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

描述（由申请人提供）：Wiskott-Aldrich综合征是一种由WASP基因突变引起的广泛临床严重程度的x连锁免疫缺陷，导致WAS蛋白（WASP）的完全表达丧失或突变WASP的表达。WASp是EVH-1结构域家族蛋白的一员，仅在造血系统细胞中表达，是细胞肌动蛋白聚合的调节因子。然而，WASp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，尽管肌动蛋白正常聚合，但WAS的淋巴细胞缺陷仍然发生。我们的数据使我们推测WASp的一种新作用——作为一种接头蛋白，将T - helper-1细胞特异性信号分子聚集在一起，并将它们分配到细胞核中，在那里它们成为th1特异性转录复合物的一部分。经典Th1信号通路的正常激活发生在Th:DC相互作用期间，导致下游STAT1和T-bet的磷酸化，以及随之而来的IFN-?的转录。同样，itk依赖性的T-bet磷酸化是Th2转录因子GATA-3失活所必需的。Notch信号通路的看似不同的激活对于Th1和Th2分化也很重要。在本研究中，我们将验证这样一个假设，即WASp是一种核接头蛋白，对于正确组装Th1特异性转录复合体至关重要，该转录复合体由Th1激活的TCR和notch依赖途径（NICD、STAT1和T-bet）的成员组成，并协调Th1分化。我们进一步假设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带来的Th功能障碍的程度。在Aim 1中，我们将测试含有T-bet、NICD、STAT1和RNA Pol II的核大分子转录复合体的形成是否需要WASp，并在th1极化条件下影响Tbx21基因的转录。在Aim 2中，我们将测试致病的WASp错义突变是否会干扰这种th1 -核转录复合物的形成和功能。最后，在Aim 3中，我们将使用体内T细胞中WASp- dna相互作用的全基因组图谱来确定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）是一种由WASP蛋白缺陷引起的遗传性全身免疫缺陷，临床表现为严重复发性感染、易瘀伤和出血、自身免疫和癌症。本课题的研究将从分子水平考察WASP的功能，以确定WAS的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，以治疗这种危及生命、使人衰弱的儿童疾病。

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.