Gene expression amplification by nuclear speckle association

Abstract

Many active genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that HSPA1B BAC transgenes and endogenous Hsp70 genes turn on 2-4 min after heat shock (HS), irrespective of their distance to speckles. However, both total HSPA1B mRNA counts and nascent transcript levels measured adjacent to the transgene are approximately twofold higher for speckle-associated alleles 15 min after HS. Nascent transcript level fold-increases for speckle-associated alleles are 12-56-fold and 3-7-fold higher 1-2 h after HS for HSPA1B transgenes and endogenous genes, respectively. Severalfold higher nascent transcript levels for several Hsp70 flanking genes also correlate with speckle association at 37°C. Live-cell imaging reveals that HSPA1B nascent transcript levels increase/decrease with speckle association/disassociation. Initial investigation reveals that increased nascent transcript levels accompanying speckle association correlate with reduced exosome RNA degradation and larger Ser2p CTD-modified RNA polymerase II foci. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through "gene expression amplification."

Figure S1.

Positioning of HSPA1B BAC transgene or endogenous Hsp70 gene relative to a nuclear speckle before (37°C) or after HS. (A) Histograms showing fraction of BAC HSPA1B transgenes (lacO) in CHOK1 cell clone C7MCP or endogenous Hsp70 alleles in HAP1 cells (DNA FISH) at varying distances from the nuclear speckle before and after 30-min HS (mean ± SEM, three biological replicates, n = 100–170 per replicate). (B) Boxplots showing distribution of varying distances from speckle shown in histogram A. Mean (square inside box), median (line inside box), box (interquartile range), ends of error bars (upper and lower limit), × (top: 99%, bottom: 1%), – (top: maximum, bottom: minimum). *, P < 0.05; ****, P < 0.00001; n.s, not significant; paired Wilcoxon signed-rank test. (C) Histograms showing fractions of RNA FISH signals from the endogenous Hsp70 locus in CHOK1 or HAP1 cells at varying distances from nuclear speckles after 30-min HS (mean ± SEM, three biological replicates, n = 100–120 per replicate). (D) Boxplots showing distribution of varying distances from speckle shown in histogram C. Box format is same as B. n.s, not significant; paired Wilcoxon signed-rank test. (E) Position of BAC transgene (green, white arrowhead) and nuclear speckle (green, empty arrowhead; left) or endogenous gene DNA FISH signal (red, white arrowhead) and nuclear speckle (green, empty arrowhead; right, DAPI staining, blue) at 37°C (top) or after HS (bottom). Scale bars = 1 µm. (F) Position of RNA FISH of endogenous Hsp70 locus transcripts (red, white arrowheads) and nuclear speckle (green, empty arrowheads) after 30-min HS in CHOK1 cells (left) or HAP1 cells (right). Scale bar = 1 µm.

Figure 1.

Both HSPA1B BAC transgenes and endogenous genes induce synchronously 2–4 min after HS but show higher transcript levels when associated with nuclear speckles. (A) Transcriptional induction of HSPA1B BAC transgene in CHOK1 cells and endogenous Hsp70 genes in HAP1 cells occurs 0–4 min after HS (SEM, three replicates). n = 110–210 for HAP1, n = 95–150 for CHOK1 cells, each replicate time point. (B) Scatterplot between levels of nascent RNA signals (RNA FISH intensity at HSPA1B locus) versus numbers of mature mRNAs after 15-min HS. n = 99 and 71 for A and N. P value (A versus N) = 8.5 × 10⁻⁷ for transcript signal, 1 × 10⁻⁶ for mRNA count; paired Wilcoxon signed rank test. r_A, Pearson correlation coefficient for speckle-associated locus; r_N, for nonassociated locus. (C) smRNA FISH image after 15-min HS. Nascent pre-mRNAs (top) at HSPA1B BAC transgene (arrowhead, middle). White dashes outline cell border. The boxed region around BAC is enlarged on left. (D) Higher pre-mRNA levels (boxed regions) for speckle-associated (top) versus nonassociated (bottom) BAC transgenes. (Left) Representative images of smRNA FISH versus nuclear speckles and BAC transgene at 0, 1, and 2 h after HS. (Right) Normalized pre-mRNA intensities at speckle-associated (black, A) or nonassociated (red, N) BAC transgenes 0, 1, and 2 h after HS, with fold differences (blue) of mean (median) for A versus N. Nascent RNA intensities were normalized by mean intensity of A at 2 h. Cell numbers (A/N) = 101/101, 91/40, and 107/63 at 0, 1, and 2 h. P = 4.5 × 10⁻¹¹ at 1-h HS and 7.8 × 10⁻¹² at 2-h HS for A versus N; 9.7 × 10⁻⁴ for A at 1 versus 2 h; and 8.4 × 10⁻⁴ for N at 1 versus 2 h; paired Wilcoxon signed-rank test. (E) Left: Same as in D but for endogenous Hsp70 locus in CHOK1 cells (left two panels) versus haploid human HAP1 cells (right two panels). Nonlinear intensity scaling used for red channel. Right: Same as in D, right, for endogenous Hsp70 locus in CHOK1 (left) versus HAP1 cells (right). Nascent RNA intensities were normalized by mean intensity of A at 2 h in CHOK1 cells and HAP1 cells, respectively. CHOK1: cell numbers (A/N) = 46/33 and 50/28 at 1 and 2 h, P = 1.8 × 10⁻⁵ at 1-h HS and 7.5 × 10⁻⁸ at 2-h HS for A versus N, 0.0038 for A at 1 versus 2 h, 0.0015 for N at 1 versus 2 h; HAP1: cell numbers (A/N) =102/44 and 112/33 at 1 and 2 h, P = 2.0 × 10⁻⁹ at 1-h HS and 6.5 × 10⁻⁹ at 2-h HS for A versus N, 4.6 × 10⁻⁴ for A at 1 versus 2 h, 0.12 for N at 1 versus 2 h; paired Wilcoxon signed-rank test.

Figure S2.

Various supporting data. (A) Transcriptional induction of HSPA1B BAC transgene without MS2 repeats in CHO DG44 cells detected by smRNA FISH against HSPA1B (clone 38, percentage ± SD estimate, n = 42 to 46 each time point, magenta) and of HSPA1B BAC with MS2 repeats in CHOK1 cells detected by smRNA FISH against MS2 (clone C7MCP, percentage ± SD estimate, n = 74 to 96 each time point, green). Full induction occurs 2–4 min after HS in both. (B) Transcriptional induction of speckle-associated (A) and non–speckle-associated (N) HSPA1B BAC transgene in CHOK1 cells (clone C7MCP). Both A and N are induced within 0–4 min after HS (percentage ± SD estimate, n = 360, 211 observed for A; n = 147, 69 for N). (C) Histogram showing fraction of endogenous Hsp70 alleles in HAP1 cells upon speckle association or non–speckle association after 2- and 4-min HS (mean ± SEM, three replicates). At 2-min HS, n = ∼100, 38% activated cells on average among observed 201 to 360 cells, and at 4-min HS, n = 70 to 111, 96.8% activated cells on average among observed 70 to 120 cells for each replicate. (D) Whole-cell images of nucleus shown in Fig. 1 D (left) and intensity profile of nascent mRNA transcripts at HSPA1B BAC transgene locus (right). Integrated mature mRNA signal is proportional to nascent mRNA signal as in Fig. 1 B. Left: White dashes outline cell border. Right, top: Image of nascent transcripts in red, HSPA1B BAC in light green and nuclear speckle in green, and merge. Right, bottom: Intensity profile of nascent transcript measured along the white dash line in top image. Intensities scaled nonlinearly in images to reduce red channel intensity dynamic range (left) but linearly for line scans. (E) Nascent mRNA level change after EXOSC3 knockdown at 1-h HS. Left: Immunoblotting with EXOSC3 antibody to estimate the percentage of knockdown. The EXOSC3 protein in the knockdown samples (100%, RRP40 siRNA treated) was estimated against different percentages (25, 50, 75, and 100%) of scrambled siRNA-treated cell lysates. Lamin B1 was used as the loading control. Right: Boxplot of nascent mRNA intensity (smRNA FISH) at A and N locus for scrambled siRNA or RRP40 siRNA–treated cells with fold differences (blue) of mean for A versus N. Mean (square inside box), median (line inside box), box (interquartile range), ends of error bars (upper and lower limit), × (top: 99%, bottom: 1%), – (top: maximum, bottom: minimum). **, P < 0.01; ****, P < 0.0001; n.s, not significant; paired Wilcoxon signed-rank test. n = 107 and 39 observed for A and N in scrambled; 106 and 43 for RRP40 siRNA–treated cells. (F) RNA polII phosphorylated on serine 2 (Ser2P) and MS2MCP-mCherry signal at 1-h HS. Left: Representative images of Ser2P polII (red, bottom left and right) signals and MS2 signals (red, bottom right, and yellow, right) for BAC transgene (light green) associated (top) versus not associated (bottom) with nuclear speckles (green). Right: Boxplot of relative Ser2P normalized by mean Ser2P intensity of A with fold differences (blue) of mean for A versus N. **, P < 0.01; paired Wilcoxon signed-rank test. n = 165 and 33 observed for A and N. (G) Histograms showing fraction of endogenous Hsp70 flanking gene alleles in HAP1 cells at varying distances from the nuclear speckle (mean ± SEM, three experimental replicates, n = 128 to 289 activated loci observed in each experiment. Scale bars = 1 µm (D, enlarged panels, and F) or 5 µm (D) .

Figure 2.

Transcription amplification of speckle-associated genes flanking Hsp70 gene locus at 37°C. (A and D) Probed gene locations (gray boxes, magenta gene names) relative to Hsp70 genes in BAC construct (A) in CHOK1 cells and at endogenous locus (D) in human HAP1 cells. (B and E) Representative images of smRNA FISH (red) signals for specific BAC transgene (B) or endogenous gene (E) showing nascent transcripts associated (top) versus nonassociated (bottom) with nuclear speckles (intensities scaled differently for top and bottom). White arrowheads point to BAC (B) or nascent transcripts (E); empty arrowheads point to nuclear speckle. Scale bars = 1 µm. (C and F) Boxplots showing nascent transcript levels for three (C) or four (F) genes flanking BAC HSPA1B transgene (C) or endogenous Hsp70 locus (F) as function of speckle association (A) or nonassociation (N). Mean (square inside box), median (line inside box), box (interquartile range), ends of error bars (upper and lower limit), × (top: 99%, bottom: 1%), – (top: maximum, bottom: minimum). Intensities are normalized by the mean intensity at speckle-associated loci: fold differences (×) of the mean (median) between A versus N (black). In C, number of cells (A/N) = 112/44, 143/37, and 185/39; P = 0.0024, 0.013, and 0.016 (paired Wilcoxon signed-rank test) for VARS, LSM2, and C6orf48 BAC transgenes. in D, number of cells (A/N) = 168/64, 144/47, 123/52, and 203/86; P = 2.0 × 10⁻⁶, 1.4 × 10⁻⁵, 0.23, and 1.5 × 10⁻¹¹ (paired Wilcoxon signed-rank test) for endogenous MSH5, VARS, LSM2, and C6orf48 genes.

Figure 3.

Strict temporal correlation between speckle association and HSPA1B transcriptional amplification. (A and B) Left: Transgene location (solid arrowheads), nuclear speckles (open arrowheads), and MS2 signal versus time (minutes) after start of observation (heating on at 1 min, stable HS temperature [HS-T] reached at 5 min). Right: Distance (d) of Hsp70 transgene/nascent transcript from closest speckle (green) and nascent RNA level (red). (A) Transgene associated with speckle throughout HS (category 1, 146/438 cells). MS2 signal appears 1 min after reaching HS-T at 5 min (Video 1). (B) Transgene initially unassociated with speckle (category 2, 41/438 cells). MS2 signal appears at 10 min, ∼5 min after reaching HS-T and ∼1 min after moving to and contacting speckle (Video 2). (C) Histogram showing time of MS2 signal appearance after reaching HS-T for transgenes initially speckle associated (gray, n = 251) versus non–speckle associated (red, n = 159). MS2 signal delayed ∼3 min when not associated (mean = 6.5 min) versus associated (mean = 3.6 min). P = 2.2 × 10⁻¹⁶; paired t test. (D) Scatterplot showing timing of speckle–transgene association after reaching HS-T versus initial transgene distance to speckle versus time lag (color) between speckle–gene association and appearance of MS2 signal (n = 42 displayed). Time lags: mean = 3.8 min, typically 0–3 min after reaching HS-T.

Figure 4.

Temporal correlation between speckle dissociation and decrease in HSPA1B transcription. Display and labeling is the same as in Fig. 3 (A and B). (A) HSPA1B transgene disassociating from speckle (category 3, 28/438 cells). Nascent transcripts decrease and then disappear after transgene separates from speckle (Video 3). (B) HSPA1B transgene disassociating from speckle. d, distance of HSPA1B transgene (green) or nascent transcript (orange) from closest speckle (2/28 cells falling in category 3). Nascent transcripts accumulate in elongated connection between speckle and transgene after their separation (Video 4). (C) No speckle association of HSPA1B transgene after HS (5/438 cells). MS2 signal sometimes transiently rises slightly above background but does not persist.

Figure 5.

Statistics for classifications of relative movements between BAC transgene and nuclear speckle. (A) Cartoons of representative dynamics (categories 1–3, top) during HS and corresponding averaged plots of live-cell data (bottom, mean ± SEM, n = 21–47 movies). Distance (d, green) of HSPA1B transgene from closest speckle and nascent RNA levels (red) in arbitrary units. Category 1 (left): stable speckle association of HSPA1B transgene and transcription increase after temperature reaches 42°C, 47 movies; category 2: transcription increase as function of time after transgene association with speckle (t = 0), 24 movies; category 3: transcription decrease as function of time after transgene dissociation (t = 0) from speckle, 21 movies. (B) Numbers for categories of dynamics observed for 20 to ∼25 min with or without HS. d_max, maximum distance between transgene locus and speckle during imaging; d₁, distance between transgene locus and speckle at first time point; d_n, distance between transgene locus and speckle at any time point during imaging; F, forward relative motion bringing transgene and speckle closer; B, backward relative motion moving transgene and speckle apart; +α, additional movements. (C) Three models for correlation of nascent transcript increase with speckle association: nuclear speckle (green), gene (yellow), and nascent transcripts (red; solid lines, intact; dashed lines, degraded). Left: Current working model. Nascent transcripts increase after speckle association through a decreased, exosome-mediated nascent transcript degradation rate, and possibly an increased transcription rate. Middle: Alternative model 1. Nascent transcripts adjacent to transcription site increase due to a reduced transcript release rate; at steady state, this would not change the rate of mRNA production and is inconsistent with the increased mRNA count 15 min after HS. Right: Alternative model 2. Nascent transcript levels increase before speckle association, causing subsequent speckle association; this is inconsistent with live-cell imaging temporal ordering of events.

Figure S3.

Additional examples of speckle and transgene dynamics. Time stamp during HS (yellow), BAC transgene (white arrowheads, bright green), nuclear speckle (empty arrowheads, lighter green), and RNA MS2-tagged transcripts (red, mCherry-MCP). All scale bars = 1 µm. (A) Combined speckle and transgene movements after speckle movement toward and association with the transgene (category: F, Fig. 5 B). Dotted lines connect to relatively stationary speckles. (B) Movement of two speckles toward the transgene locus (category: F, Fig. 5 B). (C) Speckle protrusion toward the active transgene locus (category: speckle protrusion, Fig. 5 B), with the transgene then moving closer to speckle. (D) Speckle formation at the active transgene (white arrowhead) after HS at 7 min, with nascent transcripts appearing above background at 8 min (category: speckle formation, Fig. 5 B). (E) No persisting transcription for nonassociating locus (category: no association, Fig. 5 B). Arrow (15 min) shows direction of transgene movement. Transcriptional bursting is observed at both nonassociating transgene loci at 13 min. These bursting signals are observed again at 15 min at both loci. At 16 min, one transgene (left) associates (A) with a speckle and now maintains elevated transcript signal during the rest of the observation time, whereas the other transgene that is not associated (N) with speckle does not maintain an elevated transcript signal. (F) Coordinated movement of speckle and gene (category: coordinate movement of speckle and gene, Fig. 5 B). White arrows mark the direction of gene movements. Nuclear speckle and associated BAC transgene (white arrowhead) move together as a single unit before merging with a different speckle, suggesting a stable attachment of transgene and speckle (Video 5). (G and H) Movements of Hsp70 BAC transgene away from and back to nuclear speckles at 37°C. White arrows mark the direction of gene movements. BAC transgene shows two (G) or four (H) long-range movements away from and then back toward nuclear speckle. Category in G: FB + α, Fig. 5 B and Video 6; category in H: gene moving to a different speckle, Fig. 5 B.