Supporting Information

Yoon et al. 10.1073/pnas.1506058112 SI Materials and Methods Fisher Scientific); Cy3-conjugated donkey anti-rat IgG (Jackson RNA in Situ Hybridization. Mice were perfused with 4% (wt/vol) ImmunoResearch). paraformaldehyde (fix) and the interior of the snout removed and Sections were next incubated for 10 min at 37 °C in PBS soaked in fix overnight. The tissue was decalcified by incubation containing 1 mM EGS (Sigma) and 0.1% Tween20 and then for 3 d in 0.25 M EDTA/2% (wt/vol) paraformaldehyde and then washed three times for 5 min in PBS. They were next incubated embedded in OCT (Sakura). Serial coronal cryostat sections in 0.1 M HCl for 7 min to fragment DNA, washed for 5 min in (14 μm) were obtained and stored at −80 °C. PBS, and then treated with RNase A (100 mg/mL in 2× SSC) for Digoxigenin-labeled cRNA probes were prepared using cloned 30 min at 37 °C. After two 5-min washes in 2× SSC, sections were coding regions of individual Taar genes or the Nqo1 gene as equilibrated in 50% (vol/vol) formamide/2× SSC, pH 7.0. Probe templates. In situ hybridization to OE sections was performed as mix [15 μL containing 50–100 ng BAC DNA probe and 10 μg described (1), except that up to 30 μg/mL proteinase K was used mouse Cot-1 DNA in 50% formamide/2× SSC/10% (wt/vol) to permeabilize tissue sections before hybridization. If needed, dextran sulfate] was applied to each slide, and the slides were proteinase K activity was then eliminated by washing in PBS for coverslipped, sealed with rubber cement, and then incubated at 5 min at 85 °C. 80–82 °C on a slide warmer to denature the probe and cellular DNA. Slides were next incubated overnight at 37 °C in a humid Radioactive in Situ Hybridization. Radioactive in situ hybridization chamber. Sections were then washed three times in 50% was performed as described (2) with minor modifications. Mice formamide/2× SSC at 42 °C for 5 min, twice in 2× SSC at 42 °C were perfused as above and serial coronal sections (12 μm) ob- for 5 min, and then in 4× SSC at room temperature for 5 min. tained from the olfactory bulbs. Sections were treated as for Sections were then incubated with Alexa-488–labeled streptavidin nonradioactive in situ hybridization above, except that 5 μg/mL (Thermo Fisher Scientific) or with FITC-labeled anti-DIG (Sigma), proteinase K was used for permeabilization, and hybridization and nuclei then counterstained with DAPI, and the slides mounted was to 33P-labeled cRNA probes. Slides were coated with NTB-2 in 90% (vol/vol) glycerol/2.3% (wt/vol) DABCO (Sigma). Kodak emulsion, exposed for 3–4 wk at room temperature, and then developed. BAC Probes. BAC probes were prepared using a digoxigenin or biotin nick translation kit (Roche), according to the manufacturer’s Immunfluorescence DNA in Situ Hybridization (Immuno-FISH). instructions. BAC plasmids were obtained from the BACPAC Immuno-FISH was performed as described (3–5) with minor Resources Center (BPRC). BAC plasmids used for this study were modifications. Sections were postfixed for 5 min in 4% (wt/vol) as follows: RP23-43A7 (ch10: 23,642,668-23,851,574), RP23-318I18 paraformaldehyde fix in PBS, washed for 5 min in PBS, and then, (ch9: 37,485,034-37,665,416), RP23-88A24 (ch14: 54,862,839- for antigen retrieval, incubated at 80–85 °C for 23 min in 10 mM 55,005,904), RP23-6M15 (ch15: 97,961,306-98,195,573), RP23- Tris/1 mM EDTA. 155M18 (ch16: 59,174,022-59,376,005), RP24-368I6 (ch1: Sections were permeabilized with 0.5% Triton X-100 in PBS, 174,841,505-174,981,890); all chromosome mapping information washed three times in PBS, blocked with 2% (wt/vol) IgG-free was based on the NCBI37/mm9 assembly of the mouse genome. BSA/0.1% Tween20 in PBS, and then incubated with primary antibodies followed by secondary antibodies, with each antibody Immunohistochemistry and Antibodies. Immunohistochemistry was incubation followed by three 5-min washes in 0.1% Tween20 in conducted using the same initial procedures specified above for PBS. Primary antibodies: Goat anti-Lamin B1 (Santa Cruz, immuno-FISH. Primary antibodies used were against: H3K9me3 sc-6217); Rat anti-mCherry (KeraFAST, EST202). Secondary (Abcam, ab8898), H4K20me3 (Abcam, ab9053), lamin B (Santa antibodies: Alexa-647-conjugated donkey anti-goat IgG (Thermo Cruz, sc-6217), LAP2 (BD, 611000), or lamin A (Abcam, ab133256).

1. Liberles SD, Buck LB (2006) A second class of chemosensory receptors in the olfactory 4. Croft JA, et al. (1999) Differences in the localization and morphology of chromosomes epithelium. Nature 442(7103):645–650. in the human nucleus. J Cell Biol 145(6):1119–1131. 2. Ressler KJ, Sullivan SL, Buck LB (1994) Information coding in the olfactory system: ev- 5. Clowney EJ, et al. (2012) Nuclear aggregation of olfactory receptor genes governs their idence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell monogenic expression. Cell 151(4):724–737. 79(7):1245–1255. 3. Ragoczy T, Bender MA, Telling A, Byron R, Groudine M (2006) The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation. Genes Dev 20(11):1447–1457.

Yoon et al. www.pnas.org/cgi/content/short/1506058112 1of5 Fig. S1. Coexpression of mutant and functional Taar alleles in individual OSNs. OE sections from Taar5lacZ/+ (A–C)orTaar6lacZ/+ mice (D–F) were costained with a Taar9 riboprobe and with β-gal antibodies, which detect OSNs expressing the mutant Taar allele. Some β-gal+ OSNs were labeled for Taar9, indicating that cells expressing the mutant Taar5 or Taar6 allele can coexpress a functional Taar allele. (Scale bars, 30 μm.)

Yoon et al. www.pnas.org/cgi/content/short/1506058112 2of5 Fig. S2. Expression of Taar mutant alleles in Taar5 and Taar6 knockout mice. The OE distribution of OSNs expressing wild-type versus mutant Taar5 and Taar6 alleles. (A–C) OE sections from wild-type (WT) mice were hybridized with a Taar5 (A), Taar6 (B), or Nqo1 (C) riboprobe. Taar5+ neurons are seen only in the lacZ/+ dorsal, Nqo1-labeled domain whereas Taar6+ neurons are seen in both the dorsal Nqo1+ and ventral Nqo1- domains. (D–G) OE sections from Taar5 (D and lacZ/+ E)orTaar6 (F and G) mice were immunostained for β-gal (D and F) or hybridized to an Nqo1 riboprobe (E and G). β-gal+ OSNs expressing the mutant Taar5 allele are located in only the dorsal Nqo1+ domain whereas those expressing the Taar6 mutant allele are located in both the Nqo1+ dorsal domain and Nqo1- ventral domain. White dots indicate the locations of OSNs labeled with the Taar riboprobe (A and B) or immunostained for β-gal (D and F). (Scale bars, 300 μm.)

Yoon et al. www.pnas.org/cgi/content/short/1506058112 3of5 Fig. S3. The presence of a neomycin selection cassette alters the expression of a Taar5 mutant allele. The DNA construct used to generate Taar5 knockout mice contained a neomycin selection cassette. Following removal of the selection cassette, OSNs expressing the mutant Taar5 allele (β-gal+ OSNs) were seen predominantly in the dorsal OE domain, similar to OSNs expressing the wild-type Taar5 allele (see Fig. S2). However, before removal of the selection cassette, β-gal+ OSNs were seen in both the dorsal and ventral OE domains (A). This is apparent from costaining the same OE section with a Taar4 riboprobe to label Taar4-expressing OSNs, which are found predominantly in the dorsal domain (B). White dots indicate β-gal OSNs in OE regions that lack Taar4+ OSNs. (Scale bar, 300 μm.)

Table S1. Many OSNs expressing a mutant Taar allele coexpress one other Taar gene Taar5lacZ/+ Taar6lacZ/+

Animal 1 Animal 2 Animal 3 Animal 4 Animal 1 Animal 2 Animal 3 Animal 4

Sum of percentages of β-gal+ OSNs labeled by 67.6 57.1 65.0 63.4 52.4 60.5 51.3 68.3 different Taar probes (Taar2-9)* (%) Percentage of β-gal+ OSNs labeled by a mix 56.7 60.3 57.9 55.1 65.2 48.7 47.7 55.9 containing all Taar probes* (%)

+ + *20 sections for Taar5lacZ/ , 7 sections for Taar6lacZ/ .

Table S2. OE domains expressing wild-type versus mutant Taar5 and Taar6 alleles Taar5lacZ/+ Taar6lacZ/+

Taar5+ β-gal+ Taar6+ β-gal+

No. of labeled cells 297 1,071 654 1,521 No. of labeled cells in dorsal Nqo1+ OE domain 296 1,057 151 987 % of labeled cells in dorsal domain 99.7 98.7 23.1 64.9 No. of animals (male/female) 2/1 2/1 No. of sections scored per animal 20 7

The number of OSNs labeled for Taar5, Taar6,orβ-gal in the entire OE or the dorsal Nqo1+ domain.

Yoon et al. www.pnas.org/cgi/content/short/1506058112 4of5 Movie S1. Taar localization and chromocenter distribution in OSNS and other OE cells. This movie shows a 4.75-μm-deep z-stack through the OE in 0.25-μm steps. Apical sustentacular cells are located at the top and basal cells at the bottom, with OSNs occupying a large central region. DNA FISH using a Taar BAC probe (green) shows that, in OSNs, Taar alleles mostly overlap with lamin B (red), which marks the nuclear lamina, whereas this localization is seldom seen in sustentacular cells and is less often seen in basal cells. The movie also illustrates the difference in the number and size of chromocenters present in the sus- tentacular cells versus OSNs and basal cells (DAPI stain, grayscale).

Movie S1

Yoon et al. www.pnas.org/cgi/content/short/1506058112 5of5