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 imp...