Coordination of two enhancers drives expression of olfactory trace amine-associated receptors VIII

Olfactory sensory neurons (OSNs) are functionally defined by their expression of a unique odorant receptor (OR). Mechanisms underlying singular OR expression are well studied, and involve a massive cross-chromosomal enhancer interaction network. Trace amine-associated receptors (TAARs) form a distinct family of olfactory receptors, and here we find that mechanisms regulating Taar gene choice display many unique features. The epigenetic signature of Taar genes in TAAR OSNs is different from that in OR OSNs. We further identify that two TAAR enhancers conserved across placental mammals are absolutely required for expression of the entire Taar gene repertoire. Deletion of either enhancer dramatically decreases the expression probabilities of different Taar genes, while deletion of both enhancers completely eliminates the TAAR OSN populations. In addition, both of the enhancers are sufficient to drive transgene expression in the partially overlapped TAAR OSNs. We also show that the TAAR enhancers operate in cis to regulate Taar gene expression. Our findings reveal a coordinated control of Taar gene choice in OSNs by two remote enhancers, and provide an excellent model to study molecular mechanisms underlying formation of an olfactory subsystem. We constructed PiggyBac transgenic plasmids with one of the two TAAR enhancers placed upstream of the minimal promoter sequence from mouse Hsp68 (heat shock protein 68 kDa) and followed by the GFP or tdTomato reporter gene. We then generated transgenic mice by injecting PiggyBac plasmids into the pronucleus of fertilized eggs. We obtained TAAR enhancer 1-GFP and TAAR enhancer 2-tdTomato transgenic mice. Then we FCA sorted GFP-positive and GFP-negative cells from TAAR enhancer 1-GFP mice, and tdTomato-positive and tdTomato-positive cells from TAAR enhancer 2-tdTomato mice for RNA-seq analyses.嗅觉感觉神经元（Olfactory sensory neurons, OSNs）的功能由其表达唯一的气味受体（odorant receptor, OR）所定义。调控单个OR基因表达的机制已被广泛研究，其涉及大规模的跨染色体增强子互作网络。痕量胺相关受体（Trace amine-associated receptors, TAARs）是一类独特的嗅觉受体家族，本研究发现调控Taar基因选择的机制展现出诸多独有特征。表达TAAR的OSNs中Taar基因的表观遗传特征与表达OR的OSNs存在显著差异。研究团队进一步鉴定出两个在胎盘哺乳动物中保守的TAAR增强子，它们对于整套Taar基因的表达是绝对必需的：删除任一增强子会显著降低不同Taar基因的表达概率，而同时删除两个增强子则会完全消除TAAR阳性OSNs群体。此外，两个增强子均可在部分重叠的TAAR阳性OSNs中驱动转基因表达。本研究还证实，TAAR增强子以顺式作用模式调控Taar基因的表达。本研究的发现揭示了两个远端增强子协同调控OSNs中Taar基因选择的分子机制，为研究嗅觉子系统形成的分子基础提供了优质模型。我们构建了携带两个TAAR增强子之一的PiggyBac转基因质粒：将增强子序列置于小鼠Hsp68（68kDa热休克蛋白，heat shock protein 68 kDa）最小启动子序列的上游，其后连接GFP（绿色荧光蛋白，green fluorescent protein）或tdTomato报告基因。随后通过向受精卵原核注射PiggyBac质粒的方式制备转基因小鼠，成功获得TAAR增强子1-GFP与TAAR增强子2-tdTomato转基因小鼠。之后我们采用FCA分选GFP阳性与GFP阴性细胞，从TAAR增强子2-tdTomato小鼠中分选tdTomato阳性与tdTomato阳性细胞用于RNA-seq（RNA测序）转录组分析。