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Genome Res. 13:2674-2685, 2003 ©2003 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/03 $5.00 Letter The Clustered Olfactory Receptor Gene Family 262: Genomic Organization, Promotor Elements, and Interacting Transcription FactorsInstitute of Physiology, University of Hohenheim, 70593 Stuttgart, Germany
For six mouse olfactory receptor genes from family 262 which are expressed in clustered populations of olfactory sensory neurons, the genomic as well as cDNA structures were deciphered. All genes contained several exons which in some cases were alternatively spliced. Immediately upstream of the transcription start sites, sequence motif blocks were identified that are highly conserved among olfactory receptor (OR) genes which are expressed in clustered neuronal populations. By means of electrophoretic mobility shift assays, it was demonstrated that segments of the motif block region interact with proteins extracted from nuclear fractions of the olfactory epithelium. Yeast one-hybrid screenings of an olfactory cDNA library led to the identification of a set of transcription factors that specifically bind to particular elements of the motif block region. The identified factors can be categorized into two types: One group is known to be involved in transcriptional initiation, and the second group represents factors involved in pattern formations. The identified components may contribute to govern the precise topographic expression pattern of olfactory receptor genes.
The mammalian olfactory system is able to recognize and discriminate a vast array of odorous compounds. This enormous capacity of molecular recognition is thought to be based on the multiplicity and diversity of a G-protein coupled receptor family. The receptor types are encoded by a large gene family (Buck and Axel 1991  ) which in mammals (such as mouse) comprises more than a thousand genes (Zhang and Firestein 2002), and in lower vertebrates (e.g., fish) about a few dozen genes (Ngai et al. 1993). Genes encoding the olfactory receptors (ORs) are found on most chromosomes; they reside in a variety of small and large clusters which can span up to 2 Mb and can contain as many as 100 individual genes (Sullivan et al. 1996; Buettner et al. 1998; Rouquier et al. 1998; Glusman et al. 2000a, 2001; Xie et al. 2000; Lane et al. 2001; Young et al. 2002; Zhang and Firestein 2002; for recent reviews, see Mombaerts 1999a,b, 2001; Young and Trask 2002). Despite the large size of the OR gene superfamily, individual olfactory sensory neurons (OSNs) are likely to express only a single or at most a few receptor genes from the repertoire (Malnic et al. 1999; Rawson et al. 2000); moreover, only one of the two alleles appears to be expressed in each neuron (Chess et al. 1994). In the nasal epithelium of mammals, OSNs expressing the same receptor type generally are not randomly distributed throughout the olfactory epithelium; they are either restricted to one of several broad expression zones (Ressler et al. 1993; Vassar et al. 1993; Strotmann et al. 1994b) or, like the cells expressing a member of the OR37 subfamily, are clustered within a small central region of the epithelium (Strotmann et al. 1992; 1994a).
Very little is known about the molecular parameters and processes underlying the complex transcriptional regulation of olfactory receptor expression. A detailed characterization of those DNA sequences which are involved in governing the proper spatial and temporal expression pattern of a distinct OR gene is an essential prerequisite for unraveling the mechanisms controlling expression of OR genes. Candidate regions for regulatory elements are the noncoding sequences of OR genes, most notably those conserved in OR genes which share the same expression pattern. Analyses of a few OR genes have not led to a coherent picture (Asai et al. 1996 In the present study we examined the 5' genomic regions of the OR genes from the second `OR37' cluster, in an effort to identify characteristic, putative regulatory sequence motifs. A representative sequence was then employed to search for distinct transcription factors that may bind to these DNA motifs in the 5' noncoding region of the clustered OR genes.
In order to analyze whether the `mOR37' genes in cluster II ('mOR37F', `mOR37G' and `mOR37H', named mOR262-3, mOR262-7, and mOR262-8, respectively, according to the nomenclature of Zhang and Firestein 2002 ) are colocalized with additional OR genes, the sequence 300 kb up- and downstream of the genomic locus was examined. The computer searches uncovered two OR genes (mOR262-2 and mOR262-9) flanking either side of the gene cluster (Fig. 1A). These two genes are also members of family 262, but both lack the `OR37'-characteristic insertion of six amino acids (Kubick et al. 1997) in the extracellular loop E3. The overall sequence identity of mOR262-2 and mOR262-9 with the `mOR37' genes is in the range between 53% and 59%, and thus they are considered members of different subfamilies. Assessing the sequence of an additional 300-kb region on either side uncovered no further OR genes, demonstrating that these five genes (mOR262-2, -3, -7, -8, and -9) form a distinct cluster. The question of whether the two genes mOR262-2 and -9 are expressed in clustered cell populations, like their relatives, was approached using in situ hybridization experiments. Coronal sections through the nasal cavity were incubated with specific antisense RNA probes. As shown in Figure 2A,B, cells expressing mOR262-2 or -9, respectively, are in fact located within the same small circumscribed region of the epithelium which was previously shown to be attributed to `mOR37' subtypes (Strotmann et al. 1992, 1994a). Cross-hybridizations can be excluded due to their low sequence identity and the hybridization conditions. Thus, all OR genes of chromosome 4 that have been analyzed to date are expressed in cell populations which are organized in a cluster.
This raised the question of whether OR genes on this chromosome in general may be expressed in clustered cell populations. Chromosome 4 harbors two additional loci with OR genes: At position 57.7 Mb, there is a locus with only one gene (Fig. 1B); it turned out that this gene also belongs to family 262 (mOR262-1), but lacks the extended E3 domain. At a distance of 80 Mb, a fourth OR cluster is located comprising 16 genes (Zhang and Firestein 2002 The relatedness of all genes on chromosome 4 was evaluated in more detail by means of phylogenetic analyses. As can be seen in Figure 3, all OR genes with a clustered expression pattern are closely related and not intermingled by OR genes with a zonal expression pattern. The genes with a clustered expression pattern can be categorized into three subgroups (1-3 in Fig. 3). All members of family 262 with an extended E3 loop ('mOR37' genes) are positioned on one branch (branch 1); their closest relatives are those family 262 members which are linked with cluster II as well as mOR262-1 (branch 2). The two OR genes associated with cluster I (branch 3) appear to be more distantly related.
Our previous studies demonstrated that genes from cluster I (mOR262-4, -5, -6, 10, -11P, -12, and -14) share in their 5'- region a stretch of DNA with six well conserved motif blocks (Hoppe et al. 2000
Determining the full extent of the mRNA allowed us to localize the transcription start site (TSS) for all cluster II genes and mOR262-1. Due to the highly variable gene structures, it was located between approximately 750 bp (mOR262-2) and 7200 bp (mOR262-7) upstream of the translation start. In order to identify putative expression control elements within this region, 1 kb upstream of the TSS was compared between the different genes using the programs T-COFFEE and Dialign2. Within this 1 kb, a stretch of about 170 bp was identified which shared a striking homology among the six genes (Fig. 5A). The sequence was located in close proximity (about 100-200 bp) upstream of the TSSs of the respective genes and was surrounded by DNA without any significant similarities (data not shown).
This 170-bp region was next examined for the characteristic sequence motifs, most notably such motifs which were previously identified in the putative promotor regions of OR genes in cluster I (Hoppe et al. 2000 In this comparative approach, an additional highly conserved segment emerged; it is located following the motif block region and consists of an AT-rich stretch (stars in Fig. 5A). Although this DNA segment is not a typical TATA box, its position relative to the transcription site, which is in the range of -20 to -40 for the genes mOR262-1, -2, -3, and -7, is the site where a TATA box is usually positioned. However, a "classical" TATA-box core sequence could only be identified in the putative promotor region of mOR262-1 and -8. All regions were examined for the presence of alternative motifs that may contribute to the initiation of transcription. This effort led to the identification of distinctive initiator elements for most analyzed genes including mOR262-6, -9, and -14 (Fig. 5A). Figure 5B shows the organization of the motif blocks for the various genes; it becomes obvious that not only the sequences of the motif blocks are conserved, but also their relative positions, especially within the first tandem of blocks (I-III). The distance between the blocks is slightly more variable in the second tandem (IV-VI).
Database searches revealed that some of the conserved motifs represent consensus binding sequences for distinct transcription factors (Hoppe et al. 2000
Because the typical six motif blocks can be grouped into two pairs, which are organized in a tandem-like arrangement (see Fig. 5B; Hoppe et al. 2000 To determine the molecular identity of proteins that interact with these sites, we performed a yeast one-hybrid screen. The genetically modified yeast strain YM4271, which carries two copies of the motif block sequence of mOR262-6 upstream of the LacZ-reporter, was transformed with a cDNA library from the olfactory epithelium fused to the GAL4 activation domain. Two independent screening approaches led to 25 colonies, and six of them were confirmed as true positives by retransfection (Table 1).
Sequencing and subsequent BLAST searches revealed that all six genes were coding for proteins which represent distinct transcription factors. The proteins encoded by the genes were classified as strong to weak activators of gene transcription on the basis of To characterize the protein-DNA interaction of these factors in more detail, the motif block region was divided into subregions (Fig. 7A), and corresponding yeast strains were generated. The strain containing motif block IV was unemployable due to strong self-activation. The six factors which interacted with the complete motif block region were retransfected into these strains. Most factors displayed a strong activation of the reporter gene when interacting with a particular subregion (Table 1), and interestingly, the binding of Alx-3 to the subregion containing motif blocks I+II led to a significantly stronger reaction compared with the complete motif block region. In contrast to all other factors, O/E-2(0S) only binds to the complete motif block region or to subregion IV.
To define the putative interaction site for the identified transcription factors, the sequence of the mOR262-6 motif block region was assessed for stretches related to proposed consensus binding motifs for the identified factors. In fact, for each of the transcription factors, a consensus-related sequence motif could be identified in the particular subregion (Fig. 7B). For O/E-2(0S) it indeed turns out that subregion IV (Fig. 7B) contains such a consensus-related sequence motif for this factor. A comparison of the motif block regions of all clustered genes led to the identification of a consensus site for O/E-type transcription factors close to motif block III or VI, in most of the genes (Figs. 5A, 7B). For Alx-3 and Lhx-2, corresponding sites were identified in motif block I from each gene. Initiator elements or E-boxes which represent binding sites for BEN were present close to the TSS of each gene (Fig. 5A). In addition, consensus-related sites for AP-2 To localize and identify the cells in the olfactory epithelium which express the corresponding transcription factors, we performed in situ hybridization experiments, probing coronal sections through the nasal epithelium with gene-specific antisense RNA. For two of the factors, reactive cells could be visualized. The probe for O/E-2(0S) labeled cells throughout the layer of olfactory sensory neurons and basal cells, but was absent from the most apical layer, which contains the sustentacular cells (Fig. 8A). In contrast, LH-2-expressing cells were localized only in the deep layer of the epithelium (Fig. 8B), where basal cells and immature olfactory neurons are positioned. Cells reactive to both probes were not spatially restricted to a particular region of the nasal epithelium; rather they were found to be distributed throughout all turbinate structures.
In the present study we explored the genomic features of OR genes forming a small, highly related receptor family. Several conserved sequence motifs were identified upstream of the transcription start sites; these motifs are considered putative regulatory elements. We found that the region containing these elements in one representative gene in fact binds distinct transcription factors; moreover, we demonstrated that these factors are expressed in cells of the olfactory epithelium. These results suggest that at least some of the identified DNA motifs may represent important elements of the promotor region involved in the transcriptional control of these genes.
The identification of putative control elements immediately upstream of the transcription start is consistent with previous observations, suggesting that transcription of OR genes is initiated from a region up to 6.7 kb upstream of the initial exon (Asai et al. 1996
The sequence motifs identified in this study share very high homology with those at the TSSs of previously documented clustered OR genes (Hoppe et al. 2000
The existence of highly conserved sequence motifs in the putative promotor region of the mOR262 genes seems to be in contrast to other OR genes; previous studies led to the identification of short and very diverse elements within the noncoding sequences of OR genes (Glusman et al. 2000b
It has been hypothesized that distinct levels of control are required for a given neuron to choose one gene from the thousand available, and that this could be mediated by a combination of transcription factors (Chess 1998
On a first level of organization, the expression of OR genes in sensory neurons of the nasal neuroepithelium is believed to be governed by tissue-specific transcription factors (Reed 2000
Regarding the special topographic pattern of mOR262-expressing neurons, the identification of several homeobox-type transcription factors (Ptx-1, Alx-3, Lhx-2/LH-2) could be of particular relevance, because factors of this type are generally involved in pattern formation. In this context, the most likely interaction of LH-2 with motif block I is of particular interest; motif block I shows a striking homology to the so-called `rhodopsin-conserved-sequence-I' (RCSI; Papatsenko et al. 2001 The factors identified in this study represent the first set of molecularly defined transcription factors which interact with a putative control region of OR genes; they may represent essential components for the cis-element-mediated regulation of OR gene expression.
In situ Hybridization Specific antisense riboprobes for OR genes and transcription factors were generated using the following primer combinations:
The PCR reactions were performed with mouse genomic DNA as template in a Peltier PTC 200 Thermocycler (MJ Research) with the following time and temperature schedule: An initial 4-min denaturation at 96°C was followed by 35 cycles of denaturation at 96°C (1 min), annealing at 60°C (1 min), extension at 72°C (1 min), and a final extension at 72°C for 10 min; MasterTaq polymerase from Eppendorf was used for all amplifications. The PCR products were size-fractioned on agarose gels and cloned using the pGEM-T vector system according to the manufacturer's specifications (Promega). To remove the vector backbone prior to in vitro transcription, PCR reactions with T7out (5'-AATTGTAATACGACTCACTATAGG-3') and SP6out (5'-CCAAGCTATTTAGGTGACACTATA-3') primers were performed under the same conditions as above, except for primer annealing at 55°C. After separation of PCR products by agarose gel electrophoresis and purification using the E.Z.N.A. gel extraction kit (PEQLab), 250 ng of DNA was used for in vitro transcription.
Mice at 6-8-wks of age were killed by CO2 asphyxiation and decapitated. The lower jaw and top of the skull were removed. The tissue was embedded in tissue freezing medium and frozen on dry ice. Twelve-µm coronal sections through the nasal cavity were cut on a Jung CM 3000 cryostat (Leica), adhered to Starfrost microslides (Fisher), and fixed in 4% paraformaldehyde in 0.1 M NaHCO3 for 10 min. Hybridization conditions, posthybridization washes, and detection were as described (Strotmann et al. 1994b
5' RACE
For the amplification, 1 µL cDNA was employed as template in a first amplification round. PCR was performed on an MJResearch Peltier Thermocycler PTC 200 with the following temperature profile: 1 min 40 sec 94°C, 19 cycles of 30 sec 94°C, 40 sec 70°C-0.5°C/cycle, 1 min 30 sec 68°C, and an additional 25 cycles at the same conditions as above except for an annealing at 60°C. The reaction was completed by a final extension of 10 min at 68°C. For these PCR reactions 0.4 µL Titanium Taq DNA Polymerase (BD Biosciences Clontech) was used in a total 50µL reaction cocktail containing 32 ng/µL bovine serum albumin; 10 µM of each primer, 10 µM d-NTPs each, and 1x concentrated Titanium Taq buffer. Then, 1 µL of reaction products was used in a second-round PCR using the nested primers under the following PCR conditions: 2 min 94°C, 25 cycles of 30 sec at 94°C, 30 sec at 65°C, and 2 min at 68°C; the reaction was completed by a final extension of 10 min at 68°C. The reaction mixture was the same as described above except for not using BSA. PCR products were subcloned into pGEM-T vector (Promega) and sequenced using TaqI polymerase and fluorescently labeled dideoxy-terminator chemistry on an automated sequencer (model 310, Applied Biosystems).
Electrophoretic Mobility Shift Assays (EMSAs)
Preparation of double-stranded radiolabeled DNA was done either by PCR or annealing of sticky-end-generating oligonucleotides. The mOR262-6 motif block region was amplified from an appropriate BAC clone using the primer combination 5'-CGGAATTCAGACTCACAGAGAAGCGTC-3' and 5'-CG AAGCTTGTTAGTGTTCTAGTTGTTTGGG-3' and prepared by HindIII and EcoRI restriction endonuclease digestion of the cloned fragment out of pGEMT-vector. The following oligonucleotides were used for annealing reactions: 5 ' -TCTGCAACTCTTTAATTGGATTTGTGGAACACAGGC CCTAAATTCC-3' and 5'-TGTGGAATTTAGGGCCTGTGTTCCA CAAATCCAATTAAAGAGTTGC-3' containing motif blocks I-III and 5'-TTTCGGCAAACTTTAAATTGCATCTGGGATTAA GACTGGCTCCCAAACAACTAG-3' and 5'-TTCTAGTTGTTTGG GAGCCAGTCTTAATCCCAGATGCAATTTAAAGTTTGCCG-3' containing motif blocks IV-VI. Annealing was done in a 20-µL reaction containing 1 µg of each oligonucleotide for 3 min at 65°C, 5 min at 37°C, and 10 min at room temperature. 5' overhangs of all produced double-stranded DNA fragments were used for filling up 200-400 ng DNA by Klenow polymerase in a total volume of 20 µL containing 5 mM dTTP, dCTP, dGTP, 20 µCi [ Reaction mixtures (20-45 µL total) containing 10 mM Tris-HCl, pH 7.9, 30 mM NaCl, 7% glycerol, 4.3 mM MgCl2, 1 mM EDTA, pH 8.0, 0.18 mM PMSF, 1.8 mM DTT, 1.8 mg of aprotinin per mL, 1.2 mg of leupeptin per mL, 5 µg of poly(dI-dC), and 5-20 µg of protein extract were incubated on ice in the presence of 10 fmol double-stranded radiolabeled DNA. In the competition experiments, the appropriate DNA fragments were preincubated on ice with the binding reaction mixtures 10 min prior to addition of the 32P-labeled probe. After a 20-min incubation, the reaction mixtures were mixed with the loading buffer and directly subjected to electrophoresis on 5% polyacrylamide gels (29:1, acrylamide-bisacrylamide). Before loading the gel, a 1-h 15-min prerun at 120 V and 20 mA was carried out; the dried gel was analyzed by autoradiography.
Yeast One-Hybrid Screening
Cloned PCR products were isolated from pGEM-T by using the appropriate restriction digestion enzyme combination, and purified DNA was then cloned into an EcoRI/XhoI-digested pLacZi Vector (Clontech). For generating the `bait'-plasmids `pLacZi-motif block I+II, II+III, IV, and V+VI' containing two tandem copies of distinct motif blocks, the following synthesized oligonucleotides were cloned into pLacZi EcoRV/XhoI vectors after annealing to each other as described above:
One µg of the different bait plasmids was linearized with NcoI at the URA3 locus and integrated into the yeast genome of the strain YM4271 (Liu et al. 1993 The solid-state amplification of rat olfactory epithelium MATCHMAKER cDNA library (Clontech) was done with 1.5 x 106 cfu put in 1 L 2x LB-medium containing 0.3% SeaPrep Ultralow-Gelling Soft-Agarose (FMC BioProducts) and ampicillin (100 µg/mL) at a temperature of 20°-25°C. One hundred-µL aliquots of well mixed medium were plated on LBamp-plates for DNA concentration calculation. Twenty-mL aliquots of medium were pipetted in precooled tubes and incubated for 60 min on ice until gelling of the medium. After a 30-h incubation at 37°C, the medium was centrifuged for 5 min at 8000 rpm at room temperature twice. For plasmid isolation, the `plasmid-Giga-Kit' (QIAGEN) was used. The bacterial pellet was resuspended in 125 mL buffer I (10 mM EDTA, 100 µg/mL RNAse A, 50 mM Tris-HCl, pH 8.0), mixed with 125 mL buffer II (200 mM NaOH, 1% SDS), and incubated at room temperature for 5 min and mixed well with 125 mL of buffer III (2.55 M Kac, pH 4.8). Immediately after a 30-min incubation on ice and centrifugation at 18,000 rpm at 4°C, the supernatant was centrifuged again under the same conditions and then put on a Qiagen-Tip 10000 that was equilibrated with 754 mL of QBT-buffer (750 mM NaCl, 50 mM MOPS, pH 7.0, 1,15% Triton X-100, 15% ethanol) before use. Subsequently the column was washed twice with QC-buffer (1 M NaCl, 50 mM MOPS, pH 7.0, 15% ethanol), and the DNA was then eluated with 75 mL QF-buffer (1.25 M NaCl, 50 mM Tris-HCl, pH 8.5, 15% ethanol). Precipitated plasmid DNA (adding 0.7 vol isopropanol) was pelleted for 30 min at 4°C at 14,000 rpm. After subsequent washing (10 mL 70% ethanol), 5 mL TE-buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) was added to the dried DNA. The large-scale transformation with the rat olfactory epithelium MATCHMAKER cDNA library (Clontech) was carried out according to the manufacturer's specifications using 2 mg denatured salmon sperm DNA and 20 µg amplified library DNA. Colony-lift filter assays were carried out on nitrocellulose membranes (Schleicher and Schüll, 0.025 µm) according to the `Yeast Protocols Handbook' (#PT3024-1 version #PR91200) from Clontech Laboratories.
Plasmids were isolated from yeast clones by a method modified by Kaiser and Auer (1993
Sequence Analyses
Nomenclature of OR Genes
This work was supported by the Deutsche Forschungsgemeinschaft Leibniz Programm and Sonderforschungsbereich (SFB) 495. We thank Tom Lambert for critically reading the manuscript. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1372203.
1 Corresponding author.
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ABSTRACT
RESULTS
) Shifted bands which are eliminated using the competitor DNA.(
) Bands which cannot be blocked by competitor.(B) Characterization of protein interactions with sequence motif blocks I-III (lanes 1,2) and IV-VI (lanes 3,4) from mOR262-6 using EMSAs.Each lane contains 10 fmol of 32P-labeled DNA and was incubated with 10 µg of nuclear extract from olfactory epithelium.Lanes (2,4) were pre-incubated with a 100-fold excess of unlabeled competitor DNA.(
-galactosidase reaction intensity. The strongest activation was observed for a clone encoding the `pituitary homeobox 1-factor' (Ptx-1; Drouin et al. 1998
-32P] dATP, and 1 U of Klenow enzyme for 30 min at 30°C. Reactions were stopped by adding 0.2 M EDTA, pH 7.5; excess nucleotides were removed with Micro Bio-Spin P-30 columns (Bio-Rad) according to the manufacturer's specifications. 