Dynamic regulation of interleukin-7 receptor alpha chain

描述（由申请人提供）：白细胞介素-7受体a链（IL-7Ra）是IL-7和胸腺基质淋巴生成素的细胞因子受体亚基。il - 7r1衍生信号在淋巴生成和T细胞应答中具有重要作用。此外，IL-7Ra的表达是动态调节的，以适应不同细胞类型的功能需求。尽管IL-7Ra在造血干细胞（hsc）上不表达，但它是造血干细胞衍生的共同淋巴样祖细胞（CLPs）的重要标记蛋白。当CLPs分化为前B细胞时，IL-7Ra的表达保持不变，但在骨髓的未成熟B细胞和外周血的成熟B细胞中被关闭。在胸腺细胞发育过程中，大多数未成熟的双阴性胸腺细胞表达IL-7Ra，而双阳性胸腺细胞不表达IL-7Ra。IL-7Ra的表达在CD4+和CD8+单核阳性胸腺细胞中再次上调，并在外周血初代T细胞中维持。当遇到外源抗原时，幼稚T细胞被激活并成为效应T细胞，同时IL-7Ra下调。抗原被清除后，一部分效应T细胞产生具有较高IL-7Ra表达的持久记忆T细胞。已知GA结合蛋白和PU.1可以激活IL-7Ra基因的转录，但IL-7Ra在分子水平上的动态表达变化是如何实现的尚不清楚。本课题旨在确定IL-7Ra基因位点的顺式调控模块。我们的长期目标是进一步确定促进IL-7Ra动态调控的关键顺式调控元件和相关因子。重要的是，在淋巴细胞发育和T细胞应答过程中，Notch应答基因Deltex 1、Wnt效应转录因子TCF1/LEF1等许多基因与IL-7Ra同步表达变化。IL-7Ra基因调控的精确分子机制可以外推到其他同步表达的基因，从而为造血干细胞向T和B谱系的分化以及幼稚T细胞向效应T细胞和记忆T细胞的转变提供见解。这一知识可能对免疫缺陷的治疗和疫苗/佐剂的设计具有重要意义。特定的目的。鉴定在T细胞和B细胞发育和T细胞对抗原刺激的反应过程中，IL-7Ra动态表达的顺式调控模块。我们将使用含有整个IL-7Ra位点的细菌人工染色体（BAC），通过在第一个IL-7Ra编码外显子中插入GFP报告基因来产生报告基因转基因小鼠。结合Cre重组酶介导的使用不相容Lox变体的随机重组，我们将获得含有系统缺失调控区域/模块的报告基因转基因小鼠。通过建立3个转基因奠基系，我们将把200 kb的调控区域缩小到1-2 kb的调控序列，这些序列可以在造血干细胞向CLPs和B系细胞分化、胸腺细胞发育和T细胞反应过程中上调或下调IL-7Ra。

DESCRIPTION (provided by applicant): Interleukin-7 receptor a chain (IL-7Ra) is a cytokine receptor subunit for IL-7 and thymic stromal lymphopoietin. IL-7R1-derived signal has important roles in lymphopoiesis and T cell responses. In addition, IL-7Ra expression is dynamically regulated to tune into functional requirements in different cell types. Whereas IL-7Ra is not expressed on hematopoietic stem cells (HSCs), it is an important marker protein for HSC-derived common lymphoid progenitors (CLPs). IL-7Ra expression is maintained while CLPs differentiate to pro-B cells, but is turned off in immature B cells in bone marrow and mature B cells in the periphery. During thymocyte development, the most immature double negative thymocytes express IL-7Ra, but double positive thymocytes do not. IL-7Ra expression is upregulated again in single positive CD4+ and CD8+ thymocytes and maintained on naive T cells in the periphery. Upon encountering foreign antigens, naive T cells are activated and become effector T cells accompanied by down-regulation of IL-7Ra. After the antigens are cleared, a portion of effector T cells give rise to long lasting memory T cells with higher expression of IL-7Ra. GA binding protein and PU.1 are known to activate the transcription of IL-7Ra gene, however, little is known about how the dynamic expression changes of IL-7Ra are achieved on a molecular level. This research project aims to identify cis regulatory modules in the IL-7Ra gene locus. Our long-term goal is to further define critical cis regulatory elements and associated factors that contribute to the dynamic regulation of IL-7Ra. Importantly, during lymphocyte development and T cell responses, many genes such as Notch response gene Deltex 1, Wnt effector transcription factors TCF1/LEF1 show synchronized gene expression changes in concert with IL-7Ra. The precise molecular mechanism underlying IL-7Ra gene regulation could be extrapolated to other genes with synchronized expression and thus provides insights into differentiation of HSCs to T and B lineages and transition of naive T cells to effector and memory T cells. This knowledge may have important implications in treatment of immunodeficiency and vaccine/adjuvant designs. SPECIFIC AIM. To identify cis regulatory modules that contribute to the dynamic expression of IL-7Ra during T and B cell development and T cell responses to antigen stimulation. We will use a bacterial artificial chromosome (BAC) containing the entire IL-7Ra locus to generate reporter transgenic mice by inserting a GFP reporter in the first IL-7Ra-coding exon. In combination with Cre recombinase-mediated stochastic recombination using incompatible Lox variants, we will obtain reporter transgenic mice containing systematic deletion of regulatory regions/modules. By establishing as few as 3 transgenic founder lines, we will narrow down a 200 kb regulatory region to 1-2 kb regulatory sequences which can confer the up- or down- regulation of IL-7Ra during differentiation of HSCs to CLPs and B lineage cells, thymocyte development, and T cell responses. PUBLIC HEALTH RELEVANCE: Interleukin-7 receptor a chain (IL-7Ra) is an important cell surface protein that shows dynamic changes accompanying differentiation of hematopoietic stem cells to pathogen combating T and B cells. IL-7Ra also plays critical roles in maintaining homeostasis of naive and memory T cells. This project will generate insights into how IL-7Ra expression changes are tuned into functional requirements in these cells. The knowledge will provide a scientific basis for manipulating outcomes of hematopoietic stem cell differentiation and immune responses, which is important for understanding T cell malignancy, designing gene-therapy of defective T cell development or function, and formulating more effective vaccines and adjuvants.