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

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. In order to gain genetic access to TAAR-expressing OSNs, we generated Taar5-ires-Cre and Taar6-ires-Cre knockin mouse lines, in which Cre recombinase is co-transcribed with Taar5 and Taar6 gene, respectively. Each Cre line was crossed with Cre-dependent reporter lines, including lox-L10-GFP and lox-ZsGreen, to fluorescently label the specific population of TAAR OSNs. The GFP- or ZsGreen-negative cells were also sorted to serve as control cells, approximately 70-80% of which are composed of OR-expressing OSNs.嗅觉感觉神经元（olfactory sensory neurons, OSNs）的功能定义为表达单一气味受体（odorant receptor, OR）。关于单一OR表达的调控机制已有充分研究，其涉及庞大的跨染色体增强子互作网络。痕量胺相关受体（trace amine-associated receptors, TAARs）构成一类独特的嗅觉受体家族，本研究发现调控Taar基因选择的机制具有诸多独有特征。TAAR阳性OSN中Taar基因的表观遗传特征与OR阳性OSN存在显著差异。本研究进一步鉴定出两个在胎盘类哺乳动物中保守的TAAR增强子，它们对于完整Taar基因集合的表达是绝对必需的：单独缺失任一增强子都会显著降低不同Taar基因的表达概率，而同时缺失两个增强子则会完全消除TAAR阳性OSN群体。此外，两个增强子均可在部分重叠的TAAR阳性OSN中驱动转基因表达。本研究还证实，TAAR增强子以顺式作用方式调控Taar基因的表达。本研究结果揭示了两个远端增强子协同调控OSN中Taar基因选择的机制，并为研究嗅觉亚系统形成的分子机制提供了优秀的模型体系。为实现对表达TAAR的OSN的遗传学操控，本研究构建了Taar5-ires-Cre与Taar6-ires-Cre敲入小鼠品系，二者分别将Cre重组酶与Taar5、Taar6基因共转录。将每个Cre品系与Cre依赖型报告基因品系（包括lox-L10-GFP与lox-ZsGreen）杂交，以荧光标记特定的TAAR阳性OSN群体。同时分选出GFP或ZsGreen阴性的细胞作为对照细胞，其中约70%-80%为表达OR的OSN。