Download Regulation of odorant receptors: one allele at a time

Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
doi:10.1093/hmg/ddi105
R33–R39
Regulation of odorant receptors:
one allele at a time
Benjamin M. Shykind*
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons,
Columbia University, New York, NY 10032, USA
Received January 5, 2005; Revised February 16, 2005; Accepted February 23, 2005
The odorant receptors (ORs) make up the largest gene family in mammals. Each olfactory sensory neuron
chooses just one OR from the more than 1000 possibilities encoded in the genome and transcribes it from
just one allele. This process generates great neuronal diversity and forms the basis for the development
and logic of the olfactory circuit between the nose and the brain. The mechanism behind this monoallelic
regulation has been the subject of intense speculation and increasing experimental investigation, yet
remains enigmatic. Recent genetic experiments have brought the outlines of the process into sharper
relief, identifying a feedback mechanism in which the first odorant receptor expressed, generates a signal
that stabilizes its choice, thus maintaining singular selection. In the absence of this signal, the olfactory
neuron re-enters the selection process and switches to choose an alternate OR. Irreversible genetic changes
in the nuclei of olfactory neurons do not accompany OR selection, which must therefore be initiated by an
epigenetic process that may involve a stochastic mechanism.
INTRODUCTION
The nose recognizes chemical information in the environment
and converts it into meaningful neural signal, allowing the
brain to discriminate among thousands of odorants and
giving the animal its sense of smell. Since the cloning of the
olfactory receptor (OR) genes from mammals (1), a molecular
logic of olfaction has emerged. In the mouse, the genes that
encode the ORs, numbering upwards of 1000 and comprising
the largest mammalian gene family, are located in clusters
which are scattered throughout the genome (2 –4). The existence of such a large complement of receptors suggested that
each odor elicits a unique signature, defined by the interactions
with a limited number of relatively specific olfactory receptors
(1). Together these form a combination of interactions more
than sufficient to encode the 104– 105 different odors
animals may perceive (5). For the brain to determine which
odorant is present, it must determine which ORs have been
activated and two key properties of the olfactory system facilitate this task. First, each olfactory neuron expresses only one
olfactory receptor. Secondly, the axons of neurons expressing
that same OR converge to one locus (the glomerulus) in the
olfactory bulb, the first relay station of olfactory information
in the brain (6 – 8) (Fig. 1). The brain can therefore determine
which receptors have been bound by identifying which
glomeruli have been activated. This creates a sensory map
in the bulb such that odor quality may be encoded by patterns
of spatial activation. This one-to-one correspondence between
receptor and glomerulus is critical for the logic of this circuit
and is dependent on the correct regulation of the OR gene
family, one receptor per neuron.
Olfactory sensory neurons are regenerated throughout the
life of the animal. Thus OR gene selection and expression,
which can be detected as early as E11.25 (9), is not synchronized with a developmental stage but rather occurs continuously as the olfactory neurons are born and exit the cell
cycle. The mechanism of OR regulation is unknown but
recent experiments have shed light on important aspects of
the process. Here, the phenomenon of OR regulation will be
summarized, models of OR selection will be discussed and
recent data about the initiation and maintenance of this singular, mono-allelic odorant receptor choice will be reviewed.
EMERGENCE OF A REGULATORY PUZZLE
The isolation of the OR genes led to a rapid molecular dissection of the biology of olfaction and allowed two fundamental
characteristics of OR expression to be revealed by RNA in situ
hybridization studies. First, neurons expressing a given
*To whom correspondence should be addressed. Email: [email protected]
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Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
Figure 1. Odorant receptor expression and axonal projections. (A) Whole mount view of a compound heterozygous mouse, age P30, genetically modified (as
shown by the schematic constructs) to express tau-lacZ and GFP (15,48) from each allele of the P2 odorant receptor gene through an internal ribosome entry site
(i), subject to anti-b-gal and anti-GFP immunohistochemistry. Neurons express P2 monoallelically (green or red cells) in the olfactory epithelium (oe), and
project their axons back into the olfactory bulb (ob) to form a glomerulus (gl, within white box). Nuclei are counterstained by Toto-3 (blue). Images were
acquired using a Bio-Rad MRC 1024ES confocal microscope and were assembled with Adobe Photoshop 7.0. (B) High power view of (boxed area in A)
the convergence of P2 axons to a glomerulus (gl, red and green fibers). Neighboring glomeruli are indicated by asterisks.
receptor are restricted to one of four broad zones running
across the olfactory epithelium; and secondly, within a zone,
individual receptors are expressed sparsely and without
apparent pattern (10 – 12). Quantitative analysis of these in
situ hybridization experiments led to the suggestion that
each neuron in the nose expresses only one or a few
members of the gene family (11,12). Subsequent analyses of
cDNAs synthesized from single olfactory neurons revealed
that just a single OR species could be isolated from each
cell and strengthened this ‘one neuron– one receptor’ hypothesis (5,13). An intriguing additional phenomenon was uncovered when ORs were shown to be transcribed from just one
allele (13). Thus a striking regulatory challenge (hypothesized
by Buck and Axel in 1991) exists for the olfactory sensory
neuron, which must select a single receptor from just one
allele of a spatially allowed subset of a widely dispersed
gene family. The logic of the olfactory circuit rests upon
this regulatory process as does the formation of the sensory
map, which is dependent on receptor protein to guide the
path-finding axon (14,15). Aberrant expression of multiple
ORs per neuron may disrupt olfactory axon guidance and
thus prevent accurate formation of the glomerular map. Importantly, once a neuron establishes its synapse in the olfactory
bulb, it must remain committed to its OR, as any change in
receptor would change the ligand specificity of the cell and
confound the sensory map by inappropriate glomerular activation. Thus OR regulation may be divided into two phases:
initiation of selection and maintenance of the choice. What
paradigms of gene expression are behind these processes?
MODELS OF OR GENE CHOICE
Two models may be proposed to explain the selection of
single OR alleles. In a deterministic model (Fig. 2), individual
OR is chosen by the generation of a unique combination of
transactivators that activate a single receptor bearing the
appropriate cis-acting elements. In this model of gene activation an additional level of regulation, perhaps akin to
X-inactivation (16), must be invoked to account for
monoallelic expression, as the same combinatorial would activate both copies of the OR. An alternative, stochastic model
(Fig. 2) supposes that the choice of receptor is random, resulting from the selection of a machine or process that is itself
singular, able to interact with one OR allele at a time. Such
an unorthodox mechanism would operate on a cis-acting
element common to all OR genes and could generate
random OR choice. A series of transgenic experiments in
mice have lent support for the stochastic model (17 – 19).
These studies revealed that OR transgenes could recapitulate
the pattern of OR expression yet remarkably, expression was
exclusive: transgenes and endogenous copies were rarely
co-expressed in the same cell (17 –19). This phenomenon,
which had only previously been observed for the antigen
receptor genes (20), is inconsistent with a deterministic mechanism which should co-express the transgene and cognate
endogenous alleles. Exclusive expression is even observed
between OR transgenes that are differentially marked and
located in the same array (19). These data are in contrast
with observations in Drosophila, where OR transgenes are
co-expressed with their endogenous, cognate copies (21).
There are conceivable evolutionary advantages to an OR regulatory mechanism that would generate exclusive expression by
default. Such a process would immediately accommodate new
receptor genes without the evolution of transactivators with
new specificities and their cognate OR promoters. The
simple duplication and sequence variation of an OR gene
would increase the functional repertoire of receptors. Such a
mechanism could have promoted the rapid growth of the OR
gene family. Is there evidence for common OR promoter
motifs required by the stochastic model? Sequence analyses
have been equivocal. Vassalli et al. identified a weakly conserved homeodomain element upstream of the start sites of
several OR genes (18). Analysis by Hoppe et al. did not find
Human Molecular Genetics, 2005, Vol. 14, Review Issue 1
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Figure 2. Two models of receptor choice. In the deterministic model (left), individual odorant receptor genes (R1, R2, . . ., R1000) are selected by unique
combinations of transacting factors (colored spheres) that recognize cognate promoter elements (shown in red, yellow, or green) specific to the OR gene. In
the stochastic model (right), individual odorant receptor genes (R1, R2, . . ., R1000) are activated by a singular entity, represented by the blue sphere.
such elements (22) and Lane et al. failed to find common OR
motifs flanking 18 OR genes of the P2 receptor cluster (23),
but did identify a common DNA element in front of 73 pheromone receptor genes, which share similar expression phenomena with the ORs. The importance of these elements awaits
functional validation. Candidate OR regulatory factors have
been identified by one-hybrid experiments and either fail to
demonstrate specific control of OR (24) or have not yet
been validated in vivo (25).
STOCHASTIC PROCESSES
What types of stochastic processes could generate singular,
mono-allelic OR choice? The conceptual and organizational
similarities between the odorant receptor and lymphocyte
receptor gene families led to the proposal that the olfactory
and immune systems may share common regulatory processes.
This led to the suggestion that DNA recombination may play a
role in the selection of ORs (1). The demonstration that OR
expression is mono-allelic and mutually exclusive, made this
explanation for OR regulation even more alluring. One recombinational model was envisioned in which a single OR gene
would be inserted into a transcriptionally active locus by
gene conversion (1,13). This mechanism participates in yeast
mating type switching (26,27) and trypanosome variable
surface glycoprotein (VSG) expression (28). However, fluorescent in situ hybridization (FISH) studies provide evidence
against this model (29). In these experiments, the site of transcription of an OR was localized by RNA FISH using OR
intronic sequences as probes. The location of both copies of
the OR gene was concomitantly visualized by DNA FISH.
In all cases, the site of OR synthesis corresponded to one of
the two alleles, suggesting that the selected OR was being
transcribed in situ and not from a remote expression site.
However, these data can rule out neither the transposition of
OR into an active locus nor the transposition of regulatory
sequences proximal to an OR. Evidence against these types
of recombination was obtained recently by two groups that
reported the generation of cloned mice from post-mitotic
olfactory neurons (30,31). They reasoned that if the mechanism of receptor selection involve permanent genomic
changes in olfactory neurons, then a donor neuron may
bestow a restricted genetic potential upon the clone, as was
observed in lymphocytes (32). These experiments, using
nuclear transfer from olfactory neurons genetically marked
to confirm their differentiated state, produced viable, clonal
mice that appeared to express the full repertoire of ORs. In
addition, the genomic structure around the selected OR locus
remained unaltered. Odorant receptor expression thus occurs
without irreversible changes to the DNA of olfactory neurons.
Absent DNA recombination governing receptor choice, what
types of random, epigenetic processes may explain OR selection? It is possible that a unique locus or factory exists in the
nucleus which may accommodate and activate just one OR
allele at a time. Such a spatial singularity has been invoked
to explain the conceptually similar regulation of the VSG
genes in trypanosomes. This mechanism involves the gene conversion of a VSG into just one of 20 expression sites (ES) in the
genome. However, the choice of a single VSG is thought to
arise from its interaction with the expression site body (ESB),
a solitary entity in the nucleus able to interact with just one
ES at a time. Switching can occur either by the VSG gene conversion into an ES in the ESB or by the switching of new ES for
old into the ESB. OR selection could be accomplished by a
similar mechanism, in which an individual OR is transcribed
by a single nuclear entity (23,33).
An alternate mechanism of OR choice could involve a ratelimiting process. In such a kinetic singularity model, receptor
choice is presumed to be an inefficient process in which only
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a single OR allele is activated on average during a given
developmental window. The cis-acting transcriptional elements associated with each OR gene may generate rare probabilistic expression (34) of ORs in the context of a repressive
chromatin environment. Such a model has been proposed to
regulate the exclusive, monoallelic expression of antigen
receptor genes (35,36).
The possibility of singular OR expression arising from the
action of a locus control region (LCR) has been proposed
(10,13). Such a mechanism possesses characteristics that
may be both spatially and kinetically limiting. Recently, an
LCR-like OR control element has been identified (37).
Although several groups have reported the expression of OR
transgenes containing a few kilobases of 50 flanking DNA
(17,18), the MOR28 receptor cluster requires .100 kb of 50
sequence (19). Present in this 100 kb region is a 2 kb
element, termed ‘H,’ that is conserved between mice and
humans (38) and is required for expression of the MOR28
cluster (37). Interestingly, when the H-element was placed
closer to the MOR28 gene in the cluster, it was dramatically
over-represented in its zone while downstream neighboring
ORs were chosen less frequently (37). The H-element does
possess hallmarks of an olfactory LCR but may not be a
central modulator of OR regulation. No other OR loci have
been shown to possess or require such an element, and it
remains unclear whether it is responsible for individual OR
choice within the cluster or rather opens an otherwise tightly
repressed locus and allows access of a stochastic selection
machinery to the OR genes.
MAINTENANCE OF OR CHOICE
Once an OR has been chosen, how does the neuron stay committed to it and prevent the expression of additional receptors?
OR choice must be stable in the neuron after it wires to the
bulb, as commitment to one receptor is critical for olfactory
coding. It must also be stable during axon guidance, when
it determines glomerular position. The answer appears to
involve feedback repression of the selection process by the
first expressed receptor. The existence of this mechanism
was recently reported by several groups (37,39 –41) who
observed it while examining a long-standing conundrum in
the field: what is the fate of an olfactory neuron that expresses
an odorant receptor pseudogene? The OR gene family is
riddled with pseudogenes (2,4), many of which are expressed
(42). If neurons that select non-functional ORs are destined to
inactivity, then the olfactory epithelium should be a mosaic of
functional and non-functional neurons. Alternatively, such
choices might doom the cell. The Sakano and Reed groups
examined this question by generating mice bearing OR transgenes containing open reading frame deletions and truncations
and marked to co-express lacZ or GFP (green fluorescent
protein) reporter genes. Neurons that chose these mutant
ORs express additional receptors at high frequency and
project their axons diffusely in the bulb. In control experiments, cells expressing intact OR transgenes did not show
the co-expression of other ORs and their axons converged to
a discrete glomerulus. This axon-wandering phenotype
was previously observed by Wang et al. in mice bearing a
replacement of the P2 receptor with lacZ generated by homologous recombination (15). Feinstein et al. (41) replaced an
OR coding region with GFP by homologous recombination
and showed that green fluorescent neurons gained promiscuous sensitivity to applied odorants compared with control
cells with an intact OR. Taken together, these results
suggest a feedback mechanism in which a functional OR mediates the stabilization of receptor choice and maintains its
status as the sole expressed OR. A non-functional receptor
cannot generate such a signal and the cell may continue to
choose until it picks an active OR.
What is the fate of expressed but non-functional OR? The
continued expression of the defective receptor after the choice
of a functional receptor would be prima facie evidence of
bi-allelic transcription and thus inconsistent with a spatial
singularity model of OR choice. However, experiments
where the expression of the marker protein is dependent on
the continued transcription of the mutant OR will not detect
cells that shut it down (37,40,41). To circumvent this limitation we used a lineage-marking strategy in which cells that
expressed a modified mutant or wild-type OR allele would
be permanently marked by Cre-mediated activation of GFP
expression from an unlinked reporter (43). Consistent with
previous studies demonstrating that deletion of OR coding
sequences by homologous recombination leads to sustained
decrease in the frequency of cells expressing the mutant OR
(15,44,45), we observed a comparable drop in neurons expressing the Cre-substituted allele. Consistent with Sakano and
coworkers (37) and Reed and Lewcock (40) we observed
efficient selection of alternate ORs in these cells which,
when coupled with the shutdown of the mutant allele,
amounted to a switching between ORs, rather than their coexpression (39). This lineage-marking strategy also let us
examine the stability of functional OR expression. Interestingly, we observed that MOR28 demonstrated instability and
switching 10% of the time it was chosen. Thus neurons that
choose either mutant or wild-type ORs may switch, albeit
with very different rates. Further, switching to a receptor
occurs at a frequency similar to that with which it is initially
chosen in the epithelium. This suggests that switching represents a re-entry into the selection process and not is mechanistically different than initial choice. The timing of shutoff of
the mutant OR allele suggests that switching likely occurs
early in the development of sensory neurons, before the OR
is required for axon targeting (39).
Thus a model emerges (Fig. 3) in which OR selection is
mediated by a stochastic mechanism involving either a singular
selection machine or a limiting kinetic process. Initially, receptor expression is unstable, but a functional OR may mediate a
feedback stabilization that commits the cell to the receptor.
This is analogous to one mechanism of allelic exclusion of
antigen receptor genes (20). Non-functional ORs are not able
to mediate the feedback signal and switching occurs as the
defective allele is shutdown and an alternate OR is chosen.
Following receptor stabilization the neuron enters a phase
characterized by commitment to one receptor. A generalized
repression of all other OR loci at this point may further ensure
the expression of just one OR. In this model, ORs may be transcribed serially, one promoter at a time. It remains possible that
the phenomenon interpreted as OR co-expression (37,40) in fact
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Figure 3. Model of OR selection and maintenance. (A) Choice of functional receptor. OR gene is selected by a stochastic mechanism (blue sphere). Receptor
protein (green) mediates a feedback that leads to stabilization of receptor choice (symbolized by small red sphere). (B) Choice of non-functional receptor. Nonfunctional OR gene (with red ‘X’) is selected by a stochastic mechanism (blue sphere). Non-functional OR protein (yellow) fails to mediate stabilization and the
non-functional receptor gene is shutdown as switching occurs and the selection process continues until functional OR is chosen.
represents a failure of the mutant transgenes to shutdown. This
could occur if cis-acting control elements are absent from the
transgene or if it integrates into a locus in the genome that
restricts the full repertoire of OR regulation. The use of homologous modifications of OR loci may more faithfully preserve
the facets of an arguably complex regulatory process.
Switching allows the olfactory genome to carry a large
number of pseudogenes without deleterious effects. These
defective copies may serve to generate new ORs by reversion
or gene conversion events. Shutoff of defective alleles,
although not required for the generation of monospecific
olfactory neurons, may be critical to protect cells from the
effects of toxic or interfering products of mutant ORs. Alternatively, switching and shutoff may be concerted as in trypanosome VSG regulation, when alleles exchange places in
singular selection machinery. What is the explanation of
the switching observed from functional ORs? The proposed
early instability of selection inherent in the process means
that occasionally, even a functional OR will not reach the
threshold level of expression needed to generate a feedback
signal, and switching will occur.
PERSPECTIVES ON MONOALLELISM
There are both developmental and perceptual rationales for the
olfactory neuron to select just a single receptor gene, yet it is
unclear why this expression must also be monoallelic. Is
monoallelism an integral part of the mechanism of receptor
choice or a consequence of it? In a deterministic model of
OR expression, there would be no mechanistic requirement
for monoallelism. Rather, it may have arisen to increase
olfactory discriminatory power (46). In this view, sequence
variation between alleles could give rise to sensory neurons
with new odor specificities. Such variation could only be
utilized through the expression of each allele individually.
Alternatively, monoallelic expression may be critical to singular OR choice (12). If the choice of a receptor cluster was
deterministic, while the selection of receptors from within
the locus was random, then the activation of both alleles of
a locus would frequently result in the expression of two different ORs in the same cell. OR regulation would therefore be
dependent on the inactivation of one of the two OR loci.
However, the recapitulation of OR expression by small transgenes suggests that selection occurs independent of the cluster.
It is perhaps more accurate to view OR choice as stochastic.
Such a process will more likely pick individual alleles, than
randomly choose two copies of the same receptor. This
leads to the consideration of monoallelism as a consequence
and not a requirement of singular gene choice. If this is true,
then the asymmetry between OR alleles, observed as an asynchrony of replication timing in fibroblasts (13), needs consideration. The observation that an OR can switch to its
other allele at the same frequency with which it is initially
chosen (39), suggests that OR alleles are equivalent with
respect to the selection process. Thus, it is possible that the
asymmetry of OR alleles is important for some biological
process unrelated to OR selection.
FUTURE DIRECTIONS
The goal of future experimentation will be to determine the
mechanism of the stochastic process that initially selects
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single OR alleles. Biochemical approaches, with in vivo validation, could be useful to identify core promoter elements in
OR genes and isolate the regulatory factors that bind and activate them. It will then be possible to determine whether this
transcriptional apparatus forms the core of the selection
machinery and is limiting in some manner, or is abundant
and gains access to only one or a few OR genes at a time.
Genetic experiments designed to test the transcriptional
permissiveness of OR loci could provide useful insights into
the process. The similarities between OR gene choice and
the regulation of antigen receptor genes may grow even
more evident in the future. In parallel with observations
made in lymphocytes (47), it will be interesting to examine
whether chromosome dynamics and subnuclear localization
may play a role in monoallelic OR expression. Neurons and
lymphocytes may possibly be sharing a common stochastic
mechanism to select single genes.
ACKNOWLEDGEMENTS
The author wishes to thank T. Cutforth, G. Barnea,
K. Baldwin, B. Datta and L. Konopasek for critical reading
of the manuscript, A. Fleischmann for helpful discussions
and R. Axel for helpful discussions, critical reading of the
manuscript and support through a grant from the NIC/NIH
No. 2 P01 CA023767.
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