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

项目摘要/摘要 最近的报道表明，iNKT细胞由多个不同的效应亚群组成，具有特殊的 免疫角色。然而，控制替代iNKT亚集规范的分子途径仍然很差 因此限制了我们将专门的iNKT细胞用于治疗目的的能力。在此，我们 一种新的长非编码(LNC)RNA Gm15417的鉴定 胸腺iNKT发育和功能亚群分化的调控。这表示一个 在该领域取得突破，因为人们对iNKT子集规范知之甚少，尤其是没有作用 到目前为止，iNKT细胞发育过程中的lncRNAs已有报道。在本申请中，我们试图阐明细胞 以及Gm15417根据两个特定目的控制iNKT细胞发育的分子基础： 目标1：在iNKT子集中定义Gm15417的阶段特异性和TCR依赖功能 差异化：根据我们令人信服的初步数据，我们假设1)Gm15417起刹车作用 2)促进NKT1分化，同时限制NKT17 差异化。在这里，我们建议使用我们新开发的Gm15417STOP/Stop小鼠，在其中转录 Gm15417基因座被阻断，以破译：a)确切的发育阶段 Gm15417影响iNKT细胞发育，以及b)Gm15417在调节TCR信号中的潜在作用 INKT发育过程中的强度。最后，我们将测试c)Gm15417是否也起到稳定iNKT的作用 外围设备中特定于子集的功能。 目的2：了解Gm15417调控iNKT发育的机制基础：我们表明 Gm15417是控制iNKT亚群分化的基因调控网络的重要组成部分。 为了了解Gm15417如何塑造发育中的iNKT细胞转录组，我们将：1)定义基因组- Gm15417基因去除对转录和染色质构象的广泛影响 核RNA-seq(sNucRNA-seq)和ATAC-seq(sNucATAC-seq)方法。2)区分 Gm15417功能的替代假设，要么需要Gm15417正义RNA转录本，要么需要POL-II- 通过Gm15417基因座依赖转录。如果需要Gm15417成绩单，我们将确定其 利用chirp-seq、chirp RNA seq和chirp-MS研究基因组结合部位、RNA靶标和蛋白质相互作用 接近了。如果Gm15417函数不需要表达其自身的有义转录，我们将使用 CRISPRi方法通过Gm15417基因座检测转录作用。 这是第一个涉及lncRNAs调控iNKT细胞发育的研究，提供了一个主要的 该领域的概念突破。拟议中的研究将为这部小说如何 LncRNA调节iNKT的开发和功能专业化，为未来的R01提供坚实的基础， 并最终导致基于调节iNKT亚群发育和功能的新的治疗策略。

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.