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X连锁免疫缺陷，具有广泛的临床严重性，由WASP基因突变引起，导致WAS蛋白（WASp）表达完全丧失或突变的WASp表达。WASp是蛋白质EVH-1结构域家族的成员，仅在造血系统的细胞中表达，并且是细胞肌动蛋白聚合的已知调节剂。然而，WASp对肌动蛋白细胞骨架和Th功能障碍的影响的关系仍不清楚，因为在某些情况下，尽管肌动蛋白聚合正常，但WAS中的淋巴细胞缺陷仍会发生。我们的数据使我们推测WASp的一个新作用-作为一种衔接蛋白，将辅助性T细胞-1细胞特异性信号分子聚集在一起，并将它们分配到细胞核，在那里它们成为Th 1特异性转录复合物的一部分。经典Th 1信号通路的正常激活发生在Th：DC相互作用期间，导致下游STAT 1和T-bet磷酸化，以及随后的IFN-β转录。同时，T-bet的ITK依赖性磷酸化是Th 2转录因子加塔-3失活所必需的。Notch信号通路的看似不同的激活对于Th 1和Th 2分化也是重要的。在这个建议中，我们将测试的假设，WASp是一个核衔接蛋白的正确组装的Th 1特异性转录复合物的成员组成的TCR和Notch依赖的途径的Th 1活化（NICD，STAT 1，和T-bet），和编排的Th 1分化的关键。我们进一步证实，WAS临床表型的严重程度反映了WASp突变破坏这些相互作用所引起的Th功能障碍的程度。在目的1中，我们将测试WASp是否需要在核大分子转录复合物的形成中，包含T-bet，NICD，STAT 1和RNA Pol II，并影响Tbx 21基因在Th 1极化条件下的转录。在目标2中，我们将测试致病WASp错义突变是否扰乱了这种Th 1-核转录复合物的形成和功能。最后，在目标3中，我们将使用T细胞中体内WASp-DNA相互作用的全基因组作图来确定WASp结合的T细胞特异性基因。这些研究将为WASp在产生适应性免疫中的功能提供新的见解，并可能揭示WAS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（WAS）是一种遗传性全身免疫缺陷，由一种称为WASP的蛋白质缺陷引起，临床表现为严重的复发性感染、容易瘀伤和出血、自身免疫和癌症。本提案中的研究将在分子水平上检查WASP的功能，以确定WAS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，针对这种危及生命的儿童衰弱疾病。

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.