that the severity of WAS clinical phenotype mirrors the degree of Th dysfunction imparted by WASp mutations that disrupt these interactions. In Aim 1, we will test whether WASp is required in the formation of the nuclear macromolecular transcription complex, containing T-bet, NICD, STAT1, and RNA Pol II, and effects transcription of Tbx21 gene under Th1-polarizing condition. In Aim 2, we will test whether the disease-causing WASp missense mutations perturb formation and function of this Th1-nuclear transcription complex. Finally, in Aim 3 we will determine the T cell- specific genes to which WASp binds using genome-wide mapping of in vivo WASp-DNA interactions in the T-cell. These studies should provide new insights into WASp function in the generation of adaptive immunity, and may reveal new therapeutic targets in WAS and other immune deficiencies. PUBLIC HEALTH RELEVANCE: Wiskott-Aldrich syndrome (WAS) is a genetically transmitted systemic immune deficiency resulting from a defect in a protein called WASP and manifesting clinically in severe recurrent infections, easy bruising and bleeding, autoimmunity, and cancers. The studies in this proposal will examine the function of WASP at the molecular level to determine the causes of the above clinical complications of WAS. These studies should lay the foundation for designing novel therapies in the future, for this life-threatening, debilitating disease of the childhood.