Nuclear Aggregation of Olfactory
Receptor Genes Governs
Their Monogenic Expression
E. Josephine Clowney,1Mark A. LeGros,2,4Colleen P. Mosley,2Fiona G. Clowney,2
Eirene C. Markenskoff-Papadimitriou,3Markko Myllys,5Gilad Barnea,6Carolyn A. Larabell,2,4
and Stavros Lomvardas1,2,3,*
1Program in Biomedical Sciences
2Department of Anatomy
3Program in Neurosciences
University of California, San Francisco, San Francisco, CA 94158, USA
4Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
5Department of Physics, University of Jyva ¨skyla ¨, Jyva ¨skyla ¨ FI-40014, Finland
6Department of Neuroscience, Brown University, Providence, RI 02912, USA
Gene positioning and regulation of nuclear architec-
ture are thought to influence gene expression. Here,
we show that, in mouse olfactory neurons, silent
olfactory receptor (OR) genes from different chromo-
somes converge in a small number of heterochro-
matic foci. These foci are OR exclusive and form in
a cell-type-specific and differentiation-dependent
manner. The aggregation of OR genes is develop-
mentally synchronous with the downregulation of
lamin b receptor (LBR) and can be reversed by
ectopic expression of LBR in mature olfactory
neurons. LBR-induced reorganization of nuclear
architecture and disruption of OR aggregates per-
turbs the singularity of OR transcription and disrupts
the targeting specificity of the olfactory neurons. Our
observations propose spatial sequestering of heter-
ochromatinized OR family members as a basis of
monogenic and monoallelic gene expression.
Spatial compartmentalization of genes in the mammalian
nucleus is believed to serve regulatory purposes (Fraser and
Bickmore, 2007). Heterochromatin and euchromatin were origi-
nally cytological descriptions of silent and active regions of the
genome and were only later biochemically characterized (Zach-
arias, 1995). In most cell types, interactions with the nuclear
lamina locate heterochromatin at the periphery of the nucleus,
and euchromatin occupies the nuclear core (Peric-Hupkes and
van Steensel, 2010). Higher-resolution views of the nucleus
reveal additional levels of organization and compartmentaliza-
tion. For example, transcription may be restricted to specialized
nuclear regions or transcription factories where genes converge
in a nonrandom fashion (Eskiw et al., 2010). Finally, inter- and
intragenic interactions over large genomic distances create
regulatory networks that control gene expression and differenti-
ation (de Wit and de Laat, 2012; Liu et al., 2011; Montavon et al.,
Irreversible developmental decisions, such as those made by
differentiating neurons, employ diverse epigenetic mechanisms
to lock in transcriptional status for the life of a cell. Placing genes
in subnuclear compartments compatible or incompatible with
transcription could finalize these decisions. The differentiation
of olfactory sensory neurons (OSNs) provides an extreme
example of such developmental commitment; OSNs choose
one out of ?2,800 olfactory receptor (OR) alleles and subse-
quently establish a stable transcription program that assures
that axons from like neurons converge to distinct glomeruli
(Buck and Axel, 1991; Imai et al., 2010). The monoallelic nature
of OR expression (Chess et al., 1994), together with the observa-
tion that OR promoters are extremely homogeneous and share
common regulatory elements (Clowney et al., 2011), implies
that DNA sequence is not sufficient to instruct the expression
of only one allele in each neuron and that an epigenetic mecha-
nism is in place. Indeed, the discovery of OR heterochromatini-
zation argues for epigenetic, nondeterministic control of OR
choice (Magklara et al., 2011). Because active OR alleles have
different chromatin modifications from the inactive ORs (Mag-
klara et al., 2011) and associate in cis and trans with the H
enhancer (Lomvardas et al., 2006), this epigenetic regulation
might have a spatial component. Although deletion of H does
not have detectable effects on the transcription of most ORs
(Khan et al., 2011), its association with active OR alleles could
reflect the physical separation of the active OR allele from silent
OR genes and its transfer to an activating nuclear factory.
Here, we examine the significance of nuclear organization in
OR expression. Using a complex DNA FISH probe that recog-
nizes most OR loci, we demonstrate OSN-specific and
724 Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc.
tion of silent ORs. Whereas these OR-specific foci colocalize
with H3K9me3, H4K20me3, and heterochromatin protein 1
b (HP1b), the active OR alleles have minimal overlap with hetero-
chromatic markers and reside in euchromatic territories, sug-
gesting the existence of repressive and activating nuclear
compartments for OR alleles. Critical for this nuclear organiza-
tion is the downregulation and removal of lamin b receptor
(LBR) from the nuclear envelope of OSNs. Deletion of LBR
cells in the main olfactory epithelium (MOE), whereas expression
of LBR in OSNs disrupts the formation of OR foci, resulting in de-
compaction of OR heterochromatin, coexpression of a large
number of ORs, overall reduction of OR transcription, and
disruption of OSN targeting. Our analysis provides evidence for
an instructive role of nuclear architecture in monogenic olfactory
Figure 1. Visualizing the Nuclear Distribu-
tion of OR Loci
(A) Schematic of sequence-capture-based DNA
FISH probe construction.
(B) qPCR analysis of panOR library showing
enrichment for four different ORs, but not for
control genes. Error bars display SEM between
duplicate PCR wells.
(C) Microarray analysis of panOR probe. Blue bars
represent the chromosomal location of OR clus-
ters, and red represent hybridization signal inten-
sity produced by MA2C analysis using a sliding
window of 10 Kb, with minimum number of probes
20 and maximum gap of probes 1 Kb.
(D) Wide-field image of DNA FISH on MOE
sections with panOR probe. OR foci are detected
the left) and basal cells (basal layer on the right),
the DNA FISH signal is diffuse. In the zoomed-in
view on the right, we highlight the nuclear borders
of a basal cell and a neuron.
(E–H) DNA FISH on MOE sections with panOR
probe (red) and BAC probe pools (green).
OR BACs (E) colocalize with panOR, whereas
non-OR BACs (F) do not. Pooled OR BACs across
chromosome 2 (G) coalesce into optically indis-
crete signals in OSNs. Pooled OR BACs covering
clusters on eight separate chromosomes (H)
(Chr1, 2, 3, 7, 9, 14, 15, and 16) occupy the same
panOR focus. Maximum intensity Z projections of
three micron confocal stacks are shown. Borders
are drawn around nuclear edges.
See also Figure S1 and Tables S1, S2, and S4.
ORs and other AT-rich gene families
frequently associate with the nuclear
However, our DNA FISH analysis with
individual BAC probes failed to reveal
the nuclear periphery of OSNs (Lomvar-
das et al., 2006). To obtain a comprehensive view of the distribu-
tion of OR loci in OSN nuclei, we sought to generate a DNA FISH
probe that would allow the simultaneous detection of most OR
loci. First, because OR clusters reside in extremely AT-rich iso-
chores (Clowney et al., 2011; Glusman et al., 2001), we digested
genomic DNA with restriction enzymes that recognize AT-rich
sequences and collected DNA fractions with significant enrich-
ment for ORs. Next, these were amplified and subjected to
a second round of purification by sequence capture on a custom
tiling array covering OR clusters (Figure 1A) (Albert et al., 2007).
This high-density array contains oligonucleotides against the
unique sequences within the 46 OR genomic clusters, spanning
a total region of 40 MB. Two rounds of capture, elution, and
amplification produced a DNA library highly enriched for OR
sequences. Quantitative PCR analysis (qPCR) of the final ampli-
con detects only sequences from OR clusters, suggesting the
elimination of unique, non-OR DNA (Figure 1B).
Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc. 725
Figure 2. OR Foci Are Heterochromatic Aggregates from which the Active Allele Escapes
(A–C) IF for H3K9me3 (A), H4K20me3 (B), or HP1b (C) (green) combined with panOR DNA FISH (red) in MOE sections.
green), RNA polymerase II (F, blue), and H3K27-Acetyl (G, blue).
726 Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc.
To further examine the composition of this DNA library, we
analyzed its contents by whole-genome microarray hybridiza-
tion, using a tiling array covering mouse chromosomes 1 to 4.
This analysis demonstrates the sensitivity and selectivity of our
purification strategy: the probe detects 340 of 346 OR genes
located on these 4 chromosomes, 40 of ?80 non-OR genes
located cis to OR clusters (and included on the capturing array),
and 6 of ?5,000 non-OR genes (FDR < 0.05, 98.2% sensitivity,
S1B–S1D and Table S1 available online).
OSN-Specific Aggregation of OR Genes
We used this ‘‘panOR’’ library as a probe for DNA FISH experi-
in the diploid nucleus, the panOR probe detects an average of
?5 large foci in OSNs (Figure 1D). This unexpected distribution
resented in MOE sections (undifferentiated basal cells and sus-
tentacular cells) is diffuse and more consistent with a random
arrangement of the 92 OR clusters or ?2,800 alleles. Quantifica-
tion of the distribution of the DNA FISH signal in the three cell
types of the MOE across the same sections in the same experi-
ments supports this conclusion (Figure S1E–S1F): high-intensity
pixels (above 120 in the 8 bit range of 0–255) were found only in
OSNs and not in sustentacular or basal cells. To quantify the
distribution of panOR signal, we calculated standard deviation
of signal intensity across nuclear space. Average standard devi-
ation in OSNs is 42.3, indicating spotty signal distribution, and is
9.3 or 11.3 in basal and sustentacular cells, indicating smoother
distribution (n > 100 for each cell type). Finally, DNA FISH with
this probe in other neuronal types demonstrates a diffuse distri-
bution of OR loci(data notshownand Figure S2D),arguing for an
OSN-specific nuclear pattern.
The focal nature of the panOR DNA FISH signal suggests that
OR alleles from different OR clusters merge in distinct nuclear
regions during OSN differentiation. To test this, we pooled 10
OR- or 12 non-OR-BAC probes and performed two-color DNA
FISH with the panOR probe. There was extensive colocalization
between the panOR probe and OR BAC probes (Pearson’s coef-
ficient r = 0.637, Mander’s coefficient of BAC signal colocalizing
with panOR M1 = 0.835, n > 100) and little colocalization
between the panOR probe and the non-OR BACs (r = 0.187,
M1 = 0.109, n > 100) (Figures 1E and 1F and Table S2), suggest-
ing selectivity for OR loci in the composition of these aggregates
(Figure S1I). Though the panOR probe includes most OR loci,
lack of complete overlap between the panOR and the individual
OR BAC probes was expected. The panOR probe is 200-fold
more complex than each BAC, and it is outcompeted for binding
at ORs targeted by a BAC. Thus, BAC signals colocalized with
panOR signal represent OR alleles surrounded by other OR
loci labeled by the panOR probe at distances below the optical
resolution of confocal microscopy.
The combined OR BACsproduce fewerDNAFISH spots in the
OSNs (3.94 spots/nucleus/Z stack, n = 38) than in sustentacular
(9.1 spots, n = 38) or basal cells (8.52 spots, n = 30), providing an
independent verification for the extensive aggregation of these
loci: they are optically indiscrete significantly more often in
did not appear more aggregated in the OSNs (10.08 spots in
OSNs, 6.4 in sustentacular, and 7.1 in basal cells, n = 30 for
each cell type).
To explore the contribution of intra- and interchromosomal
interactions to the formation of OR foci and the colocalization
of ORs, we used two additional pools of OR BACs, one contain-
ing seven BACs targeting three clusters on chromosome 2 and
the other containing eight BACs, each targeting a cluster from
a different chromosome. These pools, when combined with
panOR probe, revealed two layers of organization in OSNs:
alleles within the same cluster coalesce into optically indiscrete
signals, whereas clusters from different chromosomes generate
and 1H). The BACs from the same chromosome produce 5.9
dots in sustentacular cells and 2.3 dots in OSNs (n = 30 for
each), whereas BACs from different chromosomes produce
equal numbers of dots in both cell types. However, multiple
OR BAC dots from different chromosomes were seen in 50%
of the panOR foci, and more than two dots per aggregate in
29% of the cells (n = 50) (Figure 1H). Moreover, maternal and
paternal alleles of the same OR cluster reside in the same OR
aggregate in ?6% of the tested OSNs (Figure S1H). Finally,
panOR foci do not colocalize with large repeat classes, pericen-
tromeric heterochromatin (PH), or other multigene families
(Figures S2A–S2C and data not shown), suggesting that the
aggregation of OR clusters produces distinct and selective OR
Spatial Segregation between Active and Silent OR
To reveal the epigenetic signature of OR foci, we combined DNA
FISH analysis with immunofluorescence (IF) against the hetero-
chromatic marks found on ORs (Magklara et al., 2011) or hetero-
chromatin-binding protein 1 b (HP1b), the only heterochromatic
HP1 member expressed in OSNs (data not shown). This analysis
HP1b (Figures 2A–2C and Table S2), but not with Pol II (Fig-
ure S2E), consistent with a heterochromatic nature of these
aggregates. This colocalization is differentiation dependent
and cell type specific; we do not detect overlap between the
two signals in basal cells in the MOE or in retinal neurons
Wethenperformed nascent RNAFISH onsections of theMOE
using intronic probes against OR genes MOR28, M50, M71, and
P2 combined with IF for H3K9me3, H4K20me3, or HP1b. In
contrast with the bulk of the panOR signal, active OR alleles
(H and I) 3D surface color plots corresponding to cells from (F) and (G), respectively. The luminance of the image is interpreted as height for the plot. Nascent OR
transcript is shown in red, HP1b in magenta, and Pol II (H) or H3K27 acetyl (I) in green.
(J) Manual colocalization counts for nascent transcript and antigens as presented in (D–G). Signals were counted as colocalized when there was some overlap
between nascent transcript and antigen. n = 150 for HP1b, 31 for H4K20me3, 12 for H3K9me3, 64 for Pol II, and 64 for H3K27-Ac.
See also Figure S2 and Table S2.
Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc. 727
Figure 3. LBR Regulates Nuclear Topology in the MOE
(A) LBR transcript levels determined by RNA-seq analysis on FAC-sorted populations from the MOE. LBR decreases from horizontal basal cells (ICAM+) to
intermediate progenitors (Ngn+) to mature OSNs (OMP+).
(B) IF against LBR in the MOE. Sustentacular and basal cells contain LBR in their nuclear envelopes, whereas OSNs do not. Occasional LBR+ cells in the neuron
layer are migrating nonneuronal cells.
panOR foci in Ichthyotic animals. (D) High-magnification image of a control and an Ichthyotic sustentacular cell.
728 Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc.
have little overlap with any of the three heterochromatic marks
(Figures 2D–2J, S2F, and S2G and Table S2). We also combined
nascent RNA FISH with IF for Pol II or H3K27-Acetyl and
H4K20me3 or HP1b (Figures 2F–2J and Table S2). These exper-
iments corroborate that the active OR allele is spatially segre-
gated from the silent ORs and resides in euchromatic territory.
LBR Organizes the Topology of OSN Nuclei
It is intriguing that most OR genes and PH are located near the
center of OSN nuclei instead of being distributed toward the
nuclear envelope (Figure S1A). This ‘‘inside-out’’ nuclear
morphology is reminiscent of the nuclear architecture reported
in homozygous Ichthyosis mice, a spontaneous LBR loss-of-
function mutant (Goldowitz and Mullen, 1982). LBR is a nuclear
envelope protein that interacts with HP1 and heterochromatin
(Hoffmann et al., 2002; Okada et al., 2005; Pyrpasopoulou
et al., 1996). RNA-seq revealed a continuous reduction in LBR
mRNA levels during differentiation from HBCs to OSNs, and IF
confirmed that whereas LBR is present in the nuclear envelope
of basal and sustentacular cells, it is absent in the neuronal
lineage of the MOE (Figures 3A, 3B, and S3A).
PanOR DNA FISH on MOE sections from the Ichthyosis mice
revealed no changes in OR aggregation in OSNs, which already
lack LBR. However, nuclear architecture and OR organization of
Ichthyotic basal and sustentacular cells approach that of wild-
type OSNs (Figures 3C, 3D, and S3B). PH forms large, centrally
located foci in both cell types, and ORs form aggregates at the
periphery of the pericentromeric foci. According to the pooled
BAC assay in the Ichthyosis mouse, the number of DNA FISH
spots is uniform among the three cell types, and the basal and
sustentacular cells have similar numbers of DNA FISH spots to
control OSNs (Figure 3E), supporting a role for LBR downregula-
tion in OR aggregation. Because ectopic OR aggregation occurs
in two cell types that do not express ORs and likely do not
contain the transcription factors responsible for OR activation,
an effect of this mutation on OR expression and OSN targeting
is unlikely and was not detected (data not shown and
Thus, we sought to perform the opposite experiment: to
restore LBR expression to OSNs instead of removing LBR from
cells that do not express ORs. We generated a tetO LBR-
IRES-GFP transgenic mouse that we crossed to OMP-IRES-
tTA mice to achieve expression of LBR in OSNs. One transgenic
line expresses the transgene in a significant proportion of OSNs
(Figure 3F). Like endogenous LBR, transgenic LBR is restricted
to the nuclear envelope without diffusing in the nucleoplasm
We used this transgenic line to analyze the effects of ectopic
LBR expression on the nuclear morphology of mOSNs. DAPI
staining becomes less intense, and PH is moved toward the
nuclear periphery of LBR+ OSNs (Figures 3G and S3D). OSNs
in these sections that do not express the transgene have
morphology similar to wild-type nuclei (Figure S3D). IF shows
HP1b recruitment to the nuclear envelope in LBR+ OSNs,
whereas centrally shifted euchromatin occupies most of the
nucleus (Figures 3H, S3E, and S3F). Thus, ectopic LBR expres-
sion in a postmitotic cell is sufficient to reverse the ‘‘inside-out’’
arrangement and to recruit PH to the nuclear periphery.
Ectopic LBR Expression Decondenses OSN
IF does not provide information about the structural and
biophysical changes occurring in OSN chromatin upon ectopic
LBR expression. To obtain this information, we imaged control
and LBR+ OSNs with soft X-ray tomography (SXT), a high-reso-
lution imaging method that is applied to fully hydrated, unfixed,
and unstained cells and measures carbon and nitrogen concen-
tration in biological samples (McDermott et al., 2009). Orthosli-
ces (computer-generated sections) and three-dimensional (3D)
reconstructions of SXT imaging of control OSNs reveal that the
more condensed (darker) chromatin is located at the center of
the nucleus, in agreement with the morphology seen by IF
condensed structures at the periphery of this PH core that are
specific forthiscelltype;only spermnuclei havechromatinparti-
cles with higher compaction values (data not shown). Although
the arrangement of these dark foci is similar to the arrangement
of theOR foci aroundthe PHcore of the OSN nucleus,DNAFISH
or IF are incompatible with SXT, and therefore it is impossible to
prove directly that these are the same structures.
SXT imaging of LBR+ OSNs shows the relocation of the most
condensed chromatin toward the nuclear membrane (Figures
4D, 4E, and 4H and Movie S2). Moreover, LBR expression
increases the nuclear volume from 105u3to 135u3and induces
the folding of the nuclear membrane and an overall change in
the nuclear shape (Figures 4C and 4F and Movies S3 and S4).
Overall, chromatin decondensation induced by LBR expression
in OSNs is quantitatively described by measurements of the
linear absorption coefficient (LAC) (McDermott et al., 2009) of
control and LBR+ OSNs (Figure 4I). This measurement, which
depicts the concentration of organic material per voxel, corrobo-
rates the loss of the densest foci upon LBR expression. Thus, if
condensed regions correspond to OR foci, ectopic LBR expres-
sion should cause decompaction of OR heterochromatin.
DNaseI sensitivity experiments (Magklara et al., 2011) in nuclei
from fluorescence-activated cell-sorted (FAC-sorted) control or
centromeric heterochromatin upon LBR expression (Figure 4J).
(E) Quantification of number of optically discrete foci formed by a pool of OR BAC probes (as in Figure 1E) in sustentacular, OSN, and basal cell types in control
and Ichthyotic MOE sections. n R 30 for all groups. p < 0.0001 for comparison between OSN and sustentacular or basal cells in control animals and for
comparison across sustentacular or across basal cells in control versus Ichthyosis tissue (Student’s t test).
OE; high-magnification image at right shows that GFP (green) and LBR (red) are coexpressed and transgenic LBR is restricted to the nuclear envelope.
(G) False-color image of DAPI staining in LBR-expressing OSNs versus control OSNs shows loss of OSN-specific PH core (gold) upon LBR expression.
(H) IF in control and LBR+ animals for H3K4-Me1 and HP1b shows reorganization of the OSN nucleus upon LBR expression.
See also Figure S3.
Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc. 729
Figure 4. Soft X-Ray Tomography of OSNs Demonstrates Chromatin Decompaction and Nuclear Reorganization upon LBR Expression
(A) An orthoslice from the tomographic reconstruction of a GFP+ neuron from an OMP-IRES-GFP mouse. PH (asterisk) surrounded by condensed, OSN-specific
foci can be seen in the center of the nucleus; only small amounts of heterochromatin are tethered to the nuclear envelope.
(B and C) Segmented nucleus (blue; obtained by manually tracing the nuclear envelope through all orthoslices of the reconstruction) seen in a 3D cutaway view
with three orthogonal orthoslices (B) shows that the pericentromeric heterochromatin is in the center of the nucleus and the nuclear envelope is not folded (C).
(D) Orthoslice from an OMP-IRES-tTA; tetO LBR-IRES-GFP mouse. The dark particles that surround the PH core in the control OSN nucleus are not present in
LBR-expressing nucleus. PH (arrow) is positioned just beneath the highly folded nuclear envelope upon LBR expression. The nuclear envelope is thicker, likely
due to the presence of LBR and the recruitment of heterochromatin.
(E and F) Three-dimensional cutaway view showing the increased nuclear volume (E) and marked folding of the nuclear envelope, which is more apparent in the
surface view (F).
(G) Still frame of Movie S1 from the control OSN shown in (A). The nucleus was segmented from the tomographic reconstruction using the 3D linear absorption
coefficient(LAC).Itis shownhere usingatransparentsurfaceview torevealthechromatin.Toaid visualization,theopacity and color oftheobtained surfacewere
730 Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc.
Ectopic LBR Expression Disrupts OR Aggregation
The spatial reorganization of HP1b, the elimination of the dark
foci detected by SXT, and the increase in DNase sensitivity of
OR chromatin suggest that ectopic LBR expression disrupts
the aggregation of OR loci. To test this, we performed DNA
FISH with the panOR probe in sections of LBR-expressing trans-
genic mice. Low-magnification images show significant effects
of ectopic LBR expression on the distribution of OR loci. In the
apical LBR+ neuronal layer, the intense OR foci dissolve; in
mapped to a 3D color field with the same dimensions as the whole-cell data set. The color field and color map were chosen to highlight the condensed chromatin
(H) Still frame of Movie S2 from the LBR+ OSN shown in (D). The nucleus was segmented as in (D), and the color coding depicts the same LAC values. The color
field and color map highlight the acentric, condensed chromatin abutting the nuclear envelope. There is also a notable reduction in the volume of condensed
chromatin (brown) and complete loss of the most condensed OSN-specific foci.
(I) Histogram of linear absorption coefficients of each voxel in control and LBR-expressing mOSNs. Dense voxels (LAC > 0.4 um?1) that are OSN specific and
depict the most compacted chromatin are lost in LBR-expressing nuclei.
(J) qPCR analysis of DNase digestion time course assay in sorted control OMP+ (OMP-IRES-GFP) or LBR+ (OMP-IRES-tTA; tetO LBR-IRES-GFP) olfactory
neurons. OR (orange, blue) and satellite (red) loci that are heterochromatinized and DNase resistant in control OSNs are DNase sensitive in LBR+ OSNs. Control
euchromatic sequence (Omp, green) is DNase sensitive in both cell types. Pale shades denote LBR+ cells, and dark shades denote control cells. Error bars
display SEM between duplicate PCR wells. Similar results were obtained from biological replicates.
See also Movies S1,S2,S3,S4.
OMPitTA; G 8 tTA; tetO LBRiGFP
Enrichment relative to decrosslinked
OMPitTA; G 8tTA
OMPitTA; G 8tTA;
4C from H Element
Enrichment vs. input
Enrichment vs. input
Figure 5. LBR Expression Disrupts OR Foci
MOE sections from control (left) and LBR-ex-
pressing transgenic mice (right). Line depicts the
borders between immature and mature OSNs.
Transgenic LBR expression driven by OMP-IRES-
tTA is restricted to the mature OSNs, where OR
foci are disrupted.
H4K20me3 (C) enrichment in FAC-sorted control
(OMP-IRES-GFP, blue bars) or LBR-expressing
(OMP-IRES-tTA; tetO LBR-IRES-GFP, red bars)
OSNs. Error bars display SEM between duplicate
PCR wells. Similar results were obtained from
(D) IF for HP1b (red) combined with panOR DNA
FISH (green) in MOE sections from LBR-express-
ing transgenic mice. The panels on the left depict
an immature neuron that has not expressed the
transgene yet, whereas the panels on the right
show an LBR+ OSN from the same section. OR
loci lose their association with HP1b upon LBR
(E) qPCR analysis comparing the enrichment of
OR sequences in a 4C library constructed by
inverse PCR from the H enhancer from wild-type
and LBR-expressing MOEs. Enrichment values
were normalized to control material decrosslinked
before ligation. Error bars display SEM between
duplicate PCR wells.
See also Figure S4 and Table S2.
contrast, immature OSNs and progeni-
torsthatdo not yet express the transgene
but have already downregulated the
endogenous LBR retain a focal OR
arrangement (Figures 5A and S4A).
To investigate whether altering the tertiary organization of
OR loci affects the epigenetic characteristics of these genes,
we examined association of OR genes with H3K9me3,
H4K20me3 remained enriched on OR loci upon LBR expression
by native ChIP-qPCR assays on FAC-sorted OSNs (Figures 5B
and 5C) and FISH-IF (Figures S4B and S4C and Table S2). In
contrast, association of OR loci with HP1b was reduced as
measured by FISH-IF (Figures 5D and Table S2). Reduction in
Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc. 731
overlap between H4K20me3 and HP1b was also observed in the
LBR+ OSNs. Thus, despite retaining heterochromatic histone
marks, OR loci lose their aggregated arrangement and their
nonhistone heterochromatic coat upon LBR expression, which
is consistent with the increased DNase sensitivity.
In wild-type OSNs, active OR alleles interact with the H
enhancer. To test whether LBR expression also abrogates inter-
chromosomal interactions between the active allele and H, we
performed circularized chromosome conformation capture (4C)
using inverse H PCR primers as previously described (Lomvar-
das et al., 2006) on LBR-expressing or control MOEs. To
increase the proportion of LBR-expressing cells in this mixed
population, we combined two tTA drivers (OMP-IRES-tTA and
Gg8 tTA). The enrichment of various OR sequences in this 4C
library was assayed by qPCR. LBR expression in OSNs results
in the loss of most H-OR associations. In LBR transgenics, H
retains only its interaction with the linked OR MOR28 (Olfr1507)
Fold change in relative
transcripts upon LBR re-
OR M50, MOR28, C6 IF MOR10, MOR83 RNA FISH
OMPitTA; tetO LBRiGFP
Figure 6. LBR Expression Inhibits OR Tran-
(A) RNA FISH and IF against pools of ORs in MOE
sections from control or LBR-expressing trans-
(B) qRT-PCR in FAC-sorted GFP+OSNs from
IRES-GFP mice. qRT-PCR values from each cell
population were normalized to actin (which is not
affected by LBR expression), and the results are
shown as fold difference (LBR-expressing OSNs/
control OSNs). Error bars display SEM between
duplicate PCR wells, and similar results were ob-
tained from biological replicates.
(C) Whole-mount X-gal staining of MOEs from
control or LBR-expressing P2-IRES-tLacZ mice.
(D) IF for b-gal and GFP in MOE sections from
positive neurons do not express the transgenic
LBR, as shown by the absence of GFP signal.
(E) RNA-seq analysis of ORs in control versus
See also Figure S5.
located 75 Kb downstream (Figure 5E).
Therefore, ectopic LBR expression in
OSNs not only prevents heterochromatic
OR aggregation, but also disrupts the
interaction between the H enhancer and
LBR Expression Inhibits OR
IF and RNA FISH experiments in MOE
sections from control and LBR-express-
ing mice revealed a 3-fold reduction in
the numbers of neurons expressing
ure 6A). Importantly, most neurons that
retain high-level OR expression do not
express transgenic LBR (data not shown
and Figure 6D). For more quantitative
measure of the effects of LBR in OR expression, we used FAC-
sorting to isolate control or LBR+ OSNs and performed quantita-
tive, reverse-transcriptase PCR (qRT-PCR). This analysis
supports that LBR expression has significant inhibitory effects
ing in MOEs from P2-IRES-tLacZ mice crossed to LBR-express-
sive effect on OR expression (Figure 6C). Neurons that retained
high b-gal protein expression often failed to express the LBR
transgene, as demonstrated by IF for b-gal and GFP in sections
of these mice (Figure 6D). Because OMP drives LBR expression
only after OR choice, this result indicates postchoice downregu-
lation of this P2 allele and the rest of the OR repertoire.
To test whether the inhibitory effects of LBR expression apply
to genes that do not follow the spatial regulation of endogenous
OR genes, we used a transgenic OR that is under the control of
the tetO promoter (tetO MOR28-IRES-tLacZ). This transgene
732 Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc.
also carries H3K9me3 and H4K20me3 (data not shown), but
unlike the endogenous ORs, its heterochromatization is not
OSN specific and is probably caused by its multicopy (16
tandem copies) insertion (Garrick et al., 1998). This transgene
does not interact with either the endogenous ORs or the H
enhancer (Figure S1I and data not shown). In agreement with
the repressive signature of this transgene, its expression is
sporadic when crossed to the OMP-IRES-tTA driver but
increases in frequency when crossed to LBR-expressing trans-
genics (Figure S5B). This is consistent with a simple mode of
gene regulation in which chromatin decompaction allows tTA
binding on the tetO promoter in more cells and transcriptional
activation at higher frequency; such a linear and straightforward
model does not apply to the endogenous ORs.
To determine the genome-wide effects of LBR expression in
OSNs, we performed RNA-seq from whole MOE preparations.
To increase the proportion of LBR+ OSNs in this mixed popula-
tion, we combined two tTA drivers (OMP-IRES-tTA and Gg8
tTA). In agreement with the observations in the FAC-sorted
neurons, LBR expression in OSNs induces an ?8-fold downre-
gulation of total OR expression (Figure 6E). Though OR expres-
Figure 7. LBR Expression Induces OR Co-
expression and Ectopic Targeting to the
(A) Schematic of single-celldegenerate OR digest.
(B) Agarose gel electrophoresis of degenerate OR
PCR amplicons from single-cell cDNA libraries
prepared from sorted control (OMP-IRES-GFP) or
LBR-overexpressing (OMP-IRES-tTA; tetoLBR-
IRES-GFP) OSNs. Amplicons digested with DraI
(marked with D), HinfI (marked with H), MseI
(marked with M), or undigested (marked with U)
from five control and five LBR+ cells are shown.
(C) IF for b-gal (red) and ORs M50, M71, and C6
(green) inMOE sections from OMP-IRES-tTA; Gg8
tTA; tetOLBR-IRES-GFP; tetOMOR28-IRES-LacZ
mice. Two percent of the neurons expressing one
of the three endogenous ORs (n > 1,000) are b-gal
of control and LBR-expressing P2-IRES-tLacZ
mice. Medial P2 glomerular region is shown.
Axons from the LBR-expressing neurons are also
GFP positive (green). Nuclei are counterstained
See also Figure S6 and Table S3.
sion is downregulated, most of the genes
detected in OSNs (?14,000 genes) are
not affected by ectopic LBR expression
significantly upregulated, Cuffdiff FDR
0.05, genes with at least 10 reads). Inter-
estingly, expression of some markers of
immature OSN and progenitor cell popu-
lations increases in these animals (Fig-
ure S5A). Overall, these changes suggest
a partial transition toward a less-differen-
the lack of increased apoptosis in the transgenic MOE
Ectopic LBR Expression Disrupts the Singularity of OR
Downregulation of OR transcription in LBR+ neurons is counter-
intuitive considering that the accessibility of OR chromatin
tion of long-range interactions with activating enhancers, like H,
could contribute to this downregulation. Moreover, although
global decompaction of OR chromatin might make all of the
OR alleles transcriptionally competent, it is possible that OSNs
cannot support transcription of ?2,800 OR alleles at the levels
of a singularly transcribed OR. To test this, we performed
single-cell RT-PCR with degenerate OR primers, followed by
restriction enzyme digestion and electrophoresis (Buck and
Axel, 1991; Figures 7A and S6A). We obtained 10 single-cell
cDNA libraries from each genotype (see Extended Experimental
Procedures) and examined OR representation by DraI, HinfI, and
Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc. 733
The complexity of the degenerate OR amplicons is different
between control and LBR+ OSNs. In every LBR+ OSN, the
base pair sum of the individual digestion products exceeds the
length of the undigested PCR, whereas control amplicons
contain only one product (Figure 7B, showing five amplicons
from each genotype. Similar results were obtained for the other
five amplicons and with MboI digestion [data not shown]).
Sequencing 10 clones each from two control and two LBR-ex-
pressing amplicons verifies that the OR transcriptome is more
complex in LBR+ OSNs. In both control amplicons, all 10 clones
were identical, and the sequence of the cloned OR matches the
digestion pattern. In the case of the LBR+ neurons, 10/10 and
9/10 clones of each amplicon were unique and different from
each other. Moreover, the sequences of these clones did not
match their digestion pattern, suggesting that these libraries
are very complex and the observed distinct bands represent
comigrating bands of similar size digested from a large number
of different ORs. To verify this, we sequenced 96 independent
colonies from a third LBR+ single-cell library and identified 46
different ORs from 11 chromosomes without any zonal restric-
tions (Table S3). Thus, LBR expression in OSNs violates the
‘‘one receptor per neuron’’ rule and induces coexpression of
a large number of ORs. To exclude that multiple ORs are ex-
pressed in low levels also in control OSNs but are masked by
the highly expressed chosen allele, we pooled equal volumes
of the single-cell RT reaction from a control and an LBR+ neuron
and performed degenerate PCR and digest. Although the OR
amplicon from the control neuron dominates the reaction, the
ectopically coexpressed ORs from the LBR-expressing neuron
are still detectable upon digestion (Figure S6B).
For a non-PCR based confirmation for OR coexpression in
LBR+ OSNs, we performed IF for ORs M50, M71, and C6 in
tetO-LBR/tetO-MOR28 double transgenics. Most OSNs that
retain detectable OR levels by IF are LBR negative; thus, in
these OSNs, OR coexpression is extremely rare. To overcome
this, we exploited the frequent expression of transgenic
MOR28 in the LBR-expressing mice. IF in MOE sections from
double transgenics revealed double-positive OSNs (Figure 7C),
which are not detected in the absence of LBR, as previously
shown (Fleischmann et al., 2008; Nguyen et al., 2007). Most
likely, these OSNs are shutting down the endogenous OR in
response to LBR expression while activating the decondensing
The identity of the expressed OR allele instructs the targeting
reason, we examined the targeting of neurons expressing the
P2-IRES-tLacZ allele in LBR transgenics by IF for b-gal in olfac-
tory bulb sections. b-gal protein is stable in the axons long after
the cytoplasmic signal has faded (Figure S6D); thus, we can use
this approach to examine the targeting consequences of LBR
expression. In control mice, the b-gal-positive fibers coalesce
in distinct glomeruli (Figure 7D), with very few axons targeting
wrong glomeruli (extreme example shown in Figure S6C).
However, upon LBR expression, b-gal-positive fibers extend to
an extraordinary number of glomeruli (?30 per hemisphere per
mouse). We detected extra distinct glomeruli and stray fibers
both near wild-type P2 glomeruli and in ectopic positions in the
bulb (Figures 7D and S6C).
We examined the role of nuclear architecture in monogenic OR
expression. Using a complex OR-specific DNA FISH probe, we
showed that OR genes converge into approximately five
distinct and seemingly exclusive foci surrounding the PH core
of the OSN nucleus. These foci contain frequently superim-
posed OR locifromthesamechromosomeandopticallydiscrete
OR clusters from different chromosomes. The OR allele tran-
scribed in each OSN is absent from these foci. Although low-
frequency interactions between OR clusters from chromosome
7 occur in embryonic liver and brain (Simonis et al., 2006), the
widespread and differentiation-dependent interchromosomal
aggregation and focal organization of the whole-OR subgenome
may be unique to the OSN lineage. Thus, our experiments
suggest that a primary epigenetic signature is reinforced by
secondary and tertiary repressive organization: intrachromoso-
in OSNs. The importance of this elaborate arrangement is shown
LBR and PH as Organizers of OR Aggregation
A loss-of-function LBR mutation results in ectopic OR aggrega-
tion in basal and sustentacular cells. Conversely, LBR expres-
sion in OSNs reverses the nuclear morphology and disrupts
OR foci. Thus, regulation of LBR expression governs the spatial
aggregation of OR genes in the MOE. LBR could act directly on
ORs (through binding to HP1) and indirectly by recruiting peri-
centromeric heterochromatin to the nuclear envelope. ORs
were not recruited to the nuclear envelope as efficiently as PH.
The smaller size of OR clusters and their genomic embedding
in euchromatin might make them less mobile than the acrocen-
tric PH, which is robustly recruited to the nuclear periphery.
Moreover, gene relocation to the nuclear envelope requires
cell division (Zullo et al., 2012), which does not occur in OSNs.
In any case, in wild-type OSNs, PH could provide a platform
on which OR aggregates are formed upon LBR downregulation,
and in LBR+ OSNs, PH relocation might help to untangle OR
aggregates. The final biochemical outcome of this rearrange-
ment is decompaction of OR heterochromatin, demonstrated
by reduction of LAC values in SXT and increased DNaseI
Nonspecific effects of ectopic LBR expression cannot be
excluded, although most non-OR genes are unaffected by
ectopic LBR expression. Genes known or suspected to activate
OR expression, like Emx2, Lhx2, and Ebf family members (Fuss
and Ray, 2009), are either upregulated or unaffected by LBR
expression (Figure S5A), making secondary effects an unlikely
cause of OR downregulation. Moreover, LBR’s weak enzymatic
activity, which produces ergosterol, should not participate in OR
regulation, as the Ichthyosis mouse does not have OR expres-
sion deficits. Furthermore, the enzymes that produce the
substrate for LBR are both expressed at very low levels in
OSNs (data not shown);thus, LBR is notthe rate-limiting enzyme
in this pathway, and its upregulation would not affect ergosterol
734 Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc.
Spatial Regulation of OR Expression
The fact that disruption of OR aggregation results in coexpres-
sion of multiple OR genes indicates that this organization is crit-
OSN. It also implies that the heterochromatic marks found on
ORs, which remain enriched on these loci upon LBR induction,
are not sufficient to prevent basal transcription in the absence
of higher-order folding of these chromatin regions. As in the
phenomenon of transcriptional squelching, however (Gill and
Ptashne, 1988), we made the counterintuitive observation that
LBR induction in OSNs causes a significant overall reduction of
OR transcription while allowing the coexpression of multiple
alleles. This suggests that the process of OR choice is concep-
tually more complicated than, for example, the regulation of
the tetO MOR28 transgene, and the extreme number of OR
alleles might be a contributing factor. We propose that the
unprecedented number of genes that share similar transcription
factor binding motifs (Clowney et al., 2011) makes the effective
cloaking of most of these alleles imperative for the high-level
transcription of one allele. Thus, the heterochromatinization of
most OR loci and their aggregation into large nuclear foci not
only assures their effective silencing, but also conceals thou-
sands of transcription factor binding sites that could sequester
activating proteins from the chosen allele. Finally, the identifica-
tion of multiple ORs in each LBR+ neuron may reflect a contin-
uous switching process (Shykind et al., 2004) caused by the
downregulation of the initially chosen OR and the inability to
make a new, productive OR choice.
Genomic competition may not be the only reason for OR
downregulation upon LBR expression. An equally elaborate
network of interchromosomal interactions could be involved in
the activation of a single OR allele. Consequently, escape from
the heterochromatic foci might not be sufficient for activated
OR transcription. The OR gene might also need to be reposi-
tioned to a specialized, transcription-competent interchromo-
nos, 2008). Consistent with this is the fact that the active OR
allele is often found adjacent to the heterochromatic foci. This
could imply that OR aggregation not only silences OR alleles,
but also organizes some of them—probably those located on
theperiphery offoci—for activation. Poising or organizing aggre-
gated ORs for future activation may provide a reason behind the
selectivity of these foci for OR sequences. Thus, a nuclear over-
haul induced by LBR expression would also disrupt activating
interactions between long-distance enhancers and the chosen
OR allele, resulting in OR downregulation. The observation that
LBR expression disrupts the trans interactions between H and
ORs is consistent with such a model. Although there is no
genetic evidence for the requirement of simple trans interactions
of interchromosomal interactions might govern OR activation, as
is the case for OR silencing.
Nuclear Reorganization in Development
Differences in nuclear topology can be seen in many sensory
epithelia (data not shown), and regulation of LBR expression
may orchestrate some of them. Although reorganization of the
nucleus might serve additional functions (Solovei et al., 2009),
it could be critical for the execution of tissue-specific differentia-
tion modules and may permanently lock in gene expression
programs as they occur. Thus, at the highest level of chromatin
organization, the epigenetic ‘‘landscape’’ becomes a physical
landscape where particular genes and regulatory sequences
arehidden or exposed in accordance with the cell type and func-
tion. Future experiments will reveal whether spatial regulatory
mechanisms similar to the ones described here apply to less
extreme developmental decisions that do not involve choosing
one out of a thousand alleles.
Mice were housed under standard conditions in accordance with IACUC
iments were performed on postnatal day 6 (p6)–p10 animals. DNA FISH exper-
iments shown here were performed on p14–p21 animals, and staining patterns
were confirmed in younger (p7) and older (6 week) animals. IF, X-gal, sorting,
RNA-seq, SXT, and biochemical experiments were performed in 4- to 8-week-
old animals. Fortheconstruction ofthetetO-LBR-IRES-GFP mouseand strains
used in this paper, see the Extended Experimental Procedures.
Captured DNA FISH Probe Construction and Microarray Analysis
in schematic Figure 1A and detailed in the Extended Experimental Procedures
and Table S1.
DNA FISH, Immuno-DNA FISH
DNA FISH experiments were performed as described previously (Lomvardas
et al.,2006)with modifications described inthe Extended ExperimentalProce-
dures and Tables S4 and S5.
Microscopy and Image Analysis
Confocal images were collected on a Zeiss LSM700. Channels have been
pseudocolored here for consistency and visibility. Details can be found in
the Extended Experimental Procedures.
Neurons were dissociated using papain dissolved in neurobasal A medium
supplemented with HEPES, glutamine, and methylcellulose for 30–45 min,
after which the reaction was stopped with addition of albumin. Cells were
washed, filtered, loaded into capillaries, and imaged as described previously
(Uchidaet al., 2009). Statistical analyseswere carried out using the Amira soft-
ware package (Mercury Computer Systems).
Immunostaining and Antibodies
IF wasperformedunder standard conditions on MOE cryosections; antibodies
used are described in Table S5.LBR IF was performed with acustom antibody
against mouse LBR (Olins et al., 2009). See also the Extended Experimental
DNase Assay and Native ChIP
DNase assay and native ChIP were performed as described previously (Mag-
klara et al., 2011). See also Table S6.
4C was performed as described previously (Lomvardas et al., 2006). After
inverse PCR, products were analyzed for enrichment by qPCR.
(Magklara et al., 2011). qRT-PCR primer sets are listed in Table S6. For RNA
FISH, RNA-seq, X-gal staining, and single-cell RT-PCR analysis, see the
Extended Experimental Procedures.
Cell 151, 724–737, November 9, 2012 ª2012 Elsevier Inc. 735
Microarray analysis of panOR probe has been deposited in GEO under acces-
Supplemental Information includes Extended Experimental Procedures, six
figures, six tables, and four movies and can be found with this article online
We would like to thank Dr. Nicholas Ryba for the Gg8 tTA transgenic mice;
Drs. Monica Zwerger and Harald Herrmann for the anti-LBR antibody; David
Lyons and Drs. Richard Axel, Keith Yamamoto, and David Agard for input
and suggestions;andDr.NiraoShahandtheLomvardas labforcritical reading
of the manuscript. Also, we are grateful to Dr. Allan Basbaum for making the
Shidduch between S.L. and C.A.L. E.J.C. and E.C.M.-P. are supported by
fellowships from the National Science Foundation (NSF GRFP). This project
was funded by the Roadmap for Epigenomics grant 5R01DA030320-02 and
the McKnight Endowment for Neurosciences. National Center for X-Ray
Tomography is funded by grants from the National Center for Research
Resources (5P41RR019664-08) and the National Institute of General Medical
Sciences (8 P41 GM103445-08) from the National Institutes of Health and
US Department of Energy, Office of Biological and Environmental Research
(DE-AC02-05CH11231). G.B. is a Pew scholar and is supported, in part, by
NIH grant 5R01MH086920.
Received: January 19, 2012
Revised: May 18, 2012
Accepted: September 26, 2012
Published: November 8, 2012
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