Molecular functions of Tcf-1 in DP thymocytes

项目摘要 胸腺发育对形成健康和平衡的T细胞免疫力具有重要作用。像其他 它涉及线性和三维染色质的协调变化 允许阶段特定转录事件的组织。几乎在T细胞的每个阶段都有一个中央调节器 在分化中起作用的是DNA结合蛋白Tcf-1。这里提出的研究将阐明Tcf-1如何协调 表观遗传和转录调节因子指导CD 4 + CD 8 + DP胸腺细胞分化的作用。Tcf- 1通过与其保守的DNA结合直接调节染色质景观和转录谱 序列，或与其他调节蛋白间接相关。DP胸腺细胞表达两种形式的Tcf- 1，结合β-连环蛋白的全长Tcf-1 p45蛋白，和不结合β-连环蛋白的短Tcf-1 p38同种型。1、另一个 也在胸腺细胞中表达的Tcf/Lef调节因子家族成员与Tcf-1具有重叠功能。 此外，Tcf-1在DP胸腺细胞阶段与HLH结构域DNA结合蛋白HEB合作 通过在全基因组范围内共享约7000个DNA结合位点。Tcf-1和HEB两者在细胞中的存在， 共享位点对于促进染色质可接近性和调节基因转录是必要的。直接Tcf-1- HEB与T细胞分化基因增强子区域中的保守基序结合促进T细胞分化基因的表达。 表情相比之下，Tcf-1-HEB募集到缺乏保守基序的位点，在细胞的启动子区域， 周期基因降低它们的表达和细胞增殖。这种相反的转录结果可能涉及 Tcf-1-HEB在不同调控复合物中向DNA的募集配合物的组成 包含Tcf-1和HEB，每个复合物中涉及的特定Tcf-1同种型，以及 Tcf-1和Lef-1之间的冗余性仍有待阐明。之间复杂的功能合作 这些因素可能还涉及Tcf-1和Lef-1在其结合位点使DNA螺旋弯曲的能力， 可以调节3D染色质构象，并定义协同因子之间的接近度， 监管要素。这些发现和现有文献为以下假设提供了强有力的前提， DP胸腺细胞的发育是通过Tcf-1亚型与蛋白复合物的合作实现的， 3D染色质结构差异调节基因表达。提出了两个具体目标。要求1 将确定Tcf-1 p45和Tcf-1 p33如何在不同的调控背景下与Lef-1和HEB合作。 复合物，以建立DP胸腺细胞的表观遗传和转录谱，并控制其 发展进程目的2：阐明Tcf-1 p45、Tcf-1 p33、Lef-1在HEB形成中的作用。 染色质构象，以及这种功能如何促进DP胸腺细胞发育。拟议的研究 有望突出DP胸腺细胞分子调控的全新层面。

PROJECT SUMMARY Thymic development is highly responsible for shaping a healthy and balanced T cell immunity. Like other developmental processes it involves coordinated changes in the linear and three-dimensional chromatin organization that allow stage specific transcription events. A central regulator at nearly every stage of T cell differentiation is the DNA binding protein Tcf-1. Studies proposed here will elucidate how Tcf-1 coordinates the action of epigenetic and transcription regulators to instruct the differentiation of CD4+CD8+ DP thymocytes. Tcf- 1 modulates the chromatin landscapes and transcription profiles directly through binding to its conserved DNA sequence, or indirectly in association with other regulatory proteins. DP thymocytes express two forms of Tcf- 1, the full length Tcf-1p45 protein that binds β-catenin, and a short Tcf-1p38 isoform that does not. Lef-1, another member of the Tcf/Lef family of regulators, also expressed in thymocytes has overlapping functions with Tcf-1. In addition Tcf-1 cooperates with the HLH domain DNA binding protein HEB at the DP thymocyte stages through the sharing of ~7000 DNA binding sites genome wide. The presence of both Tcf-1 and HEB at the shared sites is necessary to promote chromatin accessibility and regulate gene transcription. The direct Tcf-1- HEB binding to their conserved motifs in enhancer regions of T cell differentiation genes promotes their expression. By contrast Tcf-1-HEB recruitment to sites lacking conserved motifs, in promoter regions of cell- cycle genes reduces their expression and cell proliferation. Such opposing transcription outcomes likely involve Tcf-1-HEB recruitment to DNA in the context of distinct regulatory complexes. The composition of complexes containing Tcf-1 and HEB, the specific Tcf-1 isoform involved in each complex, and which functions are redundant between Tcf-1 and Lef-1, still remain to be elucidated. The intricate functional co-operation between these factors likely also involves the ability of Tcf-1 and Lef-1 to bend the DNA helix at their binding site which may modulate the 3D chromatin conformation, and define the proximity between co-operating factors and regulatory elements. These findings and the existing literature provide strong premise for the hypothesis that DP thymocyte development is enabled by the cooperation of Tcf-1 isoforms with protein complexes that shape the 3D chromatin structure to differentially regulate gene expression. Two specific aims are proposed. Aim 1 will determine the how Tcf-1p45 and, Tcf-1p33, co-operate with Lef-1 and HEB in the context of distinct regulatory complexes to establish the epigenetic and transcription profile of DP thymocytes and control their developmental progression. Aim 2: will elucidate the roles of Tcf-1p45, Tcf-1p33, Lef-1 with HEB shaping the chromatin conformation, and how this function promotes DP thymocyte development. The proposed studies are expected to highlight a completely new layer in the molecular regulation of DP thymocytes.