Role of nuclear factor TOX in lymphocyte development

项目摘要/摘要 直到最近，先天免疫效应器功能的多样性才变得明显， 多个新的天然淋巴样细胞亚型的发现，超出了人们长期以来认识的自然 杀手细胞。ILC来源于骨髓中的共同淋巴祖细胞(CLP)，以及许多 参与ILC发育的转录调节因子也在T细胞的发育中发挥作用 在胸腺里。其中包括TOX(胸腺细胞选择相关的HMG-box蛋白)，一种核DNA 结合蛋白，是HMG-box蛋白超家族的成员。 我们之前的工作表明，TOX是胸腺中CD4T细胞谱系发育所必需的， 并适用于早期ILC世系规范。使用TOX报告品系的小鼠，我们现在已经识别出骨骼 骨髓祖细胞可能处于从CLP到共同祖细胞再到所有辅助性先天细胞的过渡阶段 淋巴样细胞(CHILP)。CHILP高度表达转录调控因子Id2，但我们已经鉴定 TOX+Id2lo可能是前体细胞，提示TOX的上调可能先于 奇尔普。我们建议在体外和体内测试这些细胞的细胞命运潜力，以确定什么是ILC 如果他们失去了T细胞潜能，他们可以形成血统。同样，我们会问，ILC的血统是否 规范甚至先于毒理上调。有了这些知识，我们建议确定 在这些细胞类型中的转录组，在单细胞水平上，并确定TOX对基因的影响 表情。Notch信号也被认为在早期ILC谱系规范中起作用。我们建议 短暂的Notch信号是区分ILC和T细胞谱系承诺的关键。使用体外培养技术 允许将CLP分化为多种ILC亚型的模型系统，我们将研究细胞命运和 时间受限的Notch信号的转录后果。作为TCF7(编码TCF-1)，一个键凹槽 信号靶基因，可能在TOX下游，我们问Tcf7的表达是否能补偿Tcf7的丢失 毒理。最后，我们用体外和体内的方法研究了TOX的潜在作用机制。 问TOX是否作为新的RSF ISWI染色质重塑复合体的一部分发挥功能，并提出 建立一种新的小鼠品系以辅助鉴定TOX的基因靶点和蛋白结合伙伴 多个单元格上下文。

Project Summary/Abstract Only recently has the extent of the diversity of innate immune effector functions become apparent, with the discovery of multiple novel subtypes of innate lymphoid cells (ILCs), beyond long recognized natural killer cells. ILCs derive from common lymphoid progenitors (CLP) in the bone marrow, and a number of transcriptional regulators that are involved in ILC development also play roles in the development of T cells in the thymus. Among these is TOX (thymocyte-selection associated HMG-box protein), a nuclear DNA binding protein and member of the HMG-box superfamily of proteins. Our previous work has shown that TOX is required for CD4 T cell lineage development in the thymus, and for early ILC lineage specification. Using a TOX reporter strain of mouse we have now identified bone marrow progenitor cells that may be in transition from CLP to the common progenitor to all helper-like innate lymphoid cells (CHILP). CHILP highly express the transcriptional regulator Id2, but we have identified Tox+Id2lo putative precursor cells, suggesting that upregulation of TOX likely precedes the formation of CHILP. We propose to test the cell fate potential of these cells in vitro and in vivo, to determine what ILC lineages they can form and if they have lost T cell potential. Similarly, we will ask if ILC lineage specification even precedes Tox upregulation. Armed with this knowledge, we then propose to determine the transcriptome in these cell types, at the single cell level, and to determine the influence of TOX on gene expression. Notch signaling is also thought to play a role in early ILC lineage specification. We propose that transient Notch signaling is key to distinguish ILC from T cell lineage commitment. Using an in vitro model system that allows differentiation of CLP to multiple ILC subtypes, we will study the cell fate and transcriptional consequences of a temporally limited Notch signal. As Tcf7 (encoding TCF-1), a key Notch signaling target gene, is likely downstream of TOX, we ask if expression of Tcf7 can compensate for loss of TOX. Finally, we address the underlying mechanism of action of TOX, using in vitro and in vivo approaches to ask if TOX functions as part of a novel RSF ISWI chromatin-remodeling complex, and propose to produce a novel mouse strain to aid in identification of gene targets and protein binding partners of TOX in multiple cell contexts.