Table_13_Transcriptional Profiling of Monocytes Deficient in Nuclear Orphan Receptors NR4A2 and NR4A3 Reveals Distinct Signalling Roles Related to Antigen Presentation and Viral Response.xlsx

The nuclear receptor sub-family 4 group A (NR4A) family are early response genes that encode proteins that are activated in several tissues/cells in response to a variety of stressors. The NR4A family comprises NR4A1, NR4A2 and NR4A3 of which NR4A2 and NR4A3 are under researched and less understood, particularly in the context of immune cells. NR4A expression is associated with multiple diseases e.g. arthritis and atherosclerosis and the development of NR4A-targetting molecules as therapeutics is a current focus in this research field. Here, we use a combination of RNA-sequencing coupled with strategic bioinformatic analysis to investigate the down-stream effects of NR4A2 and NR4A3 in monocytes and dissect their common and distinct signalling roles. Our data reveals that NR4A2 and NR4A3 depletion has a robust and broad-reaching effect on transcription in both the unstimulated state and in the presence of LPS. Interestingly, many of the genes affected were present in both the unstimulated and stimulated states revealing a previously unappreciated role for the NR4As in unstimulated cells. Strategic clustering and bioinformatic analysis identified both distinct and common transcriptional roles for NR4A2 and NR4A3 in monocytes. NR4A2 notably was linked by both bioinformatic clustering analysis and transcription factor interactome analysis to pathways associated with antigen presentation and regulation of MHC genes. NR4A3 in contrast was more closely linked to pathways associated with viral response. Functional studies further support our data analysis pointing towards preferential/selective roles for NR4A2 in the regulation of antigen processing with common roles for NR4A2 and NR4A3 evident with respect to cell migration. Taken together this study provides novel mechanistic insights into the role of the enigmatic nuclear receptors NR4A2 and NR4A3 in monocytes.

核受体亚家族4A组（nuclear receptor sub-family 4 group A, NR4A）家族属于早期应答基因，其编码的蛋白可在多种组织或细胞中响应各类应激原而被激活。NR4A家族包含NR4A1、NR4A2与NR4A3三个成员，其中NR4A2与NR4A3的研究尚不充分，功能机制尚未阐明，尤其在免疫细胞领域的相关研究更为稀缺。NR4A的表达与关节炎、动脉粥样硬化等多种疾病密切相关，开发靶向NR4A的治疗性分子已成为该研究领域的前沿热点。本研究结合RNA测序（RNA-sequencing）与系统性生物信息学分析，探究了NR4A2与NR4A3在单核细胞中的下游调控效应，并解析了二者共有的与独特的信号传导功能。研究数据显示，敲低NR4A2与NR4A3可在静息状态与脂多糖（Lipopolysaccharide, LPS）刺激状态下，对细胞转录组产生广泛且显著的调控影响。值得注意的是，静息与刺激两种状态下均受调控的基因数量众多，这揭示了NR4A家族在静息细胞中此前未被揭示的重要功能。通过系统性聚类分析与生物信息学解析，我们明确了NR4A2与NR4A3在单核细胞中既存在共同的转录调控功能，也具有各自独特的作用通路。其中，生物信息学聚类分析与转录因子互作组（transcription factor interactome）分析均证实，NR4A2与抗原呈递及主要组织相容性复合体（major histocompatibility complex, MHC）基因的调控通路紧密关联。与之相反，NR4A3则更多与病毒应答通路相关联。功能实验进一步验证了本研究的数据分析结果：NR4A2在抗原加工过程中发挥优先性或选择性调控作用，而NR4A2与NR4A3则在细胞迁移方面存在共同的调控功能。综上，本研究为神秘的核受体NR4A2与NR4A3在单核细胞中的功能提供了全新的机制性见解。