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.