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, 9 (3), 884-92

Heterochromatin-mediated Gene Silencing Facilitates the Diversification of Olfactory Neurons

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Heterochromatin-mediated Gene Silencing Facilitates the Diversification of Olfactory Neurons

David B Lyons et al. Cell Rep.

Abstract

An astounding property of the nervous system is its cellular diversity. This diversity, which was initially realized by morphological and electrophysiological differences, is ultimately produced by variations in gene-expression programs. In most cases, these variations are determined by external cues. However, a growing number of neuronal types have been identified in which inductive signals cannot explain the few but decisive transcriptional differences that cause cell diversification. Here, we show that heterochromatic silencing, which we find is governed by histone methyltransferases G9a (KMT1C) and GLP (KMT1D), is essential for stochastic and singular olfactory receptor (OR) expression. Deletion of G9a and GLP dramatically reduces the complexity of the OR transcriptome, resulting in transcriptional domination by a few ORs and loss of singularity in OR expression. Thus, our data suggest that, in addition to its previously known functions, heterochromatin creates an epigenetic platform that affords stochastic, mutually exclusive gene choices and promotes cellular diversity.

Figures

Figure 1
Figure 1. H3K9 methyltransferases G9a and GLP are needed for OR expression and ensure neuronal diversity in the MOE
(A) Circos plot(Krzywinski et al., 2009) depicting fold change in all olfactory receptor (OR) genes calculated as log2 of G9a/GLP double knockout (dKO) (OR RPKM + 1) / G9a heterozygote (OR RPKM +1). Chromosomes are shown as an ideogram with differently colored lines. Each OR cluster is delineated by a grey line dividing the chromosome ideogram such that the relative size of each cluster is maintained. Position of Olfr231 is denoted on Chr.1. y-axis ranges from −7 to 7. Axis marks are every 0.7. (B) Non-chemoreceptor genes were plotted as a function of their expression levels in control and dKO. Genes up- or downregulated log2 2-fold or more are shown in blue and are listed in Table S1. (C) RNA fluorescent in situ hybridization (FISH) of Olfr231 in Cre-negative control MOEs (left) and G9a-GLP dKO (right). DAPI nuclear stain shown in blue.
Figure 2
Figure 2. Coexpression of multiple ORs in OSNs lacking G9a and GLP
(A) Barplot illustrating relative expression level of 5 most highly expressed ORs in G9a/GLP dKO used to make RNA probes for FISH below. (B) 2-color FISH with Olfr231 probe in red and 10 other ORs in green including the 4 next-most highly expressed ORs in dKO (Olfr878, −1339, −464, −1361), 4 other highly upregulated ORs (Olfr446, −419, −433, −420) and 2 control probes that are readily detected in non-mutant E18.5 MOE (OIfr686, −556). Left is control, right is dKO. DAPI is removed from bottom panels. (C) Magnified view of inset as shown in (b) to highlight red/green coexpressing OSNs (arrowheads). Count totals for dKO are coexpressing cells: 49; Olfr231+ cells: 698; Olfr878 pool+: 350. Totals for control are: coexpressing cells: 0, Olfr231+: 4, Olfr878 pool+: 81.
Figure 3
Figure 3. Heterochromatin is reduced at OR clusters in G9a/GLP dKO
(A and B) DNA FISH and immunofluoresence for OR gene cluster DNA (green; “pan-OR”) and H3K9me3 (red) in MOE cryosections at E18.5 of control and G9a/GLP dKO, respectively. Upper left: merged image; upper right: pan-OR with DAPI; lower left: pan-OR with H3K9me3; lower right: H3K9me3 with DAPI. (C) Summary of measurements from pan-OR aggregate radial analysis (top) and peripheral signal quantification (bottom) (n=100 pan-OR foci for both). Cartoon depicts source of measurements for these analyses, whereby pan-OR aggregates were defined as the area containing signal between 25–100% maximum intensity. (D) H3K9me3 signal intensity in two different regions of the nucleus illustrate a specific loss of H3K9me3 from OR gene foci in the dKO. PH: pericentromeric heterochromatin; OR: olfactory receptor gene aggregates. Error bars are standard deviation from the mean (for both genotypes: nPH=50; nOR=150). (E and G) Image of pan-OR and H3K9me3 in control and dKO, respectively, with reference line used for signal quantification (white, top panel); intensity plot corresponding to pixels intersecting reference line (bottom panel). (F and H) Pixel intensity plot for entire image plane (inset) (control and dKO, respectively). H3K9me3 intensity is plotted along y-axis; pan-OR is plotted on x-axis. Hotter colors correspond to greater frequency of occurrence. For C, D, E, and G, morphologically identified OSNs were used for measurements whereas F and H are quantifications of entire fields of cells in an MOE section.
Figure 4
Figure 4. Reduction of H3K9me3 at OR genes allows LSD1-independent OR activation
(A) Olfr231 ISH in control (Cre-) MOE (leftmost) (average ~2 Olfr231+ cells per section n=20 sections) compared with (from left to right) LSD1 KO (average 0 Olfr231+ cells per section n=20), G9a KO (average ~38 Olfr231+ cells per section n=20 sections), and LSD1/G9a dKO (average ~4 Olfr231+ cells per section n=20) (for GLP KO: see Fig. S4). (B) 8-oxoguanosine DNA immunoprecipitation in G9a heterozygote (cont.) and G9a KO MOE at E18.5. Values represent the mean of technical duplicates; error bars are mean +/−SEM. (C) Percentage of total OR RPKM that the top ten most highly expressed ORs represent for each genotype as shown (all are Foxg1-Cre+, each color corresponds only to rank, not Olfr gene identity). Flox is abbreviated “fl”. (D) Lorenz curve depicting OR expression across all OR genes in the G9a-GLP dosage series (if all OR genes express at equal levels, Gini=0 and curve is perfect diagonal; see Fig. S2C–D). Curve depicts cumulative fraction of OR expression as a function of cumulative OR genes detected. (“Het”: flox/+; “KO”: flox/flox.)

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