The clustered olfactory receptor gene family 262: genomic organization, promotor elements, and interacting transcription factors

Abstract

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.

Figure 1

(A) Overview of the genomic organization of OR genes located at cluster mOR37-II (gene names are according to Zhang and Firestein [2002]; mOR37 notations are given in brackets). Genes marked in bold are receptors from family 262 (Zhang and Firestein 2002) which lack the insertion of amino acids in the E3 loop.Arrows indicate the transcriptional orientations.Intergenic distances are given below in kilobases; numbers are rounded off to the nearest decimal place.(B) Distribution of the genomic loci accommodating OR genes on mouse chromosome 4.Positions are given in megabases (Mb) and chromosomal bands.The length of bars represents the number of OR genes at each locus.

Figure 2

In situ hybridizations of digoxigenin-labeled antisense riboprobes specific for representative OR genes from chromosome 4 on adjacent coronal sections through the mouse nasal cavity.Neurons expressing a gene from family 262 are detectable exclusively in three distinct areas of the sensory epithelium, located on endoturbinates II, III, and on ectoturbinate 3 (A,B,C).A representative gene from family 259 is distributed throughout the dorsal zone of the epithelium.(A) mOR262-2, (B) mOR262-9, (C) mOR262-1, (D) mOR259-9.Boxed area in (A) is shown in higher magnification in (B,C).ns, nasal septum.Scale bars = 500 μm in (A,D) and 200 μm in (B,C).

Figure 3

Phylogenetic tree based on the nucleotide sequences of representative OR genes located on chromosome 4.Family 262 is segregated on three different branches (1-3).Their cluster affiliation is indicated by shading: black, `mOR37' cluster I; medium gray, `mOR37' cluster II; light gray, single gene at position 57.7 Mb. The mouse melanocyte-stimulating hormone receptor (mMSH-R) was used as an outgroup.Pseudogenes are marked with an asterisk.Because the gene mOR37A was not given a new name in the study by Zhang and Firestein (2002), we named it mOR262-14.

Figure 4

Gene structures of mOR-262 genes from cluster II based on 5′ RACE experiments.All genes are aligned according to the initial methionine (position 0 kb).Boxes represent exons; lines below indicate intronic regions (lengths are given in kilobases).White blocks upstream of the initial exons mark the extension of conserved sequence stretches.Alternatively spliced isoforms are indicated by sequential numbers.Regions which are not yet sequenced are indicated with number of Ns.Numbers in brackets indicate the length of the coding sequence in bp.

Figure 5

(A) Nucleotide sequence alignment of the conserved sequences in the 5′ regions of OR genes expressed in clustered neuron populations. The alignment was generated using T-COFFEE and manually edited. Identical nucleotide positions in all sequences are shaded in black, highly conserved positions are shaded in gray. Numbered frames (I-VI) indicate characteristic sequence motif blocks; the repeated motif block I found in mOR262-9 is also boxed. Stars indicate an additional AT-rich stretch located close to the most upstream TSSs (arrows). Putative initiator elements are underlined; TATA boxes and TATA-box-like sequence stretches are indicated in italics. Olf-1-like sequence motifs are framed by dotted lines, E-boxes and E-box-like sequences by solid lines. The numbers indicate the distance in bp to the nearest TSS. (B) Array of the conserved sequence motifs identified in the 5′ region of clustered OR genes. Nucleotides spacing the sequence motifs are indicated by the double dotted lines. An X is included at positions where no motif block could be identified in one particular sequence, to uphold the conserved distance of the remaining blocks. The resulting consensus sequence of the conserved motif blocks I-VI is given below.

Figure 5

(A) Nucleotide sequence alignment of the conserved sequences in the 5′ regions of OR genes expressed in clustered neuron populations. The alignment was generated using T-COFFEE and manually edited. Identical nucleotide positions in all sequences are shaded in black, highly conserved positions are shaded in gray. Numbered frames (I-VI) indicate characteristic sequence motif blocks; the repeated motif block I found in mOR262-9 is also boxed. Stars indicate an additional AT-rich stretch located close to the most upstream TSSs (arrows). Putative initiator elements are underlined; TATA boxes and TATA-box-like sequence stretches are indicated in italics. Olf-1-like sequence motifs are framed by dotted lines, E-boxes and E-box-like sequences by solid lines. The numbers indicate the distance in bp to the nearest TSS. (B) Array of the conserved sequence motifs identified in the 5′ region of clustered OR genes. Nucleotides spacing the sequence motifs are indicated by the double dotted lines. An X is included at positions where no motif block could be identified in one particular sequence, to uphold the conserved distance of the remaining blocks. The resulting consensus sequence of the conserved motif blocks I-VI is given below.

Figure 6

(A) Characterization of protein interactions with sequence motif blocks I-VI from the putative promotor region of mOR262-6 using EMSAs. Each lane contains 10 fmol of ³²P-labeled motif block DNA and was incubated with different nuclear extracts.(Lanes 1,2) 5 μg protein from olfactory epithelium; (lanes 3,4) 10 μg from cortex; (lanes 5,6) 20 μg from liver.Lanes (2,4,6) were pre-incubated with a 30-fold excess of unlabeled competitor DNA.(•) 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.(•) Shifted bands resulting from interactions of proteins with either motif blocks I-III (left) or motif blocks IV-VI (right).

Figure 7

(A) Representation of oligonucleotides spanning the different segments of the motif blocks I-VI which were used in the yeast one-hybrid studies.The oligonucleotide containing motif block IV (dotted line) could not be utilized due to auto-activation.(B) Scheme of interactions for distinct transcription factors with the motif block region from mOR262-6.The positions of the respective motif blocks are indicated by boxes.The transcription factors are positioned at sites of their most likely interaction (underlined sequence).The consensus binding sequences from the literature are indicated below the motif block region in italics.

Figure 8

Expression patterns of O/E-2 (A) and LH-2 (B) in the olfactory epithelium revealed by in situ hybridization.Coronal sections were incubated with digoxigenin-labeled antisense riboprobes.The O/E-2 message is found in the layer of olfactory sensory neurons and basal cells. O/E-2 expression is absent from the sustentacular cell layer. LH-2-expressing cells are restricted to the deep layer of the olfactory epithelium, close to the basal membrane.sus, sustentacular cell layer; OSN, olfactory sensory neuron layer; B/IN, layer of basal cells and immature olfactory neurons.Scale bar, 50 μm.