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

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.


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All studies must disclose on these points even when the disclosure is negative. The data supporting the findings of this study are included within the article and its Supplemental files. Reagents are available from the corresponding author upon reasonable request. The source data underlying Figure 1B-D, 2D, 2F, 4F-H, 5F, 6F, 7C, 7E, 8C, and Supplementary Figure 1B, 3A, 5D, 5E, 6E, 6F, 7D, 8C, 9A-C are provided as a Source Data file. The raw and processed sequencing data were deposited in the Gene Expression Omnibus with the accession number GSE163778 (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE163778).
A sample size of at least three independent biological replicates was selected for RNA-seq, in situ, and immunohistochemistry experiments in the figures. This size was selected because multiple statistical approaches have been developed to allow identification of significantly changed genes from more than two biological replicates of high throughput sequencing data (e.g. DEseq2) and experimental data (e.g. one-way ANOVA). Wherever possible, additional biological replicates were included. Sample sizes were based on previous publications with similar experimental systems (Tetsuo Iwata et al., Nature Communications, 2017).
For in situ and immunohistochemistry expeiments, the damaged tissues were excluded from the imaging and quantification. This exclusion was pre-established. No other data were excluded.
At least three independent biological replicates were performed for each experiment. Replicate experiments yielded the same results.
No experiments were performed with live animals. For the purpose of sorting reporter-positive and reporter-negative cells, animals of similar age were grouped by genotype and sorted together.
Animals were used as the source of tissue and primary cells, so knowledge of genotype was required for proper handling and cell sorting. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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TAAR4, TAAR5, TAAR6 -the three antibodies have been validated and stated in Johnson et al., PNAS, 2012. Caspase-3 -Manufacturer states that the antibody is validated for immunofluorescence in Staurosporine treated HT-29 cells. GFP -Manufacturer states that the antibody is validated for immunofluorescence in GFP-transfected NIH3T3 cells. tdTomato -Manufacturer states that the antibody is validated for immunofluorescence in previous publication (Jinfei D Ni et al., eLife, 2019). H3K9me3 -Manufacturer states that the antibody is validated for ChIP in 293T cells. Donkey Anti-Chicken IgY conjugated to Alexa Fluo 488 -Manufacturer states that antibody is validated for immunofluorescence. Donkey Anti-Rabbit IgG conjugated to Alexa Fluo 488 -Manufacturer states that antibody is validated for immunofluorescence. Donkey Anti-Guinea Pig IgG conjugated to Cy3 -Manufacturer states that antibody is validated for immunofluorescence.
Study did not involve specimens collected from the field.
All mouse experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine and the Institutional Animal Care and Use Committee (Department of Laboratory Animal Science, Shanghai Jiao Tong University School of Medicine, animal protocol number A-2016-049).
The raw and processed ChIP-seq data were deposited in the Gene Expression Omnibus with the accession number GSE163778 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163778).