Functional Interrogation of an Odorant Receptor Locus Reveals Multiple Axes of Transcriptional Regulation

A transgenic approach in mice allows the functional interrogation of an odorant receptor locus in vivo and reveals characteristics of its monogenic and monoallelic expression.
The odorant receptor (OR) genes constitute the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion, through an unknown mechanism that likely exploits positive and negative regulation. We devised a genetic strategy in mice to examine OR selection by determining the transcriptional activity of an exogenous promoter homologously integrated into an OR locus. Using the tetracycline-dependent transactivator responsive promoter (teto), we observed that the OR locus imposes spatial and temporal constraints on teto-driven transcription. Conditional expression experiments reveal a developmental change in the permissiveness of the locus. Further, expression of an OR transgene that suppresses endogenous ORs similarly represses the OR-integrated teto. Neurons homozygous for the teto-modified allele demonstrate predominantly monoallelic expression, despite their potential to express both copies. These data reveal multiple axes of regulation, and support a model of initiation of OR choice limited by nonpermissive chromatin and maintained by repression of nonselected alleles.
Author Summary Odorant receptor (OR) gene choice is a paradigmatic example of transcriptional regulation in which each olfactory sensory neuron selects a single OR from a repertoire of over 1,000 genes. Two mechanistic models of OR choice have been proposed. One postulates the existence of a specialized transcriptional machinery that selects just one OR allele, while a second, kinetic model proposes that OR chromatin is intrinsically nonpermissive, such that inefficient activation during a critical window of time restricts expression to a single OR allele. Here, we used a transgenic approach in mice in which we inserted a conditionally regulated exogenous promoter into an OR locus by homologous recombination in embryonic stem cells. The resulting novel mouse lines allowed the functional interrogation of the OR locus in vivo during development of the olfactory epithelium, enabling us to directly test models of OR choice. Using this experimental strategy we found that OR loci are indeed slow to activate and that the subsequent phenomenon of spatial restriction of OR expression is accomplished by repression. We also observed a developmental shutdown of OR loci concomitant with expression of the OR repertoire. Together, these experiments provide prima facie evidence for a kinetic model of initiation of OR gene choice, coupled with repression of nonselected OR alleles.