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• W2045974768 abstract "Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from different chromosomes converge in a small number of heterochromatic foci. These foci are OR exclusive and form in a cell-type-specific and differentiation-dependent manner. The aggregation of OR genes is developmentally 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 perturbs the singularity of OR transcription and disrupts the targeting specificity of the olfactory neurons. Our observations propose spatial sequestering of heterochromatinized OR family members as a basis of monogenic and monoallelic gene expression.PaperClip/cms/asset/b92ebf36-04ea-4811-ba3a-d5a240c69c68/mmc9.mp3Loading ...(mp3, 3.07 MB) Download audio Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from different chromosomes converge in a small number of heterochromatic foci. These foci are OR exclusive and form in a cell-type-specific and differentiation-dependent manner. The aggregation of OR genes is developmentally 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 perturbs the singularity of OR transcription and disrupts the targeting specificity of the olfactory neurons. Our observations propose spatial sequestering of heterochromatinized OR family members as a basis of monogenic and monoallelic gene expression. In olfactory neurons, OR genes converge in specialized heterochromatic foci The active OR allele in each neuron is transcribed outside of these foci Lamin B receptor (LBR) downregulation allows OR aggregation in olfactory neurons Ectopic LBR expression disrupts OR foci and causes OR coexpression Spatial compartmentalization of genes in the mammalian nucleus is believed to serve regulatory purposes (Fraser and Bickmore, 2007Fraser P. Bickmore W. Nuclear organization of the genome and the potential for gene regulation.Nature. 2007; 447: 413-417Crossref PubMed Scopus (577) Google Scholar). Heterochromatin and euchromatin were originally cytological descriptions of silent and active regions of the genome and were only later biochemically characterized (Zacharias, 1995Zacharias H. Emil Heitz (1892-1965): chloroplasts, heterochromatin, and polytene chromosomes.Genetics. 1995; 141: 7-14PubMed Google Scholar). 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, 2010Peric-Hupkes D. van Steensel B. Role of the nuclear lamina in genome organization and gene expression.Cold Spring Harb. Symp. Quant. Biol. 2010; 75: 517-524Crossref PubMed Scopus (60) Google Scholar). Higher-resolution views of the nucleus reveal additional levels of organization and compartmentalization. For example, transcription may be restricted to specialized nuclear regions or transcription factories where genes converge in a nonrandom fashion (Eskiw et al., 2010Eskiw C.H. Cope N.F. Clay I. Schoenfelder S. Nagano T. Fraser P. Transcription factories and nuclear organization of the genome.Cold Spring Harb. Symp. Quant. Biol. 2010; 75: 501-506Crossref PubMed Scopus (36) Google Scholar). Finally, inter- and intragenic interactions over large genomic distances create regulatory networks that control gene expression and differentiation (de Wit and de Laat, 2012de Wit E. de Laat W. A decade of 3C technologies: insights into nuclear organization.Genes Dev. 2012; 26: 11-24Crossref PubMed Scopus (541) Google Scholar; Liu et al., 2011Liu Z. Scannell D.R. Eisen M.B. Tjian R. Control of embryonic stem cell lineage commitment by core promoter factor, TAF3.Cell. 2011; 146: 720-731Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar; Montavon et al., 2011Montavon T. Soshnikova N. Mascrez B. Joye E. Thevenet L. Splinter E. de Laat W. Spitz F. Duboule D. A regulatory archipelago controls Hox genes transcription in digits.Cell. 2011; 147: 1132-1145Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). 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 subsequently establish a stable transcription program that assures that axons from like neurons converge to distinct glomeruli (Buck and Axel, 1991Buck L. Axel R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.Cell. 1991; 65: 175-187Abstract Full Text PDF PubMed Scopus (3691) Google Scholar; Imai et al., 2010Imai T. Sakano H. Vosshall L.B. Topographic mapping—the olfactory system.Cold Spring Harb. Perspect. Biol. 2010; 2: a001776Crossref PubMed Scopus (61) Google Scholar). The monoallelic nature of OR expression (Chess et al., 1994Chess A. Simon I. Cedar H. Axel R. Allelic inactivation regulates olfactory receptor gene expression.Cell. 1994; 78: 823-834Abstract Full Text PDF PubMed Scopus (881) Google Scholar), together with the observation that OR promoters are extremely homogeneous and share common regulatory elements (Clowney et al., 2011Clowney E.J. Magklara A. Colquitt B.M. Pathak N. Lane R.P. Lomvardas S. High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation.Genome Res. 2011; 21: 1249-1259Crossref PubMed Scopus (52) Google Scholar), implies that DNA sequence is not sufficient to instruct the expression of only one allele in each neuron and that an epigenetic mechanism is in place. Indeed, the discovery of OR heterochromatinization argues for epigenetic, nondeterministic control of OR choice (Magklara et al., 2011Magklara A. Yen A. Colquitt B.M. Clowney E.J. Allen W. Markenscoff-Papadimitriou E. Evans Z.A. Kheradpour P. Mountoufaris G. Carey C. et al.An epigenetic signature for monoallelic olfactory receptor expression.Cell. 2011; 145: 555-570Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Because active OR alleles have different chromatin modifications from the inactive ORs (Magklara et al., 2011Magklara A. Yen A. Colquitt B.M. Clowney E.J. Allen W. Markenscoff-Papadimitriou E. Evans Z.A. Kheradpour P. Mountoufaris G. Carey C. et al.An epigenetic signature for monoallelic olfactory receptor expression.Cell. 2011; 145: 555-570Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and associate in cis and trans with the H enhancer (Lomvardas et al., 2006Lomvardas S. Barnea G. Pisapia D.J. Mendelsohn M. Kirkland J. Axel R. Interchromosomal interactions and olfactory receptor choice.Cell. 2006; 126: 403-413Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar), 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., 2011Khan M. Vaes E. Mombaerts P. Regulation of the probability of mouse odorant receptor gene choice.Cell. 2011; 147: 907-921Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), 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 recognizes most OR loci, we demonstrate OSN-specific and differentiation-dependent intra- and interchromosomal aggregation of silent ORs. Whereas these OR-specific foci colocalize with H3K9me3, H4K20me3, and heterochromatin protein 1 β (HP1β), the active OR alleles have minimal overlap with heterochromatic markers and reside in euchromatic territories, suggesting the existence of repressive and activating nuclear compartments for OR alleles. Critical for this nuclear organization is the downregulation and removal of lamin b receptor (LBR) from the nuclear envelope of OSNs. Deletion of LBR causes ectopic aggregation of OR loci in basal and sustentacular cells in the main olfactory epithelium (MOE), whereas expression of LBR in OSNs disrupts the formation of OR foci, resulting in decompaction 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 receptor expression. ORs and other AT-rich gene families frequently associate with the nuclear lamina (Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). However, our DNA FISH analysis with individual BAC probes failed to reveal a significant distribution of OR loci toward the nuclear periphery of OSNs (Lomvardas et al., 2006Lomvardas S. Barnea G. Pisapia D.J. Mendelsohn M. Kirkland J. Axel R. Interchromosomal interactions and olfactory receptor choice.Cell. 2006; 126: 403-413Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). To obtain a comprehensive view of the distribution 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 isochores (Clowney et al., 2011Clowney E.J. Magklara A. Colquitt B.M. Pathak N. Lane R.P. Lomvardas S. High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation.Genome Res. 2011; 21: 1249-1259Crossref PubMed Scopus (52) Google Scholar; Glusman et al., 2001Glusman G. Yanai I. Rubin I. Lancet D. The complete human olfactory subgenome.Genome Res. 2001; 11: 685-702Crossref PubMed Scopus (525) Google Scholar), we digested genomic DNA with restriction enzymes that recognize AT-rich sequences and collected DNA fractions with significant enrichment 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., 2007Albert T.J. Molla M.N. Muzny D.M. Nazareth L. Wheeler D. Song X. Richmond T.A. Middle C.M. Rodesch M.J. Packard C.J. et al.Direct selection of human genomic loci by microarray hybridization.Nat. Methods. 2007; 4: 903-905Crossref PubMed Scopus (539) Google Scholar). 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 amplicon detects only sequences from OR clusters, suggesting the elimination of unique, non-OR DNA (Figure 1B). To further examine the composition of this DNA library, we analyzed its contents by whole-genome microarray hybridization, 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, 98.4% OR cluster specificity, p ≤ 10−72) (Figures 1C and Figures S1B–S1D and Table S1 available online). We used this “panOR” library as a probe for DNA FISH experiments on sections of the MOE. Although there are 92 OR clusters in the diploid nucleus, the panOR probe detects an average of ∼5 large foci in OSNs (Figure 1D). This unexpected distribution is specific for OSNs: OR distribution in other cell populations represented in MOE sections (undifferentiated basal cells and sustentacular cells) is diffuse and more consistent with a random arrangement of the 92 OR clusters or ∼2,800 alleles. Quantification of the distribution of the DNA FISH signal in the three cell types of the MOE across the same sections in the same experiments 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 deviation 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 distribution of OR loci (data not shown and 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 coefficient 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), suggesting 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 BACs produce fewer DNA FISH 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 OSNs than in basal and sustentacular cells. Non-OR BAC probes 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 containing 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 distinguishable signals inside the same panOR focus (Figures 1G 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, pericentromeric 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 gene territories. To reveal the epigenetic signature of OR foci, we combined DNA FISH analysis with immunofluorescence (IF) against the heterochromatic marks found on ORs (Magklara et al., 2011Magklara A. Yen A. Colquitt B.M. Clowney E.J. Allen W. Markenscoff-Papadimitriou E. Evans Z.A. Kheradpour P. Mountoufaris G. Carey C. et al.An epigenetic signature for monoallelic olfactory receptor expression.Cell. 2011; 145: 555-570Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) or heterochromatin-binding protein 1 β (HP1β), the only heterochromatic HP1 member expressed in OSNs (data not shown). This analysis reveals overlap between the OR foci, H3K9me3, H4K20me3, and HP1β (Figures 2A–2C and Table S2), but not with Pol II (Figure 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 (Figure S2D). We then performed nascent RNA FISH on sections of the MOE using intronic probes against OR genes MOR28, M50, M71, and P2 combined with IF for H3K9me3, H4K20me3, or HP1β. In contrast with the bulk of the panOR signal, active OR alleles 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 HP1β (Figures 2F–2J and Table S2). These experiments corroborate that the active OR allele is spatially segregated from the silent ORs and resides in euchromatic territory. 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, 1982Goldowitz D. Mullen R.J. Nuclear morphology of ichthyosis mutant mice as a cell marker in chimeric brain.Dev. Biol. 1982; 89: 261-267Crossref PubMed Scopus (23) Google Scholar). LBR is a nuclear envelope protein that interacts with HP1 and heterochromatin (Hoffmann et al., 2002Hoffmann K. Dreger C.K. Olins A.L. Olins D.E. Shultz L.D. Lucke B. Karl H. Kaps R. Müller D. Vayá A. et al.Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Huët anomaly).Nat. Genet. 2002; 31: 410-414Crossref PubMed Scopus (264) Google Scholar; Okada et al., 2005Okada Y. Suzuki T. Sunden Y. Orba Y. Kose S. Imamoto N. Takahashi H. Tanaka S. Hall W.W. Nagashima K. Sawa H. Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles.EMBO Rep. 2005; 6: 452-457Crossref PubMed Scopus (63) Google Scholar; Pyrpasopoulou et al., 1996Pyrpasopoulou A. Meier J. Maison C. Simos G. Georgatos S.D. The lamin B receptor (LBR) provides essential chromatin docking sites at the nuclear envelope.EMBO J. 1996; 15: 7108-7119Crossref PubMed Scopus (140) Google Scholar). 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 downregulation 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 Figure S3C). 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 (Figure 3F). 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 HP1β 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 expression in a postmitotic cell is sufficient to reverse the “inside-out” arrangement and to recruit PH to the nuclear periphery. 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-resolution imaging method that is applied to fully hydrated, unfixed, and unstained cells and measures carbon and nitrogen concentration in biological samples (McDermott et al., 2009McDermott G. Le Gros M.A. Knoechel C.G. Uchida M. Larabell C.A. Soft X-ray tomography and cryogenic light microscopy: the cool combination in cellular imaging.Trends Cell Biol. 2009; 19: 587-595Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Orthoslices (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 (Figures 4A, 4B, and 4G and Movie S1). We also detect extremely condensed structures at the periphery of this PH core that are specific for this cell type; only sperm nuclei have chromatin particles with higher compaction values (data not shown). Although the arrangement of these dark foci is similar to the arrangement of the OR foci around the PH core of the OSN nucleus, DNA FISH 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 105u3 to 135u3 and 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., 2009McDermott G. Le Gros M.A. Knoechel C.G. Uchida M. Larabell C.A. Soft X-ray tomography and cryogenic light microscopy: the cool combination in cellular imaging.Trends Cell Biol. 2009; 19: 587-595Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) of control and LBR+ OSNs (Figure 4I). This measurement, which depicts the concentration of organic material per voxel, corroborates the loss of the densest foci upon LBR expression. Thus, if condensed regions correspond to OR foci, ectopic LBR expression should cause decompaction of OR heterochromatin. DNaseI sensitivity experiments (Magklara et al., 2011Magklara A. Yen A. Colquitt B.M. Clowney E.J. Allen W. Markenscoff-Papadimitriou E. Evans Z.A. Kheradpour P. Mountoufaris G. Carey C. et al.An epigenetic signature for monoallelic olfactory receptor expression.Cell. 2011; 145: 555-570Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) in nuclei from fluorescence-activated cell-sorted (FAC-sorted) control or LBR+ OSNs confirms a significant decompaction of OR and pericentromeric heterochromatin upon LBR expression (Figure 4J). The spatial reorganization of HP1β, 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 transgenic 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 contrast, immature OSNs and progenitors that do 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, and HP1β in LBR+ OSNs. H3K9me3 and 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 HP1β was reduced as measured by FISH-IF (Figures 5D and Table S2). Reduction in overlap between H4K20me3 and HP1β 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 interchromosomal interactions between the active allele and H, we performed circularized chromosome conformation capture (4C) using inverse H PCR primers as previously described (Lomvardas et al., 2006Lomvardas S. Barnea G. Pisapia D.J. Mendelsohn M. Kirkland J. Axel R. Interchromosomal interactions and olfactory receptor choice.Cell. 2006; 126: 403-413Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar) 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 Gγ8 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) 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 unlinked ORs. IF and RNA FISH experiments in MOE sections from control and LBR-expressing mice revealed a 3-fold reduction in the numbers of neurons expressing particular ORs in the transgenic mice (Figure 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 quantitative, reverse-transcriptase PCR (qRT-PCR). This analysis supports that LBR expression has significant inhibitory effects on OR expression (Figure 6B). Similarly, whole-mount X-gal staining in MOEs from P2-IRES-τLacZ mice crossed to LBR-expressing transgenics shows reduced X-gal signal, supporting a repressive effect on OR expression (Figure 6C). Neurons that retained high β-gal protein expression often failed to express the LBR transgene, as demonstrated by IF for β-gal and GFP in sections of these mice (Figure 6D). Because OMP drives LBR expression only after OR choice, this result indicates postchoice downregulation 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-τLacZ). This transgene 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., 1998Garrick D. Fiering S. Martin D.I. Whitelaw E. Repeat-induced gene silencing in mammals.Nat. Genet. 1998; 18: 56-59Crossref PubMed Scopus (533) Google Scholar). This transgene does not interact with either the endog" @default.
• W2045974768 created "2016-06-24" @default.
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• W2045974768 creator A5016787055 @default.
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• W2045974768 date "2012-11-01" @default.
• W2045974768 modified "2023-09-29" @default.
• W2045974768 title "Nuclear Aggregation of Olfactory Receptor Genes Governs Their Monogenic Expression" @default.
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