Table_14_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（NR4A）家族属于即刻早期应答基因，其所编码的蛋白可在多种组织或细胞中响应各类应激刺激后被激活。NR4A家族包含NR4A1、NR4A2与NR4A3三个成员，其中NR4A2和NR4A3的研究程度较低，相关机制尚不明晰，在免疫细胞中的研究尤为匮乏。NR4A的表达与多种疾病相关，例如关节炎与动脉粥样硬化；开发靶向NR4A的治疗性分子，亦是当前该研究领域的热点方向。本研究结合RNA测序（RNA-sequencing）与系统性生物信息学分析手段，探究NR4A2与NR4A3在单核细胞中的下游调控效应，并解析二者共通与独特的信号通路功能。研究数据表明，NR4A2与NR4A3的功能缺失会对未刺激状态及脂多糖（LPS）刺激状态下的基因转录产生显著且广泛的影响。有趣的是，受二者调控的诸多基因在未刺激与刺激状态下均有表达，这揭示出NR4A家族在未刺激状态的单核细胞中存在此前未被认知的功能。系统性聚类分析与生物信息学鉴定结果显示，NR4A2与NR4A3在单核细胞中兼具共通与独特的转录调控功能。值得注意的是，通过生物信息学聚类分析与转录因子互作组分析，均发现NR4A2与抗原呈递及主要组织相容性复合体（MHC）基因调控相关通路存在紧密关联。与之相反，NR4A3则更多与病毒应答相关通路相关联。功能实验进一步验证了本研究的数据分析结果，表明NR4A2在抗原加工调控中发挥选择性调控功能，而NR4A2与NR4A3在细胞迁移过程中则存在共通的调控作用。综上，本研究为阐释NR4A2与NR4A3这两种尚未被充分认知的核受体在单核细胞中的功能提供了全新的机制层面见解。