Abstract
Gene regulatory networks drive the specific transcriptional programmes responsible for the diversification of cell types during the development of multicellular organisms. Although our knowledge of the genes involved in these dynamic networks has expanded rapidly, our understanding of how transcription is spatiotemporally regulated at the molecular level over a wide range of timescales in the small volume of the nucleus remains limited. Over the past few decades, advances in the field of single-molecule fluorescence imaging have enabled real-time behaviours of individual transcriptional components to be measured in living cells and organisms. These efforts are now shedding light on the dynamic mechanisms of transcription, revealing not only the temporal rules but also the spatial coordination of underlying molecular interactions during various biological events.
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References
Semrau, S. & van Oudenaarden, A. Studying lineage decision-making in vitro: emerging concepts and novel tools. Annu. Rev. Cell Dev. Biol. 31, 317–345 (2015).
Hager, G. L., McNally, J. G. & Misteli, T. Transcription dynamics. Mol. Cell 35, 741–753 (2009).
Voss, T. C. & Hager, G. L. Dynamic regulation of transcriptional states by chromatin and transcription factors. Nat. Rev. Genet. 15, 69–81 (2014).
Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).
Eng, C. L. et al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature 568, 235–239 (2019).
Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).
Lubeck, E., Coskun, A. F., Zhiyentayev, T., Ahmad, M. & Cai, L. Single-cell in situ RNA profiling by sequential hybridization. Nat. Methods 11, 360–361 (2014).
Rodriques, S. G. et al. Slide-seq: a scalable technology for measuring genome-wide expression at high spatial resolution. Science 363, 1463–1467 (2019).
Stahl, P. L. et al. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353, 78–82 (2016).
VanHorn, S. & Morris, S. A. Next-generation lineage tracing and fate mapping to interrogate development. Dev. Cell 56, 7–21 (2021).
Eze, U. C., Bhaduri, A., Haeussler, M., Nowakowski, T. J. & Kriegstein, A. R. Single-cell atlas of early human brain development highlights heterogeneity of human neuroepithelial cells and early radial glia. Nat. Neurosci. 24, 584–594 (2021).
Bhaduri, A. et al. An atlas of cortical arealization identifies dynamic molecular signatures. Nature 598, 200–204 (2021).
Di Bella, D. J. et al. Molecular logic of cellular diversification in the mouse cerebral cortex. Nature 595, 554–559 (2021).
Wagh, K., Stavreva, D. A., Upadhyaya, A. & Hager, G. L. Transcription factor dynamics: one molecule at a time. Annu. Rev. Cell Dev. Biol. 39, 277–305 (2023).
Dahal, L., Walther, N., Tjian, R., Darzacq, X. & Graham, T. G. W. Single-molecule tracking (SMT): a window into live-cell transcription biochemistry. Biochem. Soc. Trans. 51, 557–569 (2023).
Garcia, H. G., Berrocal, A., Kim, Y. J., Martini, G. & Zhao, J. Lighting up the central dogma for predictive developmental biology. Curr. Top. Dev. Biol. 137, 1–35 (2020).
Sato, H., Das, S., Singer, R. H. & Vera, M. Imaging of DNA and RNA in living eukaryotic cells to reveal spatiotemporal dynamics of gene expression. Annu. Rev. Biochem. 89, 159–187 (2020).
Pichon, X., Robert, M. C., Bertrand, E., Singer, R. H. & Tutucci, E. New generations of MS2 variants and MCP fusions to detect single mRNAs in living eukaryotic cells. Methods Mol. Biol. 2166, 121–144 (2020).
Le, P., Ahmed, N. & Yeo, G. W. Illuminating RNA biology through imaging. Nat. Cell Biol. 24, 815–824 (2022).
Johansson, H. E. et al. A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein. Proc. Natl Acad. Sci. USA 95, 9244–9249 (1998).
Shav-Tal, Y. et al. Dynamics of single mRNPs in nuclei of living cells. Science 304, 1797–1800 (2004).
Chubb, J. R., Trcek, T., Shenoy, S. M. & Singer, R. H. Transcriptional pulsing of a developmental gene. Curr. Biol. 16, 1018–1025 (2006).
Lionnet, T. et al. A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat. Methods 8, 165–170 (2011).
Garcia, H. G., Tikhonov, M., Lin, A. & Gregor, T. Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning. Curr. Biol. 23, 2140–2145 (2013).
Campbell, P. D., Chao, J. A., Singer, R. H. & Marlow, F. L. Dynamic visualization of transcription and RNA subcellular localization in zebrafish. Development 142, 1368–1374 (2015).
Lee, C., Shin, H. & Kimble, J. Dynamics of notch-dependent transcriptional bursting in its native context. Dev. Cell 50, 426–435.e4 (2019).
Larson, D. R., Zenklusen, D., Wu, B., Chao, J. A. & Singer, R. H. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332, 475–478 (2011). This study analyses the detailed kinetics of the transcription cycle, including initiation, elongation and termination. It provides direct evidence that search behaviours of TFs mechanistically account for transcriptional bursting kinetics.
Hocine, S., Raymond, P., Zenklusen, D., Chao, J. A. & Singer, R. H. Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat. Methods 10, 119–121 (2013).
Bothma, J. P. et al. Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos. Proc. Natl Acad. Sci. USA 111, 10598–10603 (2014).
Lucas, T. et al. Live imaging of bicoid-dependent transcription in Drosophila embryos. Curr. Biol. 23, 2135–2139 (2013).
Zoller, B., Little, S. C. & Gregor, T. Diverse spatial expression patterns emerge from unified kinetics of transcriptional bursting. Cell 175, 835–847.e25 (2018).
Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14, 796–806 (2007).
Fiers, W. et al. Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene. Nature 260, 500–507 (1976).
Valegard, K., Liljas, L., Fridborg, K. & Unge, T. The three-dimensional structure of the bacterial virus MS2. Nature 345, 36–41 (1990).
Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).
Chao, J. A., Patskovsky, Y., Almo, S. C. & Singer, R. H. Structural basis for the coevolution of a viral RNA-protein complex. Nat. Struct. Mol. Biol. 15, 103–105 (2008).
Chen, J. et al. High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis. Proc. Natl Acad. Sci. USA 106, 13535–13540 (2009).
Lange, S. et al. Simultaneous transport of different localized mRNA species revealed by live-cell imaging. Traffic 9, 1256–1267 (2008).
Daigle, N. & Ellenberg, J. LambdaN-GFP: an RNA reporter system for live-cell imaging. Nat. Methods 4, 633–636 (2007).
Takizawa, P. A. & Vale, R. D. The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. Proc. Natl Acad. Sci. USA 97, 5273–5278 (2000).
Brodsky, A. S. & Silver, P. A. Identifying proteins that affect mRNA localization in living cells. Methods 26, 151–155 (2002).
Nguyen, D. H., DeFina, S. C., Fink, W. H. & Dieckmann, T. Binding to an RNA aptamer changes the charge distribution and conformation of malachite green. J. Am. Chem. Soc. 124, 15081–15084 (2002).
Babendure, J. R., Adams, S. R. & Tsien, R. Y. Aptamers switch on fluorescence of triphenylmethane dyes. J. Am. Chem. Soc. 125, 14716–14717 (2003).
Bouhedda, F., Autour, A. & Ryckelynck, M. Light-up RNA aptamers and their cognate fluorogens: from their development to their applications. Int. J. Mol. Sci. 19, 44 (2017).
Zhang, J. et al. Tandem spinach array for mRNA imaging in living bacterial cells. Sci. Rep. 5, 17295 (2015).
Cawte, A. D., Unrau, P. J. & Rueda, D. S. Live cell imaging of single RNA molecules with fluorogenic Mango II arrays. Nat. Commun. 11, 1283 (2020).
Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).
Strack, R. L., Disney, M. D. & Jaffrey, S. R. A superfolding spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat. Methods 10, 1219–1224 (2013).
Filonov, G. S., Moon, J. D., Svensen, N. & Jaffrey, S. R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J. Am. Chem. Soc. 136, 16299–16308 (2014).
Song, W. et al. Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex. Nat. Chem. Biol. 13, 1187–1194 (2017).
Autour, A. et al. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nat. Commun. 9, 656 (2018).
Garcia, J. F. & Parker, R. MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA 21, 1393–1395 (2015).
Haimovich, G. et al. Use of the MS2 aptamer and coat protein for RNA localization in yeast: a response to MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA 22, 660–666 (2016).
Garcia, J. F. & Parker, R. Ubiquitous accumulation of 3′ mRNA decay fragments in Saccharomyces cerevisiae mRNAs with chromosomally integrated MS2 arrays. RNA 22, 657–659 (2016).
Heinrich, S., Sidler, C. L., Azzalin, C. M. & Weis, K. Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. RNA 23, 134–141 (2017).
Li, W., Maekiniemi, A., Sato, H., Osman, C. & Singer, R. H. An improved imaging system that corrects MS2-induced RNA destabilization. Nat. Methods 19, 1558–1562 (2022).
Wu, B. et al. Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev. 29, 876–886 (2015).
Tutucci, E. et al. An improved MS2 system for accurate reporting of the mRNA life cycle. Nat. Methods 15, 81–89 (2018).
Tyagi, S. & Kramer, F. R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).
Bratu, D. P., Cha, B. J., Mhlanga, M. M., Kramer, F. R. & Tyagi, S. Visualizing the distribution and transport of mRNAs in living cells. Proc. Natl Acad. Sci. USA 100, 13308–13313 (2003).
Vargas, D. Y., Raj, A., Marras, S. A., Kramer, F. R. & Tyagi, S. Mechanism of mRNA transport in the nucleus. Proc. Natl Acad. Sci. USA 102, 17008–17013 (2005).
Turner-Bridger, B. et al. Single-molecule analysis of endogenous beta-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons. Proc. Natl Acad. Sci. USA 115, E9697–E9706 (2018).
Chan, S. H. et al. Brd4 and P300 confer transcriptional competency during zygotic genome activation. Dev. Cell 49, 867–881.e8 (2019).
Nelles, D. A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).
Wang, S., Su, J. H., Zhang, F. & Zhuang, X. An RNA-aptamer-based two-color CRISPR labeling system. Sci. Rep. 6, 26857 (2016).
Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature 550, 280–284 (2017).
Yang, L. Z. et al. Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems. Mol. Cell 76, 981–997.e7 (2019).
Wang, H. et al. CRISPR-mediated live imaging of genome editing and transcription. Science 365, 1301–1305 (2019).
Colognori, D., Trinidad, M. & Doudna, J. A. Precise transcript targeting by CRISPR–Csm complexes. Nat. Biotechnol. 41, 1256–1264 (2023).
Chen, H. & Larson, D. R. What have single-molecule studies taught us about gene expression? Genes Dev. 30, 1796–1810 (2016).
Liu, Z., Lavis, L. D. & Betzig, E. Imaging live-cell dynamics and structure at the single-molecule level. Mol. Cell 58, 644–659 (2015).
English, B. P. et al. Single-molecule investigations of the stringent response machinery in living bacterial cells. Proc. Natl Acad. Sci. USA 108, E365–E373 (2011).
Hansen, A. S. et al. Robust model-based analysis of single-particle tracking experiments with Spot-On. eLife 7, e33125 (2018).
Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).
Lukinavicius, G. & Johnsson, K. Switchable fluorophores for protein labeling in living cells. Curr. Opin. Chem. Biol. 15, 768–774 (2011).
Wang, L., Frei, M. S., Salim, A. & Johnsson, K. Small-molecule fluorescent probes for live-cell super-resolution microscopy. J. Am. Chem. Soc. 141, 2770–2781 (2019).
Grimm, J. B. & Lavis, L. D. Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels. Nat. Methods 19, 149–158 (2022).
Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).
Shen, H. et al. Single particle tracking: from theory to biophysical applications. Chem. Rev. 117, 7331–7376 (2017).
Speil, J. et al. Activated STAT1 transcription factors conduct distinct saltatory movements in the cell nucleus. Biophys. J. 101, 2592–2600 (2011). This study demonstrates that the motion of the STAT1 TF in the nucleus consists of several modes, characterized by slow, intermediate and fast diffusion. The slowest diffusing fraction is shown to represent chromatin-bound TFs as the residence time of that fraction robustly increases upon activation.
Mazza, D., Abernathy, A., Golob, N., Morisaki, T. & McNally, J. G. A benchmark for chromatin binding measurements in live cells. Nucleic Acids Res. 40, e119 (2012).
Gebhardt, J. C. et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat. Methods 10, 421–426 (2013).
Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat. Methods 10, 60–63 (2013).
Chen, J. et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156, 1274–1285 (2014). This study characterizes the temporal organization of the enhancer-binding pluripotency regulators (SOX2 and OCT4) in living ESCs.
Izeddin, I. et al. Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus. eLife 3, e02230 (2014).
Xie, X. S., Yu, J. & Yang, W. Y. Living cells as test tubes. Science 312, 228–230 (2006).
Elf, J., Li, G. W. & Xie, X. S. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194 (2007).
Schwille, P. Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem. Biophys. 34, 383–408 (2001).
Digman, M. A. & Gratton, E. Lessons in fluctuation correlation spectroscopy. Annu. Rev. Phys. Chem. 62, 645–668 (2011).
Elson, E. L. Fluorescence correlation spectroscopy: past, present, future. Biophys. J. 101, 2855–2870 (2011).
Wu, B., Buxbaum, A. R., Katz, Z. B., Yoon, Y. J. & Singer, R. H. Quantifying protein-mRNA interactions in single live cells. Cell 162, 211–220 (2015).
Kim, S. A., Heinze, K. G., Bacia, K., Waxham, M. N. & Schwille, P. Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry. Biophys. J. 88, 4319–4336 (2005).
Bacia, K. & Schwille, P. Practical guidelines for dual-color fluorescence cross-correlation spectroscopy. Nat. Protoc. 2, 2842–2856 (2007).
Gandin, V. et al. Cap-dependent translation initiation monitored in living cells. Nat. Commun. 13, 6558 (2022).
Michelman-Ribeiro, A. et al. Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy. Biophys. J. 97, 337–346 (2009).
Reisser, M. et al. Inferring quantity and qualities of superimposed reaction rates from single molecule survival time distributions. Sci. Rep. 10, 1758 (2020).
Ball, D. A. et al. Single molecule tracking of Ace1p in Saccharomyces cerevisiae defines a characteristic residence time for non-specific interactions of transcription factors with chromatin. Nucleic Acids Res. 44, e160 (2016).
Loffreda, A. et al. Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity. Nat. Commun. 8, 313 (2017).
Reisser, M. et al. Single-molecule imaging correlates decreasing nuclear volume with increasing TF–chromatin associations during zebrafish development. Nat. Commun. 9, 5218 (2018).
Hipp, L. et al. Single-molecule imaging of the transcription factor SRF reveals prolonged chromatin-binding kinetics upon cell stimulation. Proc. Natl Acad. Sci. USA 116, 880–889 (2019).
Nguyen, V. Q. et al. Spatiotemporal coordination of transcription preinitiation complex assembly in live cells. Mol. Cell 81, 3560–3575.e6 (2021). This study characterizes the molecular interplay involved in the formation of PIC in yeast by using SPT to detect general TFs, Mediator and Pol II.
Zhang, Z. et al. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis. Genes Dev. 30, 2106–2118 (2016).
Cho, W. K. et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412–415 (2018). This study characterizes the organization and dynamics of the Mediator coactivator and Pol II in living ESCs using light sheet imaging.
Millan-Zambrano, G., Burton, A., Bannister, A. J. & Schneider, R. Histone post-translational modifications — cause and consequence of genome function. Nat. Rev. Genet. 23, 563–580 (2022).
Hayashi-Takanaka, Y. et al. Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling. Nucleic Acids Res. 39, 6475–6488 (2011).
Sato, Y. et al. Genetically encoded system to track histone modification in vivo. Sci. Rep. 3, 2436 (2013).
Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).
Kerppola, T. K. Visualization of molecular interactions by fluorescence complementation. Nat. Rev. Mol. Cell Biol. 7, 449–456 (2006).
Mazzocca, M., Fillot, T., Loffreda, A., Gnani, D. & Mazza, D. The needle and the haystack: single molecule tracking to probe the transcription factor search in eukaryotes. Biochem. Soc. Trans. 49, 1121–1132 (2021).
Normanno, D. et al. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher. Nat. Commun. 6, 7357 (2015).
Darzacq, X. & Tjian, R. Weak multivalent biomolecular interactions: a strength versus numbers tug of war with implications for phase partitioning. RNA 28, 48–51 (2022).
Coulon, A., Chow, C. C., Singer, R. H. & Larson, D. R. Eukaryotic transcriptional dynamics: from single molecules to cell populations. Nat. Rev. Genet. 14, 572–584 (2013).
Rhee, H. S. & Pugh, B. F. Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature 483, 295–301 (2012).
Sainsbury, S., Bernecky, C. & Cramer, P. Structural basis of transcription initiation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 129–143 (2015).
Dultz, E. et al. Quantitative imaging of chromatin decompaction in living cells. Mol. Biol. Cell 29, 1763–1777 (2018).
Kim, J. M. et al. Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin. eLife 10, e69387 (2021).
Kenworthy, C. A. et al. Bromodomains regulate dynamic targeting of the PBAF chromatin-remodeling complex to chromatin hubs. Biophys. J. 121, 1738–1752 (2022).
Biswas, J., Li, W., Singer, R. H. & Coleman, R. A. Imaging organization of RNA processing within the nucleus. Cold Spring Harb. Perspect. Biol. 13, a039453 (2021).
Tantale, K. et al. A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting. Nat. Commun. 7, 12248 (2016).
Liu, J. et al. Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage. PLoS Comput. Biol. 17, e1008999 (2021).
Cho, W. K. et al. RNA Polymerase II cluster dynamics predict mRNA output in living cells. eLife 5, e13617 (2016).
Donovan, B. T. et al. Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting. EMBO J. 38, e100809 (2019).
Li, J. et al. Single-molecule nanoscopy elucidates RNA polymerase II transcription at single genes in live cells. Cell 178, 491–506.e28 (2019). In this study, target-locked 3D stimulated emission depletion is used to enable simultaneous single-molecule detection of Pol II assembly and nascent mRNA production at the endogenous OCT4 and Nanog loci in living mESCs.
Popp, A. P., Hettich, J. & Gebhardt, J. C. M. Altering transcription factor binding reveals comprehensive transcriptional kinetics of a basic gene. Nucleic Acids Res. 49, 6249–6266 (2021).
Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).
Chong, S. et al. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361, eaar2555 (2018).
Guo, Y. E. et al. Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572, 543–548 (2019).
Cisse, I. I. et al. Real-time dynamics of RNA polymerase II clustering in live human cells. Science 341, 664–667 (2013).
Liu, Z. et al. 3D imaging of Sox2 enhancer clusters in embryonic stem cells. eLife 3, e04236 (2014).
Dufourt, J. et al. Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs. Nat. Commun. 9, 5194 (2018).
Chen, H. et al. Dynamic interplay between enhancer–promoter topology and gene activity. Nat. Genet. 50, 1296–1303 (2018).
Fukaya, T., Lim, B. & Levine, M. Enhancer control of transcriptional bursting. Cell 166, 358–368 (2016).
Kawasaki, K. & Fukaya, T. Functional coordination between transcription factor clustering and gene activity. Mol. Cell 83, 1605–1622.e9 (2023).
Lim, B., Heist, T., Levine, M. & Fukaya, T. Visualization of transvection in living Drosophila embryos. Mol. Cell 70, 287–296.e6 (2018).
Levo, M. et al. Transcriptional coupling of distant regulatory genes in living embryos. Nature 605, 754–760 (2022).
Mateo, L. J. et al. Visualizing DNA folding and RNA in embryos at single-cell resolution. Nature 568, 49–54 (2019).
Benabdallah, N. S. et al. Decreased enhancer-promoter proximity accompanying enhancer activation. Mol. Cell 76, 473–484.e7 (2019).
Zuin, J. et al. Nonlinear control of transcription through enhancer-promoter interactions. Nature 604, 571–577 (2022).
Espinola, S. M. et al. Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development. Nat. Genet. 53, 477–486 (2021).
Alexander, J. M. et al. Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity. eLife 8, e41769 (2019).
Furlong, E. E. M. & Levine, M. Developmental enhancers and chromosome topology. Science 361, 1341–1345 (2018).
Hafner, A. & Boettiger, A. The spatial organization of transcriptional control. Nat. Rev. Genet. 24, 53–68 (2023).
Brandao, H. B., Gabriele, M. & Hansen, A. S. Tracking and interpreting long-range chromatin interactions with super-resolution live-cell imaging. Curr. Opin. Cell Biol. 70, 18–26 (2021).
Palacio, M. & Taatjes, D. J. Merging established mechanisms with new insights: condensates, hubs, and the regulation of RNA polymerase II transcription. J. Mol. Biol. 434, 167216 (2022).
Boehning, M. et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 25, 833–840 (2018).
Nagashima, R. et al. Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II. J. Cell Biol. 218, 1511–1530 (2019).
Mir, M. et al. Dense Bicoid hubs accentuate binding along the morphogen gradient. Genes Dev. 31, 1784–1794 (2017).
Tsai, A. et al. Nuclear microenvironments modulate transcription from low-affinity enhancers. eLife 6, e28975 (2017).
Uchino, S. et al. Live imaging of transcription sites using an elongating RNA polymerase II-specific probe. J. Cell Biol. 221, e202104134 (2022).
Forero-Quintero, L. S. et al. Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene. Nat. Commun. 12, 3158 (2021).
Rowley, M. J. & Corces, V. G. Organizational principles of 3D genome architecture. Nat. Rev. Genet. 19, 789–800 (2018).
Hansen, A. S., Pustova, I., Cattoglio, C., Tjian, R. & Darzacq, X. CTCF and cohesin regulate chromatin loop stability with distinct dynamics. eLife 6, e25776 (2017).
Nora, E. P. et al. Molecular basis of CTCF binding polarity in genome folding. Nat. Commun. 11, 5612 (2020).
Hansen, A. S., Amitai, A., Cattoglio, C., Tjian, R. & Darzacq, X. Guided nuclear exploration increases CTCF target search efficiency. Nat. Chem. Biol. 16, 257–266 (2020). This study characterizes the cluster formation and binding kinetics of CTCF.
Gabriele, M. et al. Dynamics of CTCF- and cohesin-mediated chromatin looping revealed by live-cell imaging. Science 376, 496–501 (2022). In this study, the chromatin looping within the FBN2 TAD in mESCs is directly visualized using super-resolution live-cell imaging, and the dynamics of the loops are quantified.
Mach, P. et al. Cohesin and CTCF control the dynamics of chromosome folding. Nat. Genet. 54, 1907–1918 (2022).
Luan, J. et al. Distinct properties and functions of CTCF revealed by a rapidly inducible degron system. Cell Rep. 34, 108783 (2021).
Xie, L. et al. 3D ATAC–PALM: super-resolution imaging of the accessible genome. Nat. Methods 17, 430–436 (2020).
Xie, L. et al. BRD2 compartmentalizes the accessible genome. Nat. Genet. 54, 481–491 (2022).
Kent, S. et al. Phase-separated transcriptional condensates accelerate target-search process revealed by live-cell single-molecule imaging. Cell Rep. 33, 108248 (2020).
Henikoff, S. & Shilatifard, A. Histone modification: cause or cog. Trends Genet. 27, 389–396 (2011).
Lee, J. S., Smith, E. & Shilatifard, A. The language of histone crosstalk. Cell 142, 682–685 (2010).
Stasevich, T. J. et al. Regulation of RNA polymerase II activation by histone acetylation in single living cells. Nature 516, 272–275 (2014). This study characterizes the temporal order of histone acetylation deposition and transcriptional activation at single specific loci.
Sato, Y. et al. Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis. Development 146, dev179127 (2019).
Zhang, T., Zhang, Z., Dong, Q., Xiong, J. & Zhu, B. Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells. Genome Biol. 21, 45 (2020).
Blackledge, N. P. et al. Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation. Cell 157, 1445–1459 (2014).
Youmans, D. T., Schmidt, J. C. & Cech, T. R. Live-cell imaging reveals the dynamics of PRC2 and recruitment to chromatin by SUZ12-associated subunits. Genes Dev. 32, 794–805 (2018).
Tatavosian, R. et al. Live-cell single-molecule dynamics of PcG proteins imposed by the DIPG H3.3K27M mutation. Nat. Commun. 9, 2080 (2018).
Hojfeldt, J. W. et al. Accurate H3K27 methylation can be established de novo by SUZ12-directed PRC2. Nat. Struct. Mol. Biol. 25, 225–232 (2018).
Reveron-Gomez, N. et al. Accurate recycling of parental histones reproduces the histone modification landscape during DNA replication. Mol. Cell 72, 239–249.e5 (2018).
Laugesen, A., Hojfeldt, J. W. & Helin, K. Molecular mechanisms directing PRC2 recruitment and H3K27 methylation. Mol. Cell 74, 8–18 (2019).
Huseyin, M. K. & Klose, R. J. Live-cell single particle tracking of PRC1 reveals a highly dynamic system with low target site occupancy. Nat. Commun. 12, 887 (2021).
Zhen, C. Y. et al. Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. eLife 5, e17667 (2016).
Biswas, S. et al. HP1 oligomerization compensates for low-affinity H3K9me recognition and provides a tunable mechanism for heterochromatin-specific localization. Sci. Adv. 8, eabk0793 (2022).
Loda, A., Collombet, S. & Heard, E. Gene regulation in time and space during X-chromosome inactivation. Nat. Rev. Mol. Cell Biol. 23, 231–249 (2022).
Masui, O. et al. Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation. Cell 145, 447–458 (2011). This study identifies the temporal interaction between paired homologous X chromosomes, suggesting the mechanism of choice of the X chromosome to be silenced.
Yoshimura, H. Live cell imaging of endogenous RNAs using pumilio homology domain mutants: principles and applications. Biochemistry 57, 200–208 (2018).
Ng, K. et al. A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells. Mol. Biol. Cell 22, 2634–2645 (2011).
Ha, N. et al. Live-cell imaging and functional dissection of xist RNA reveal mechanisms of X chromosome inactivation and reactivation. iScience 8, 1–14 (2018).
Tjalsma, S. J. D. et al. H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensable for inducing gene silencing. EMBO Rep. 22, e51989 (2021).
Rodermund, L. et al. Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading. Science 372, eabe7500 (2021). This study uses RNA-SPLIT to enable time-resolved analysis of Xist RNA molecules at a super-resolution, allowing for pulse–chase detection of single-molecule Xist RNA during XCI.
Dolgosheina, E. V. et al. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem. Biol. 9, 2412–2420 (2014).
Hayashi-Takanaka, Y., Yamagata, K., Nozaki, N. & Kimura, H. Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase. J. Cell Biol. 187, 781–790 (2009).
Rajan, M. et al. Generation of an alpaca-derived nanobody recognizing γ-H2AX. FEBS Open Bio 5, 779–788 (2015).
Sato, Y. et al. A genetically encoded probe for live-cell imaging of H4K20 monomethylation. J. Mol. Biol. 428, 3885–3902 (2016).
Suzuki, M. et al. In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration. Genes Cell 21, 358–369 (2016).
Kurita, K. et al. Live imaging of H3K9 acetylation in plant cells. Sci. Rep. 7, 45894 (2017).
Villasenor, R. et al. ChromID identifies the protein interactome at chromatin marks. Nat. Biotechnol. 38, 728–736 (2020).
Delachat, A. M. et al. Engineered multivalent sensors to detect coexisting histone modifications in living stem cells. Cell Chem. Biol. 25, 51–56.e6 (2018).
Dos Santos Passos, C., Choi, Y. S., Snow, C. D., Yao, T. & Cohen, R. E. Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells. J. Cell Biol. 220, e201911130 (2021).
Chung, C. I. et al. Intrabody-based FRET probe to visualize endogenous histone acetylation. Sci. Rep. 9, 10188 (2019).
Sasaki, K., Ito, T., Nishino, N., Khochbin, S. & Yoshida, M. Real-time imaging of histone H4 hyperacetylation in living cells. Proc. Natl Acad. Sci. USA 106, 16257–16262 (2009).
Ito, T. et al. Real-time imaging of histone H4K12-specific acetylation determines the modes of action of histone deacetylase and bromodomain inhibitors. Chem. Biol. 18, 495–507 (2011).
Nakaoka, S., Sasaki, K., Ito, A., Nakao, Y. & Yoshida, M. A genetically encoded FRET probe to detect intranucleosomal histone H3K9 or H3K14 acetylation using BRD4, a BET family member. ACS Chem. Biol. 11, 729–733 (2016).
Peng, Q. et al. Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors. Proc. Natl Acad. Sci. USA 115, E11681–E11690 (2018).
Sasaki, K. et al. Visualization of the dynamic interaction between nucleosomal histone H3K9 tri-methylation and HP1α chromodomain in living cells. Cell Chem. Biol. 29, 1153–1161.e5 (2022).
Lungu, C., Pinter, S., Broche, J., Rathert, P. & Jeltsch, A. Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites. Nat. Commun. 8, 649 (2017).
Ohmuro-Matsuyama, Y., Kitaguchi, T., Kimura, H. & Ueda, H. Simple fluorogenic cellular assay for histone deacetylase inhibitors based on split-yellow fluorescent protein and intrabodies. ACS Omega 6, 10039–10046 (2021).
Vincenz, C. & Kerppola, T. K. Different polycomb group CBX family proteins associate with distinct regions of chromatin using nonhomologous protein sequences. Proc. Natl Acad. Sci. USA 105, 16572–16577 (2008).
Acknowledgements
The authors thank R. A. Coleman and Z. J. Liu for critical reading and discussion of the manuscript. They also thank members of the Singer laboratory for their comments and discussions. This work has been completed with funding from the US NIH grants R01-NS083085 and 1R35 GM136296 (to R.H.S.), and from the World Premier International Research Center Initiative (WPI), Ministry of Education, Culture, Sports, Science and Technology, Japan (to H.S).
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H.S., D.-W.H. and A.M. researched data for the article, H.S. and D.-W.H. contributed substantially to discussion of the content, H.S., D.-W.H. and A.M. wrote the manuscript, all authors edited and reviewed the manuscript before submission and H.S. and A.M. drafted the figures and tables.
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Hwang, DW., Maekiniemi, A., Singer, R.H. et al. Real-time single-molecule imaging of transcriptional regulatory networks in living cells. Nat Rev Genet 25, 272–285 (2024). https://doi.org/10.1038/s41576-023-00684-9
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DOI: https://doi.org/10.1038/s41576-023-00684-9
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