Defining role of Long non-coding RNA (LncRNA) Gm15417 in iNKT development and subset differentiation

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ABSTRACT Recent reports have shown that iNKT cells are comprised of multiple distinct effector subsets with specialized immune roles. However, the molecular pathways that govern alternate iNKT subset specification remain poorly understood, hence limiting our capacity to utilize specialized iNKT cells for therapeutic purposes. Herein we report the identification of a novel Long noncoding (Lnc) RNA, Gm15417, that mediates physiological regulation of iNKT development and functional subset differentiation in the thymus. This represents a breakthrough in the field, since iNKT subset specification is poorly understood, and in particular no role of lncRNAs in iNKT cell development has so far been reported. In this application we seek to elucidate the cellular and molecular basis by which Gm15417 controls iNKT cell development according to two specific aims: Aim 1: Defining stage-specific and TCR-dependent function of Gm15417 in iNKT subset differentiation: Based on our compelling preliminary data, we hypothesize that 1) Gm15417 acts as a brake on iNKT development, and 2) promotes NKT1 differentiation, while limiting NKT17 differentiation. Here we propose to use our newly developed Gm15417STOP/STOP mice, in which transcription of the Gm15417 locus is blocked, to decipher: a) the precise developmental stage at which Gm15417 impacts iNKT cell development, and b) the potential role of Gm15417 in modulating TCR signal intensity during iNKT development. Finally, we will test c) whether Gm15417 also plays a role in stabilizing iNKT subset-specific functions in the periphery. Aim 2: Understanding mechanistic basis for Gm15417 regulation of iNKT development: We show that Gm15417 is an important component of the gene regulatory network that controls iNKT subset differentiation. To understand how Gm15417 shapes the developing iNKT cell transcriptome, we will: 1) define the genome- wide effect of Gm15417 gene ablation on transcription and chromatin conformation using paired single- nucleus RNA-seq (sNucRNA-seq) and ATAC-seq (sNucATAC-seq) approaches. 2) distinguish between alternate hypotheses of Gm15417 function, either requiring the Gm15417 sense RNA transcript, or Pol-II- dependent transcription through the Gm15417 locus. If the Gm15417 transcript is required, we will identify its genomic binding sites, RNA targets and protein interactors using ChIRP-seq, ChIRP RNA seq and ChIRP-MS approaches. If Gm15417 function does not require expression of its own sense transcript we will use the CRISPRi approach to test role of transcription through the Gm15417 locus. This is the first study to implicate LncRNAs in regulation of iNKT cell development, providing a major conceptual breakthrough for the field. The proposed studies will yield valuable insights into how this novel lncRNA regulates iNKT development and functional specialization, providing a solid foundation for a future R01, and ultimately lead to novel therapeutic strategies based on modulating iNKT subset development and function.