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Single-gene imaging links genome topology, promoter–enhancer communication and transcription control

Abstract

Transcription activation by distal enhancers is essential for cell-fate specification and maintenance of cellular identities. How long-range gene regulation is physically achieved, especially within complex regulatory landscapes of non-binary enhancer–promoter configurations, remains elusive. Recent nanoscopy advances have quantitatively linked promoter kinetics and ~100- to 200-nm-sized clusters of enhancer-associated regulatory factors (RFs) at important developmental genes. Here, we further dissect mechanisms of RF clustering and transcription activation in mouse embryonic stem cells. RF recruitment into clusters involves specific molecular recognition of cognate DNA and chromatin-binding sites, suggesting underlying cis-element clustering. Strikingly, imaging of tagged genomic loci, with ≤1 kilobase and ~20-nanometer precision, in live cells, reveals distal enhancer clusters over the extended locus in frequent close proximity to target genes—within RF-clustering distances. These high-interaction-frequency enhancer-cluster ‘superclusters’ create nano-environments wherein clustered RFs activate target genes, providing a structural framework for relating genome organization, focal RF accumulation and transcription activation.

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Fig. 1: Specific interactions with acetylated targets and Sox DNA motifs are needed for Brd4 and Sox2 clustering, respectively, while IDRs are dispensable.
Fig. 2: Specific interactions with acetylated targets and Sox DNA motifs are needed for Brd4 and Sox2 clustering, respectively, at the Pou5f1 locus, while IDRs are dispensable.
Fig. 3: High detection sensitivity enables imaging of tagged genomic loci and measuring of genomic interactions with nanometer precision in live cells.
Fig. 4: Imaging genomic interactions in the extended Pou5f1 locus reveals frequent proximity of distal enhancer clusters and the target gene.
Fig. 5: A shared transcription nanoscale environment created by RF clustering underlies apparent transcription coordination between Nanog sister chromatids.

Data availability

Datasets that support the findings in the paper are available in the Zenodo repository, https://doi.org/10.5281/zenodo.3960997. Source data are provided with this paper.

Code availability

Custom-written analysis code is available in the Zenodo repository, https://doi.org/10.5281/zenodo.3960997.

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Acknowledgements

We thank J. Gao, C.-Y. Han, D. Shola and C. Yang (Rockefeller University Gene Targeting Resource Center) for 24 × MS2 targeting at the Sox2 locus. We thank B. Gu and J. Wysocka (Stanford University) for providing reagents and advice regarding dCas9 imaging. We thank L. Lavis (HHMI-Janelia) for dye-labeling reagents. This work is supported by a NYSTEM Postdoctoral Training Award (C32599GG; J.L.), the JST PRESTO program (Japan) (JPMJPR15F2; H.O.) and JSPS KAKENHI (Japan) (JP18H05531 and JP19K06612; H.O.) and partially by JST CREST (Japan) (JPMJCR16G1; H.O.), the Louis V. Gerstner, Jr. Young Investigators Fund (A.P.), a National Cancer Institute grant (P30 CA008748), a National Institutes of Health (NIH) Director’s New Innovator Award (1DP2GM105443-01; A.P.) and the National Institute of General Medical Sciences of NIH (1R01GM135545-01 and 1R21GM134342-01; A.P.).

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Contributions

A.P. conceived, designed and supervised the study. J.L. developed and validated the experimental systems. H.O. and T.Y. established Pou5f1 and Nanog 24 × MS2-edited mESCs and materials and methods for live-cell MS2-MCP transcription imaging. J.L., A.H., Y.H., L.C. and A.P. performed experiments and analyzed the data. G.W. developed image-analysis code. A.P. wrote the manuscript.

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Correspondence to Alexandros Pertsinidis.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Quantification of Brd4 and Sox2 clustering throughout mESC nuclei.

(a, b) Normalized nuclear level for endogenous as well as ectopically expressed WT and mutant SNAP-tagged proteins. SNAP-SiR fluorescence was quantified over the whole nucleus, and then the data were normalized to the mean nuclear level of the endogenous SNAP-tagged factors. (a) Endogenous, as well as ectopically expressed WT, BDmut and IDRdel Brd4. Data points are from 2 independent experiments with total n= 29, 18, 21, and 16 cells for endogenous, WT, Bdmut, and IDRdel, respectively. (b) Endogenous, as well as ectopically expressed WT, 2M, TAD, and 2D Sox2. Data points are from 2 independent experiments with total n= 43, 15, 21, 18, and 20 cells for endogenous, WT, 2M, TAD, and 2D, respectively. (c, d) Scatter plots of total intensity vs. detection significance (p-value from a Wilcoxon rank-sum test) for each identified nuclear cluster. Representative data from 1 experiment are shown. (c) WT and mutant Brd4 (n=9, 8, 11, and 10 cells, for Endogenous, WT, Bdmut and IDRdel, respectively). (d) WT and mutant Sox2 (n=14, 13, 11, 10, and 11 cells, for Endogenous, WT, HMG point mutant (Sox2M) and HMG, and TAD deletion mutants (Sox2 TAD and Sox2D), respectively). Dashed lines in (c,d): p-value=0.001 cutoff. Box-plots in (a, b): boxes indicate inter-quartile range (IQR: 25%75% intervals) and the median line, whiskers indicate 1.5× the IQR; ‘×’ symbols indicate 1% and 99% percentiles; square symbols indicate the mean. Data for graphs in (a-d) are available as source data online.

Source data

Extended Data Fig. 2 Pol II and RF clustering at the endogenous Sox2 locus in mESCs.

(a) Live-cell imaging (2 μm × 2 μm ROIs, 0.8 s/scan) shows co-localized MCP-mNeonGreen and SiR-Rpb1, -Sox2, -Brd4, and –Med19 foci (yellow arrows). Bottom panels: relative MCP-Rpb1, -Sox2, -Brd4, and -Med19 xy coordinates. (b) 2D distances between MCP and factors (mean ± SD): Rpb1 102 ± 56 nm, Sox2 167 ± 81nm, Brd4 143 ± 80 nm, Med19 115 ± 57 nm (n = 12, 16, 32, and 23 cells, respectively). (c) Relative peak amplitude (A0), background level (B), r.m.s. width sxy = (sxsy)0.5, and integrated signal (πA0sxsy), for the Rpb1, Sox2, Brd4, and Med19 foci, respectively. Peak parameters are obtained by fitting to an elliptical 2D Gaussian function of the form I(x,y) = B + A0·exp[-x2/(2sx)2-y2/(2sy)2]. In the peak amplitude graph, the right-hand y-axis shows the number of molecules, calibrated based on the Pol II counts obtained by target-locking. (d and e) Organization of SNAP-Brd4 at the Sox2 locus. (d) Live-cell imaging reveals co-localized SNAP-tagged Brd4 foci with MCP-mNeonGreen-tagged nascent RNA, for WT and IDRdel, but not for BDmut Brd4. Yellow arrows indicate colocalized Brd4 clusters. (e) Quantification of Brd4 clustering at the Sox2 locus. Each data point corresponds to a single time-point measurement in a single cell (n=23, 8, 13, and 12 total cells for endogenous, WT, BDmut, and IDRdel Brd4, respectively). The left y-axis shows the size of individual clusters, normalized to the average cluster size of endogenous Brd4 at the Sox2 locus (~20 molecules). The right y-axis shows the absolute number of Brd4 molecules per cluster. The x-axis shows the nuclear level, estimated from the fitted local background level in the Sox2 ROI, normalized to the average nuclear level of endogenous Brd4. Solid lines: apparent binding curves by fitting to the function y=Ax/(x+K). Grey bar: endogenous Brd4 expression level. Box-plots in (b, c): boxes indicate inter-quartile range (IQR: 25%75% intervals) and the median line, whiskers indicate 1.5× the IQR; ‘×’ symbols indicate 1% and 99% percentiles; square symbols indicate the mean. Data for graphs in (b, c, e) are available as source data online.

Source data

Extended Data Fig. 3 Precision and reproducibility of dCas9 imaging and 2D distance measurements.

(a) Correlation between dCas9-Halo-JF646 – MCP-mNeongreen 2D distance measurements from 2 independent experiments in the extended Pou5f1 locus. Points: mean distances, error bars: S.E.M.. Solid line: linear fit. Pearson’s r = 0.93, indicates the reproducibility of the measurements. Total n= (29, 34, 23, 13, 32, 21, 17, 20, and 34) and (20, 12, 16, 17, 14, 17, 17, 13, and 16) individual transcription sites were measured in experiments 1 and 2, respectively, for the -80kb, -36kb, -16kb, -5kb, +21kb, +40kb, +90kb, +108kb, and +190kb genomic regions, respectively. (b) Absolute deviation between n=2 independent experiments. The mean absolute deviation is 20.8 nm. (c) xy scatter plots of the positions of the 9 tagged loci relative to the Pou5f1 transcription site. Dashed circles indicate the cutoff 2D distance (200 nm), used in Fig. 4c. Data for graphs in (a-c) are available as source data online.

Source data

Extended Data Fig. 4 Imaging genomic interactions in the extended Sox2 locus reveals frequent proximity of distal enhancer clusters and the target gene.

(a) Organization of the extended Sox2 locus from previously published proximity ligation (Hi-C, 5-C; accession numbers GSM2533818-21, GSM883649) and ChIP-Seq assays. ECs: enhancer clusters, characterized by presence of Sox2, p300 and H3K27ac ChIP signals. Accession numbers of ChIP-seq datasets used: GSM1910642, ENCFF001LJC, ENCFF001LJI, ENCFF001LIR, and ENCFF001KDN for Sox2, p300, Pol II, Ctcf, and H3K27ac, respectively. (b) Average distances and (c) proximity probabilities between the Sox2 transcription site (visualized with the 3′UTR MS2-tagged nascent RNA) and 5 regions between +20 kb and +300 kb from Sox2. (b) Data points: mean, error bars: S.E.M.. (c) Gray area: 95% binomial distribution confidence intervals. (b,c) Pooled data from 2 independent experiments with total n= 26, 24, 21, 38, and 27 individual transcription sites for the +21kb, +55kb, +98kb, +159kb, and +256kb genomic regions, respectively. In (b) Dashed line, indicates the 180 nm cutoff, corresponding to roughly the distance between RF clusters and the Pou5f1 transcription site. The 180nm distance cutoff is also used for the proximity probabilities shown in (c). (d) Cartoon of Sox2, highlighting frequent proximity of distal ECs to the Sox2 transcription site. Data for graphs in (b, c) are available as source data online.

Source data

Extended Data Fig. 5 A shared transcription nano-scale environment created by RF clustering underlies apparent transcription coordination between Sox2 sister chromatids.

(a) Bursting of sister chromatids at Sox2. Images show a resolvable MCP-mNeongreen doublet during the burst. (b) Representative live-cell imaging of the organization of nascent RNA and Brd4 in doublet Sox2 transcription sites. Brd4 is concentrated in the space between the two resolved transcription sites. (c and d) Localization of Brd4 clusters relative to the doublet Sox2 transcription sites (n=11 cells from 2 independent experiments). (c) Scatter plot with the MCP doublet axis aligned along x and the doublet mid-point at the origin. Each doublet is plotted twice, once for each possible orientation. Simulated Brd4 cluster localizations are based on the experimental distribution of Brd4 clusters in singlet Sox2 transcription sites (Extended Data Fig. 2a, b). (d) Distribution of distances along x for the experimental data in (c), as well as 105 simulations of n=11 doublets with independent Brd4 clusters. The experimental Brd4 distribution clearly shows Brd4 density in-between the MCP doublet, contrary to the expected bi-modal distribution if the two transcription sites each have an independent Brd4 cluster. (e) The measured mean absolute deviation of the Brd4 clusters along the axis of the MCP doublet, 157±27 nm (mean±S.E.M., n=11 cells from 2 independent experiments), is significantly smaller than the expected 228±23 nm (mean±S.D., 105 simulations of n=11 doublets with independent Brd4 clusters, p=0.0013 estimated from the numerical simulations). Data for graphs in (c, d, e) are available as source data online.

Source data

Supplementary information

Supplementary Information

Supplementary Figure 1.

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Supplementary Video 1

Bursting of sister chromatids at Nanog. Maximum-intensity projection of MPCP-mNeonGreen showing a transcription burst with apparent coordination between resolvable Nanog sister chromatids. Original data consist of 2.25-μm z stacks (250-nm z steps) obtained at 10 s per stack. Movie is played at 25× speed (4 frames per s).

Supplementary Video 2

Bursting of sister chromatids at Sox2. Maximum-intensity projection of MPCP-mNeonGreen showing a transcription burst with apparent coordination between resolvable Sox2 sister chromatids. Original data consist of 4.75-μm z stacks (250-nm z steps) obtained at 20 s per stack. Movie is played at 12.5× speed (4 frames per s).

Supplementary Table 1

Sequences of gRNAs for imaging the extended Sox2 locus.

Supplementary Table 2

Sequences of gRNAs for imaging the extended Pou5f1 locus.

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Li, J., Hsu, A., Hua, Y. et al. Single-gene imaging links genome topology, promoter–enhancer communication and transcription control. Nat Struct Mol Biol 27, 1032–1040 (2020). https://doi.org/10.1038/s41594-020-0493-6

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