An epigenetic trap stabilizes singular olfactory receptor expression

Abstract

The molecular mechanisms regulating olfactory receptor (OR) expression in the mammalian nose are not yet understood. Here, we identify the transient expression of histone demethylase LSD1 and the OR-dependent expression of adenylyl cyclase 3 (Adcy3) as requirements for initiation and stabilization of OR expression. As a transcriptional coactivator, LSD1 is necessary for desilencing and initiating OR transcription, but as a transcriptional corepressor, it is incompatible with maintenance of OR expression, and its downregulation is imperative for stable OR choice. Adcy3, a sensor of OR expression and a transmitter of an OR-elicited feedback, mediates the downregulation of LSD1 and promotes the differentiation of olfactory sensory neurons (OSNs). This novel, three-node signaling cascade locks the epigenetic state of the chosen OR, stabilizes its singular expression, and prevents the transcriptional activation of additional OR alleles for the life of the neuron.

Figure 1

Transient LSD1 expression is required for OR expression (A) mRNA-seq reads per million mapped per thousand basepairs of exon model (RPKM) for LSD1 and LSD2 in the mature versus immature/globose basal cells (Ngn1+). (B) LSD1 immunofluorescence (IF, red) in the Ngn1-GFP+ MOE at PND30. (C) LSD1 and OMP 2-color RNA in situ hybridization (ISH) at PND5. DAPI nuclear stain is shown in blue. (D) Removal of LSD1 over developmental time with 3 different MOE-specific Cre recombinase mouse lines. (E) OR ISH probe pool for 8 Class II OR genes in Foxg1-cre; LSD1flox/+ and Foxg1;cre;flox/flox (Class I OR ISH is shown in Figure S1). (F) MOR28-IRES-Cre mediated Cre reporter (green) in MOE with MOR28 immunofluorescence (red); coexpressing cells are stably expressing MOR28 in the absence of LSD1 (G) Class II OR ISH in OMP-IRES-Cre; LSD1 flox/+ and flox/flox MOE at PND1. (H) Olfactory bulbs of MOR28-IRES-Cre; LSD1+/+ and MOR28-IRES-Cre; LSD1flox/flox animals at PND30 with a 2-color membrane-bound Cre-reporter: mT before Cre; mG after Cre (mT/mG; Muzumdar et al. 2007). See also Figure S1.

Figure 2

Early deletion of LSD1 with Foxg1-Cre causes massive reduction in OR gene expression and developmental arrest at a differentiation stage synchronous to the onset of OR transcription. (A) mRNA-seq RPKM for each Refseq OR in mouse genome from E18.5 MOE sample. Each spoke of a given color is the value for that OR in MOE of that genotype (red: Foxg1-Cre; LSD1flox/+; green: Foxg1-Cre; Lsd1flox/flox). External doughnut represents relative chromosomal location of each OR gene. Summary boxplot is shown within Circos plot; student’s paired t-test used for significance testing. (B) RPKM values of the 2 known transcriptional activators of OR genes in the LSD1 heterozygote and knockout. (C-F) Chromogenic ISH for developmental markers in LSD1 heterozygote (top panels) and knockout (bottom panels), respectively: Neurod1, GAP43, NCAM1, OMP. (G) IF for Adcy3 at same embryonic stage, DAPI nuclear stain is shown in blue. See also Figure S2.

Figure 3

Ectopic expression of transgenic MOR28 in the LSD1 KO MOE can rescue the loss of Adcy3 expression. (A) Model summary of findings from misexpression study. Using two tTA drivers, one active in the immature neuron (Gγ8-tTa), and one active in the mature neuron (OMPitTA), it is possible to express high levels MOR28 in the LSD1 KO MOE, in a sporadic fashion. We find that OR expression is followed by the onset of Adcy3 protein expression. (B) Xgal staining in sections of LSD1 KO MOE shows infrequent transgenic MOR28 expression under the control of two tTA drivers. Whole-mount image is shown in Figure S3. (C) Foxg1-Cre; tetO-MOR28-lacZ MOE at E18.5 with either Lsd1 flox/+; OMPitTA (left panels) or Lsd1 flox/flox; Gγ8-tTa; OMPitTA (right panels). Adcy3 IF (green); Beta-galactosidase IF (red); and merge. (D) LSD1 dosage positively correlates with Adcy3 immunoreactivity. Adcy3+ cells in E18.5 MOE were quantified per unit area in ImageJ. Y axis units are Adcy3+ cells per micron of MOE area considered. Error bars show standard error of 2 quantified regions of MOE from one experiment.

Figure 4

Adcy3 removal triggers upregulation of LSD1 protein levels and increase in OR gene switching. (A) PND21 sections with IF for LSD1 in Adcy3+/− (top) and −/− (bottom), respectively. See Figure S4 for quantification. (B,C) Fluorescent RNA ISH for immature (GAP43) and mature (OMP) neurons in Adcy3+/− (top) and −/− (bottom), respectively (D) Beta-gal (green) and LSD1 (red) IF in Adcy3+/− (top) and −/− (bottom), respectively. A lacZ reporter is knocked into Adcy3 locus. See supplemental experimental procedures for details. (E) PND21 sections from Adcy3+/− (top) and −/− (bottom), stained with OR (green, MOR28 and M50) and LSD1 (red) antibodies. (F) RNA-seq RPKM values for all expressed Refseq ORs (n=1072) and OR pseudogenes (n=48) in corresponding genotype from PND21 MOE. RPKMs of unexpressed intact and pseudogene ORs were excluded. See also Fig S4. (G) PND3 RNA ISH for M50 and MOR28 as in (E). IF for ORs and LSD1 shown in supplemental Figure 4 (H) PND2 MOE from MOR28-IRES-Cre; Cre-reporter mice in Adcy3 wildtype or knockout background. Cre IF (magenta) and mT/mG reporter (green). (I) Quantification of experiment in (H). Single (CRE or GFP positive) and double positive cells from 10 sections of PND2 wild type and Adcy3 KO mice were counted and plotted as ratio of single to double positive. SE represents variation across sections, single animal, 10 sections quantified. P value=0.007, calculated with Student's unpaired T-test.

Figure 5

Ectopic expression of transgenic LSD1 in the mature neuron layer causes reversible destabilization of OR expression. (A) Model summarizing results in adult MOE regarding the expression pattern of ORs and LSD1 under different genetic manipulations:. OR-expressing OSNs are prevalent in LSD1-negative layer regardless of genotype. Weakly OR-expressing OSNs are present in OMPitTA;tetO-LSD1 mice before dox but robust expression returns following dox and the reduction of LSD1 misexpression. (B) Adult OMP-tTA; tetO-LSD1 mice were raised until 3 weeks and either placed on doxycycline for 3 weeks to shut off tTA activation, or maintained on dox-free food. Control littermate mice (OMPitTA only) were also placed on doxycycline for 3 weeks. 6 week old MOE were harvested and IF was performed for LSD1 (red top panel) or Olfr49 (C6) (red two bottom panels with or without DAPI). See also Figure S5. (C) Misexpressing LSD1 in the MOE with OMPitTA reduced OR expression in the MOE. Chromogenic ISH OR pool (15 OR probes total) in OMPitTA (left) and OMPitTA; tetO-LSD1(right). (D) P2-lacZ (top left) and P2-lacZ;OMPitTA; tetO-LSD1 (bottom left) MOE and bulb following whole mount X-gal staining in PND25 mice. Olfactory bulb sections (right) from the same genotype are shown with bet-agalactosidase IF (green). Despite the low levels of beta-gal at the cell bodies due to switching, the protein appears stable at the axons (Clowney et al, 2013), which allows the visualization of additional glomeruli.

Figure 6

LSD1 generates stable 8-oxodG at active OR genes. (A) 8-oxodG DIP was performed on sonicated genomic DNA (gDNA) from LSD1 wildtype, heterozygote, and knockout MOE at E18.5. (B) 8oxodG-DIP-qPCRs from gDNA of P2-GFP sorted cells from PND30 mice. (C) PND30 MOE of MOR28-del-Cre; Cre-reporter mouse, with Cre IF (magenta) and mT/mG Cre-reporter (Green are cells that have expressed Cre to levels sufficient to recombine reporter locus). DAPI nuclear stain is blue. (D) 8oxodG-DIP-qPCRs from Cre-reporter-positive neuron gDNA at PND30, as shown in (C). (E) DIP-seq analysis of an E18.5 wild-type 8-oxodG library. Expression quartiles from the RPKM values generated from Fig. 1 mRNA-seq. y-axis is 8-oxodG RPKM. Boxplots show mean 8-oxodG RPKM for each expression quartile demarcated by horizontal red bar. (F, G) 8oxodG-DIP-qPCRs from gDNA from whole MOE of LSD1 overexpressing mice and Adcy3 knockout mice, respectively. Error bars are standard error from 2 PCR replicates from one representative experiment. See also Figure S6 for control experiments.

Figure 7

A three node signaling cascade combined with a feedback signal that generates epigenetic memory (A) Stabilization of OR expression is achieved by an Adcy3-dependent "trap" such that the functional chosen OR cannot be turned off once LSD1 is downregulated by its induction of Adcy3. This trap is caused by removing LSD1 from the signaling circuit which allows stable transcription to ensue (represented by dashed line that reflects the indirect OR stabilization by Adcy3). (B) Pseudogene ORs (ORΨ) are unable to activate Adcy3 and thus OSNs that have chosen these ORs maintain the ability to re-choose and use LSD1 to transcriptionally silence the ORΨ. (C) Alternatively, LSD1 may not silence directly the previously chosen OR, but causes its repression by activating and additional OR allele.