1. Enhancer Control of Transcriptional Bursting
Takashi Fukaya, Bomyi Lim, Michael Levine 
Cell 
Volume 166, Issue 2, Pages (July 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Enhancer Location Influences Transcriptional Dynamics
(A) The sna locus contains the proximal primary enhancer adjacent to the promoter, as well as a distal shadow enhancer located in the neighboring Tim17b2 locus.
(B) Schematic representation of yellow reporter gene containing the 100-bp minimal sna promoter and 24x MS2 RNA stem loops within the 5′ UTR. The 1.5-kb sna shadow enhancer was placed either 1 kb upstream (5′ sna shadow enhancer; top) or 7.5 kb downstream (3′ sna shadow enhancer; bottom) of the promoter. This reporter gene also contains a linked snaPr-PP7-yellow in symmetric location that will be discussed in Figures 4 and 5.
(C and D) Representative traces of transcription activity in individual nuclei. Snapshots are taken from the nucleus that was used to measure the corresponding traces. The intensity of green false-color is proportional to the signal strength of the transcription focus at a given time.
(E) Average values of total transcripts produced per nucleus. The output was measured by quantifying the area under the curve for individual traces. A total of 614 and 907 nuclei, respectively, were measured from three individual embryos for the reporter genes with 5′ and 3′ sna shadow enhancer. Error bars represent the SD of the mean of three biological replicates. Asterisk indicates p < 0.01.
See also Figure S1.
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4. Figure 2 Bursting Frequencies Correlate with Enhancer Strength
(A) Schematic representation of MS2-yellow reporter gene containing the 2.8-kb sna primary enhancer. The enhancer is located 7.5 kb downstream of the sna promoter.
(B and C) A representative trace of transcription activity of MS2-yellow reporters with sna primary (B) and sna shadow (C) enhancers.
(D) Schematic representation of MS2-yellow reporter genes with rho NEE, Kr CD2, and IAB5 enhancers. In all cases, the enhancer is located 7.5 kb downstream of the sna promoter.
(E) Average values of amplitude, frequency, and output per nucleus. Totals of 907, 788, 450, 799, and 413 nuclei from three independent embryos were analyzed for the reporter genes containing the sna shadow, sna primary, rho NEE, IAB5, and Kr CD2 enhancers, respectively. Error bars represent the SD of the mean of three biological replicates. Output of sna shadow enhancer is the same as the plot in Figure 1E. Asterisk indicates p < 0.01 compared to sna shadow enhancer. Note that these transgenes also contain a linked snaPr-PP7-yellow reporter gene in symmetric location (see Figure 5).
See also Figures S2 and S3.
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5. Figure 3 Differential Bursting Frequencies of Segmentation Genes
(A) Distribution of bursting frequencies in transgenic embryo expressing snaPr-MS2-yellow with the 3′ sna shadow enhancer (Figure 1B, bottom). Each nucleus is colored with respect to the total number of transcriptional bursts detected throughout nc 14. The image is oriented with anterior to the left and ventral view facing up.
(B) Representative traces of transcription activity in individual nuclei from each domain shown in (A). Each domain spans about half the total limits of the sna expression pattern.
(C) Boxplots showing the distribution of burst frequencies and output per nucleus in each domain shown in (A). 149 and 152 nuclei were analyzed in domains 1 and 2, respectively. The box indicates the lower (25%) and upper (75%) quantile and the solid line indicates the median. Whiskers extend to 10th and 90th percentile of each distribution.
(D) Distribution of bursting frequencies in the embryo expressing snaPr-MS2-yellow with the Kr CD2 enhancer (Figure 2D). The image is oriented with anterior to the left and dorsal up.
(E) Representative traces of transcription activity in individual nuclei from each domain shown in (D). Each domain spans about half the total limits of the Kr expression pattern.
(F) Boxplots showing the distribution of burst frequencies and output in each domain shown in (D). 110 and 114 nuclei were analyzed in domains 1 and 2, respectively. The box indicates the lower (25%) and upper (75%) quantile and the solid line indicates the median. Whiskers extend to 10th and 90th percentile of each distribution. Asterisk indicates p < 0.01.
See also Figures S4 and S5.
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6. Figure 4 Promoter Competition Diminishes Bursting Frequencies
(A) Schematic representation of snaPr-MS2-yellow and snaPr-PP7-yellow reporter genes with the sna shadow enhancer. snaPr-PP7-yellow is located either 7.6 kb (symmetric, top) or 1 kb (asymmetric, bottom) from the enhancer. In both cases, the shared enhancer is located 7.5 kb from the snaPr-MS2-yellow reporter.
(B and C) Representative traces of transcription activity of PP7-yellow and MS2-yellow in symmetric configuration.
(D and E) Representative traces of transcription activity of PP7-yellow and MS2-yellow in asymmetric configuration (e.g., the shared enhancer is located near the PP7-yellow reporter).
(F) Average amplitude, frequency and output per nucleus in the symmetric and the asymmetric reporter constructs. 604 and 332 nuclei from three independent embryos were analyzed for MS2-yellow signals in symmetric and asymmetric configurations, respectively. Error bars represent the SD of the mean of three biological replicates. Asterisk indicates p < 0.01.
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7. Figure 5 Coordinated Transcriptional Bursts by a Shared Enhancer
(A) Schematic representation of the symmetric snaPr-MS2-yellow and snaPr-PP7-yellow reporters with different enhancers (sna shadow, sna primary, rho NEE, Kr CD2, and IAB5).
(B and C) Representative traces of transcription activity in the reporter gene with the sna shadow enhancer. Snapshots are taken from the nucleus that corresponds to the indicated traces. The intensity of green (MS2-yellow) and red (PP7-yellow) false-coloring is proportional to the signal strength of transcription focus at a given time.
(D and E) Representative traces of transcription activity of MS2-yellow and PP7-yellow reporters with sna primary (D) and Kr CD2 enhancers (E).
(F and G) Left: frequency of the coordinate transcriptional burst among total MS2-yellow (F) and PP7-yellow bursts (G). Error bars represent the SD of the mean of three biological replicates. 613, 502, 382, 369, and 803 nuclei were analyzed for sna shadow, sna primary, IAB5, rho NEE, and Kr CD2 enhancers, respectively. Right: frequency of coordinate bursts among randomly paired traces. From a pool of MS2 and PP7 traces in a given embryo, traces with comparable bursting frequency were randomly paired and the number of coordinate bursts was counted. Error bars represent the SD of the mean of three biological replicates. Totals of 613, 477, 310, 249, and 365 nuclei were analyzed for sna shadow, sna primary, IAB5, rho NEE, and Kr CD2 enhancers, respectively. Asterisk indicates p < 0.01.
See also Figures S6 and S7.
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8. Figure 6 The gypsy Insulator Alters Bursting Frequencies
(A) Schematic representation of the symmetric snaPr-MS2-yellow and snaPr-PP7-yellow reporter genes with the gypsy insulator inserted between the sna shadow enhancer and snaPr-PP7-yellow.
(B–D) Representative traces of transcription activity of MS2-yellow and PP7-yellow.
(E) Average amplitude, frequency and output per nucleus in the reporter constructs with or without the insulator. 604 and 579 nuclei from three independent embryos were analyzed for the reporters without and with the insulator, respectively. The plot shown in Figure 4F (symmetric MS2-yellow) was used for the reporter without the insulator. Error bars represent the SD of the mean of three biological replicates. Asterisk indicates p < 0.01.
(F) Frequency of coordinate transcriptional bursts among total MS2-yellow and PP7-yellow bursts. The error bars represent the SD of the mean of three biological replicates and 579 nuclei were analyzed.
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9. Figure 7 A Model for Dynamic Transitions in Chromosome Topology
(A) A model for the coordinated activation of two-linked reporter genes by a shared enhancer. A higher-order chromosomal loop domain positions the shared enhancer near both target promoters.
(B) We propose that the gypsy insulator blocks enhancer-promoter interactions by forming a chromosomal loop domain by pairing with a nearby endogenous insulator (bottom). The occasional coordinated bursts that are observed can be explained by dynamic conformational changes in chromosomal loop domains.
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10. Figure S1
MS2-yellow Reporter Gene Recapitulates an Endogenous Pattern of Gene Expression, Related to Figure 1
(A) Splicing pattern of MS2-yellow and PP7-yellow was confirmed by RT-PCR analysis.
(B) The sequence chromatograms of PCR products shown in (A). MS2-yellow and PP7-yellow show exactly same splicing pattern as endogenous yellow mRNA.
(C) Double fluorescent in situ hybridization using probes against the introns of MS2-yellow and endogenous sna.
(D) Relative MS2-yellow mRNA abundance was determined by quantitative RT-PCR using primers against intronic sequence. The mean values ± SD for three biological replicates are shown.
(E) Boxplot showing the distribution of total transcripts produced per nucleus in three independent embryos. The box indicates the lower (25%) and upper (75%) quantile. 229, 177 and 208 nuclei from three independent embryos were analyzed for 5′ sna shadow enhancer and 303, 319 and 285 nuclei were analyzed for 3′ sna shadow enhancer.
(F) Average bursting duration per nucleus. Totals of 531 and 619 nuclei from three independent embryos were analyzed for the reporter genes containing the sna shadow and sna primary enhancers, respectively. The error bars represent the standard deviation of the mean of three biological replicates.
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11. Figure S2
Transcriptional Bursts Driven by Developmental Enhancers, Related to Figure 2
(A) Schematic representation of MS2-yellow reporters with different enhancers.
(B–F) Representative traces of transcription activity of MS2-yellow reporters with sna shadow (B), sna primary (C), rho NEE (D), IAB5 (E), and Kr CD2 (F) enhancers.
(G) Average values of nascent RNAs produced per burst. Totals of 730, 834, 810, 804 and 853 nuclei from three individual embryos were analyzed for the reporter genes containing the sna shadow, sna primary, rho NEE, IAB5, and Kr CD2 enhancers, respectively. The error bars represent the standard deviation of the mean of three biological replicates. Asterisk indicates p < 0.01 compared to sna shadow enhancer.
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12. Figure S3
Distribution of Amplitude, Frequency, and Output in Three Independent Embryos, Related to Figure 2
(A–C) Boxplots showing the distribution of average values of amplitude (A), frequency (B), and output (C) per nucleus in three independent embryos. The box indicates the lower (25%) and upper (75%) quantile and the solid line indicates the median. The mean of three biological replicates is shown in Figure 2E. Following number of nuclei from three independent embryos was analyzed for each enhancer.
303, 319 and 285 nuclei for sna shadow, 232, 282 and 274 nuclei for sna primary, 137, 180 and 133 nuclei for rho NEE, 290, 261and 248 nuclei for IAB5, and 167, 126 and 120 nuclei for Kr CD2 enhancer.
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13. Figure S4
Differential Bursting Frequencies Contribute to Spatiotemporal Control of Gene Expression, Related to Figure 3
(A) Distribution of bursting frequencies in each 10 min time window during nc 14. sna shadow enhancer exhibits uniform bursting frequencies throughout the expression domain (top), while Kr CD2 enhancer exhibits dynamic frequency distributions over time (bottom).
(B) Distribution of bursting frequencies detected throughout nc 14 in the embryo expressing MS2-yellow with sna primary, rho NEE, and IAB5 enhancers. The images are oriented with anterior to the left. Ventral view is shown for sna primary enhancer. Images of rho NEE and IAB5 enhancers are oriented with dorsal to the top.
(C and D) Boxplots showing the distribution of burst frequencies and output per nucleus in each domain of rho NEE (C) and IAB5 (D) enhancers. In domains 1 and 2, 72 and 62 nuclei were analyzed for rho NEE and 86 and 89 nuclei were analyzed for IAB5, respectively. The box indicates the lower (25%) and upper (75%) quantile and the solid line indicates the median. Whiskers extend to 10th and 90th percentile of each distribution. Asterisk indicates p < 0.01.
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14. Figure S5
Core Promoter Sequence Alters the Dynamics of Transcription Activity, Related to Figure 3
(A) Schematic representation of MS2-yellow reporters with sna shadow enhancer and 100 bp minimal eve and 122 bp sog promoters. These transgenes also contain linked evePr-PP7-yellow and sogPr-PP7-yellow in symmetric location (see Figure S7).
(B) Relative MS2-yellow mRNA abundance was determined by quantitative RT-PCR using primers against intronic sequence. The mean values ± SD for three biological replicates are shown.
(C and D) Representative traces of transcription activity in the reporters with eve promoter (C) and sog promoter (D).
(E) Average values of amplitude, frequency, and output per nucleus in reporters with different promoters. 907, 761, 708 nuclei from three independent experiments were analyzed for the reporters with sna, eve and sog promoters, respectively. The error bars represent the standard deviation of the mean of three biological replicates. Plots shown in Figure 2E (sna shadow) were used for the reporter with sna promoter.
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15. Figure S6
Coordinated Bursts by Shared rho NEE and IAB5 Enhancers, Related to Figure 5
(A and B) Representative traces of transcription activity of MS2-yellow and PP7-yellow with rho NEE (A) and IAB5 (B) enhancers.
(C) Computational simulation of independent enhancer-promoter looping on the two sister chromatids. Each sister chromatid randomly produces bursts from either MS2 or PP7 reporter. The transcription activities from each chromatid were re-plotted as MS2 and PP7 trajectories over time, which is comparable to the observations from experimental data of rho NEE (∼5 bursts with ∼4 min duration; see Figure 2E). Frequencies of coordinate burst for rho NEE (Figures 5F and 5G) are shown for comparison. The error bars represent the standard deviation of the mean of three independent simulations (200 traces each). Asterisk indicates p < 0.01.
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16. Figure S7
Change in Core Promoter Sequence Alters the Frequency of Coordinate Bursts, Related to Figure 5
(A) Schematic representation of symmetric MS2-yellow and PP7-yellow reporters with minimal eve and sog promoters.
(B–E) Representative traces of transcription activity of MS2-yellow and PP7-yellow with eve (B and C) and sog promoters (D and E).
(F and G) Frequency of the coordinate bursts among all MS2-yellow (F) and PP7-yellow bursts (G). The error bars show the standard deviation of the mean of three biological replicates. 690 and 465 nuclei were analyzed for the reporters with eve promoter and sog promoter, respectively.
Cell  , DOI: ( /j.cell )
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