Molecular basis of γδ T lineage specification

项目摘要/摘要 尽管越来越多的人认识到γδT细胞在宿主防御和免疫病理中发挥的关键作用， 控制其发育和效应器功能的分子事件仍然知之甚少。我们的节目 寻求使用基因组范围的方法来填补这一知识空白，这些方法最终侧重于关键的监管节点。 在上一个资助周期中，我们提供了令人信服的证据，证明T细胞前体细胞对 αβ和γδT细胞的命运取决于T细胞受体信号强度的不同。这些信号 差异通过ID3的比例诱导来调节命运，从而导致E功能的渐进性抑制 盒DNA结合蛋白(E蛋白)。用全基因组方法定义E蛋白-DNA的变化 结合，我们产生了一些新的洞察力来控制γδT谱系规范，这可以 主题1)E蛋白特异性-E蛋白家族成员表现出不同的反应 不同强度/持续时间的TCR信号指定命运并在支持γδT细胞中发挥不同的作用 发展(Proj1/3/4)；主题2)非编码转录-E蛋白结合广泛相关 非编码RNA(LncRNA)，如ThymoD(所有项目)，重要的是，项目4开发了新的成像 研究lncRNA功能的方法，通过实时可视化lncRNA对染色体环的促进 活细胞；和主题3)人类γδ开发-项目3开发了一种新的人类多能干细胞 (PSC)用于研究人类γδT细胞发育的系统，揭示了对HEB的要求。在我们的 续期申请，我们将通过整合四个项目的互补技能来探索这些主题 领袖们。Zúñiga-Pflücker实验室将与米歇尔·安德森一起专注于E蛋白的具体作用 家庭成员，以及他们对身份中介的压抑的反应，在协调 小鼠(主题1)和人类(主题3)分化模型。他们的观察将为 为了了解E蛋白家族成员控制ThymoD表达的分子基础，a LncRNA(主题2)，它策划了αβ血统承诺的开始和αβ命运的最终丧失 γδ血统承诺的潜力。Zúñiga-Pflücker和Murre实验室的努力将使 Wiest实验室(与Dietmar Kappes)了解E蛋白家族成员和非编码转录的作用 (主题2)通过Tcf7和Zbtb7b附近的E蛋白结合位点确定IL-17产生效应器的命运 精神错乱。最后，所有项目都将使庄氏努力了解E蛋白家族成员(主题1)和 非编码转录(主题2)控制定型γδTCR复合体的产生和功能 来驱动NKγδT细胞的发育。所有项目将继续依靠核心B的基因组专业知识来评估 破坏E蛋白结合位点对基因组结构的影响，然后将这些变化联系起来 为细胞命运干杯。这四个项目的共同努力有望为E蛋白的作用提供更大的洞察力 在控制γδ的发展和功能方面，这对人类的健康和疾病至关重要。

PROJECT SUMMARY/ABSTRACT Despite the growing appreciation for the critical role played by γδ T cells in host defense and immunopathology, the molecular events controlling their development and effector function remain poorly understood. Our program seeks to fill this gap in knowledge using genome-wide approaches that ultimately focus on key regulatory nodes. During the last funding cycle, we provided compelling evidence that the commitment of T cell progenitors to the αβ and γδ T cell fates depends on differences in T cell receptor (TCR) signal strength. These signaling differences regulate fate by proportional induction of Id3, which causes graded repression of the function of E box DNA binding proteins (E proteins). Using genome-wide approaches to define changes in E protein-DNA binding, we generated a number of novel insights into the control of γδ T lineage specification, which can be distilled into 3 themes: Theme 1) E protein specificity – E protein family members exhibit distinct responses to the TCR signals of differing intensity/duration that specify fate and play distinct roles in supporting γδ T cell development (Proj1/3/4); Theme 2) Non-coding transcription - E protein binding is extensively associated long non-coding RNAs (lncRNA), such as ThymoD (all Projects), Importantly, Project 4 has developed novel imaging approaches to study lncRNA function, by visualizing lncRNA promotion of chromosome looping in real time in live cells; and Theme 3) Human γδ development - Project 3 developed a novel human pluripotent stem cell (PSC) based system for studying human γδ T cell development, which revealed a requirement for HEB. In our renewal application, we will explore these themes by integrating the complementary skills of the four project leaders. The Zúñiga-Pflücker lab, together with Michele Anderson, will focus on the specific roles of E protein family members, and their responsiveness to Id-mediated repression, in orchestrating lineage commitment in mouse (theme 1) and human (theme 3) differentiation models. Their observations will inform the efforts of the Murre lab to understand the molecular basis by which E protein family members control ThymoD expression, a lncRNA (theme 2), which orchestrates both the onset of αβ lineage commitment and the ultimate loss of αβ fate potential upon γδ lineage commitment. The efforts of both the Zúñiga-Pflücker and Murre labs will enable the Wiest lab (with Dietmar Kappes) to understand the role of E protein family members and non-coding transcription (theme 2) in specifying the IL-17 producing effector fate through E protein binding sites near the Tcf7 and Zbtb7b loci. Finally, all Projects will inform efforts of Zhuang to understand how E protein family members (theme 1) and non-coding transcription (theme 2) control the generation and function of the stereotyped γδ TCR complexes that drive NKγδT cell development. All projects will continue to rely on the genomic expertise of Core B to assess the implications of destroying E protein binding sites on the genomic architecture, and then link those changes to cell fate. The collective efforts of these four projects promise to provide great insight into the role of E proteins in controlling both γδ development and function, which is of critical importance to human health and disease.