The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice

Abstract

Background: In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice.

Aim: Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary) from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice.

Procedures: Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J), and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes) and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections.

Results: In the mouse genome there are eight intact solitary genes: Olfr19 (M12), Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a whole, our findings favor Olfr266 as a model gene to investigate odorant receptor gene choice.

Fig 1. Chromosomal distribution of the solitary genes in Muridae.

Solitary genes for Mus musculus (GRCm38.p1) and Rattus norvegicus (RGSC 3.4) are indicated in light green if intact, in dark green if pseudogenized. Grey dashed lines connect each solitary gene with its ortholog. Yellow and orange squares, amplified below the chromosomal diagram of the corresponding species, show pairs of solitary genes that (based on annotation) retain a conserved synteny in Muridae (while a non-annotated, neighboring odorant receptor gene is present for Olr390). The distance (measured in Mb) between genes of each gene pair is annotated. Chromosome bands represent Giemsa staining.

Fig 2. Solitary genes expressed in the mouse main olfactory epithelium.

In situ hybridization of the main olfactory epithelium of three-day-old mice (n = 3), showing the expression of the eight mouse solitary genes: Olfr19 (M12), Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466 and Olfr1402 (green). Nuclear counterstaining is with DAPI (magenta). The table on the right reports the average number of olfactory sensory neurons (± a standard deviation) for each solitary gene. Scale bars: 20 μm.

Fig 3. 5' RACE clones aligned against their genomic references.

For each gene, middle black line represents the genomic locus (GRCm38.p1). Transcription start sites derived from literature are labelled with capital letters (C for Clowney et al., P for Plessy et al., Y for Young et al.). Light blue arrows depict coding sequences as they appear on Ensembl. Fuchsia arrows represent sequences selected as putative promoters. On Olfr466, a pink arrow renders an alternative putative promoter derived from Plessy et al. All arrows point towards 3'-end. Above genomic loci, grey stacked lines represent spliced 5' RACE clones obtained from whole olfactory mucosa; similarly, in green below the genomic reference, clones derived from sorted olfactory sensory neurons are depicted. Thin (grey or green) lines in 5' RACE clones represent introns. Three dots (…) on a stacked line indicate that the clone is not sequenced until its 3'-end. Shortest clones are omitted for graphical reasons. The control, non-solitary, odorant receptor gene Olfr6 (M50) is boxed in orange.

Fig 4. Candidate promoter sequences of the solitary genes.

A: detail of the P element showing the sequences used to generate the position-specific weight matrix for homeodomain factor binding sites; on the main sequence, 3'-pointing arrows in salmon show position and orientation of homeodomain factor binding sites as annotated by Vassalli et al.; blue arrows, also 3'-oriented, show emipalindromes identified by EMBOSS Palindrome and used to extend sequences to be added to the matrix; grey boxes mark sequence stretches conserved between the P element and the neighboring Olfr713 (P3) promoter (Conserved P-P3) or between the P and the H element (Conserved P-H); a matrix (depicted on the right, top) was obtained aligning the three extended binding sites on P element and the single extended binding site on H element (identical to the central binding site on P element). B: on the putative promoter of Olfr266 (3'-pointing grey arrow) a homeodomain factor binding site (3'-pointing salmon arrow labelled as HDBS) is predicted; also, sequences conserved in Mammalia Eutheria are found (green boxes); more towards the 3'-end of the putative promoter, SNPs that are present in the wild for Mus musculus domesticus are found in two positions. C: multi-alignments of candidate promoter sequences (5'-3') from different mouse populations for the solitary genes plus (orange box) the non-solitary, reference odorant receptor gene Olfr6 (M50); for Olfr466 two different sequences are presented, one 5' RACE-derived (tagged with [R]) and one based on Plessy et al. (labelled with [P]); B6 stands for C57BL/6J, W stands for consensus of all wild-caught populations, F stands for French population, G stands for German population and I stands for Iranian population; in brown are marked mutant C57BL/6J bases; in violet are highlighted bases differing from consensus; thin vertical lines (light blue) on sequence alignment mark the position of variations in at least some of the 36 non-C57BL/6J laboratory strains, reporting also the fraction of strains carrying the variation (above) and the variation details (below); for Olfr266, sequences boxed in green are conserved in Mammalia Eutheria, and bases in arrowed salmon box (arrow points towards 3') are part of the predicted homeodomain factor binding site.