PROJECT SUMMARY Group 1, 2, and 3 innate lymphoid cells (ILC1, ILC2, and ILC3) are immune effector cells that contribute to tissue homeostasis and host defense against nearly all classes of pathogens, but their dysregulation also play key roles in prevalent diseases such as cancer, obesity, asthma, and colitis. The transcription factor (TF) networks that control the development and functions of the different groups of ILC have recently been identified. Yet how the chromatin accessibility landscape and the 3-dimensional (3D) genome architecture determine the development, homeostasis, and effector functions of ILC is largely unknown. Thus, the overarching goal of this proposal is to uncover how the 3D genomic and epigenetic architecture regulate the development of each ILC subset and to the development of allergic airway inflammation. It is now well-stablished that the transcriptional repressor Id2 determines the commitment and identity of the ILC lineage. As such, Id2 expression is now considered a hallmark of all ILC subsets in mice and humans. Our preliminary data indicates that Id2 expression is controlled in ILC1, but not ILC2 or ILC3, by specific long-range DNA interacting loops between specific distal cis-regulatory elements (cis-RE) and the Id2 promoter. Moreover, we showed that ablation of these promoter- cis-RE interactions in mice leads to a dramatic reduction in ILC1 in multiple tissues, while the development and functions of ILC2 and ILC3 were unaltered. Thus, our findings indicate for the first time that Id2 expression is regulated by long-range DNA interacting loops between the Id2 promoter and distal cis-RE in an ILC-subset specific manner. Moreover, it indicates that ablating these cis-RE is a powerful strategy to generate genetic tools to study the roles of each ILC subset in the context of an otherwise intact immune system. Yet how the chromatin accessibility landscape and the 3D genomic architecture determines Id2 expression specifically in ILC2 and ILC3 remains unknown. Thus, in aims 1 and 2 of this project, we will use novel genetic tools that we generated, single cell sequencing technologies, and HiC to elucidate how chromatin folding and accessibility determine the development and functions of ILC2 and ILC3 through the regulation of Id2 expression. In aim 3, we will exploit the specificity of these regulatory mechanisms to study the functions of ILC2 during allergic airway inflammation in the context of an otherwise intact immune system. Collectively, these studies will answer the long-standing question of how Id2 expression is controlled to drive the ILC fate. Moreover, it will generate an atlas of the 3D genomic landscape of each ILC subset, which that will allow us to identify unknown non-coding regulatory regions are critical for the function and development ILC1, ILC2, and ILC3.Importantly, through the identification of specific regulatory mechanisms in each ILC subset, we have created novel mouse genetic tools to study...