[en] Transcription factors (TFs) regulate gene expression by binding to specific consensus motifs within the local chromatin context. The mechanisms by which TFs navigate the nuclear environment as they search for binding sites remain unclear. Here, we used single-molecule tracking and machine-learning-based classification to directly measure the nuclear mobility of the glucocorticoid receptor (GR) in live cells. We revealed two distinct and dynamic low-mobility populations. One accounts for specific binding to chromatin, while the other represents a confinement state that requires an intrinsically disordered region (IDR), implicated in liquid-liquid condensate subdomains. Further analysis showed that the dwell times of both subpopulations follow a power-law distribution, consistent with a broad distribution of affinities on the GR cistrome and interactome. Together, our data link IDRs with a confinement state that is functionally distinct from specific chromatin binding and modulates the transcriptional output by increasing the local concentration of TFs at specific sites.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Garcia, David A; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA, Department of Physics, University of Maryland, College Park, MD 20742, USA
Johnson, Thomas A ; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA
Presman, Diego M ; Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), CONICET-Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, C1428EGA Buenos Aires, Argentina
Fettweis, Grégory ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et biologie moléculaires animales ; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA
Wagh, Kaustubh ; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA, Department of Physics, University of Maryland, College Park, MD 20742, USA
Rinaldi, Lorenzo; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA
Stavreva, Diana A; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA
Paakinaho, Ville; Institute of Biomedicine, University of Eastern Finland, Kuopio, P.O. Box 1627, 70211 Kuopio, Finland
Jensen, Rikke A M ; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
Mandrup, Susanne ; Institute for Physical Science and Technology, University of Maryland, College Park, MD 20742, USA
Upadhyaya, Arpita; Department of Physics, University of Maryland, College Park, MD 20742, USA, Institute for Physical Science and Technology, University of Maryland, College Park, MD 20742, USA. Electronic address: arpitau@umd.edu
Hager, Gordon L; Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20893, USA. Electronic address: hagerg@exchange.nih.gov
Language :
English
Title :
An intrinsically disordered region-mediated confinement state contributes to the dynamics and function of transcription factors.
We thank Tatiana Karpova and David Ball from the Optical Microscopy Core at the NCI, NIH for assistance in imaging and data processing. We thank Luke Lavis (Janelia Research Campus) for providing HALO dyes. This research was supported (in part) by the Intramural Research Program of the NIH , National Cancer Institute , Center for Cancer Research . D.M.P. was supported by CONICET . V.P. was supported by the Academy of Finland , the Cancer Foundation Finland , and the Sigrid Jusélius Foundation . R.A.M.J. was supported by the Vissing Foundation , the William Demant Foundation , the Knud Højgaard Foundation , the Frimodt-Heineke Foundation , the Director Ib Henriksen Foundation , and the Ove and Edith Buhl Olesen Memorial Foundation . S.M. acknowledges support from the Danish Independent Research Council | Natural Sciences . A.U. acknowledges support from the NCI-UMD Cancer Technology Partnership and the awards NSF PHY 1607645 and NSF PHY 1806903 .
Banaz, N., Mäkelä, J., Uphoff, S., Choosing the right label for single-molecule tracking in live bacteria: side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes. J. Phys. D Appl. Phys., 52, 2019, 064002.
Basu, S., Mackowiak, S.D., Niskanen, H., Knezevic, D., Asimi, V., Grosswendt, S., Geertsema, H., Ali, S., Jerković, I., Ewers, H., et al. Unblending of Transcriptional Condensates in Human Repeat Expansion Disease. Cell 181 (2020), 1062–1079.e30.
Ben-Avraham, Z., Havlin, S., Diffusion and Reactions in Fractals and Disordered Systems. 2000, Cambridge University Press.
Bénichou, O., Chevalier, C., Meyer, B., Voituriez, R., Facilitated diffusion of proteins on chromatin. Phys. Rev. Lett., 106, 2011, 038102.
Berry, J., Weber, S.C., Vaidya, N., Haataja, M., Brangwynne, C.P., RNA transcription modulates phase transition-driven nuclear body assembly. Proc. Natl. Acad. Sci. USA 112 (2015), E5237–E5245.
Bledsoe, R.K., Montana, V.G., Stanley, T.B., Delves, C.J., Apolito, C.J., McKee, D.D., Consler, T.G., Parks, D.J., Stewart, E.L., Willson, T.M., et al. Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 110 (2002), 93–105.
Boehning, M., Dugast-Darzacq, C., Rankovic, M., Hansen, A.S., Yu, T., Marie-Nelly, H., McSwiggen, D.T., Kokic, G., Dailey, G.M., Cramer, P., et al. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 25 (2018), 833–840.
Boija, A., Klein, I.A., Sabari, B.R., Dall'Agnese, A., Coffey, E.L., Zamudio, A.V., Li, C.H., Shrinivas, K., Manteiga, J.C., Hannett, N.M., et al. Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell 175 (2018), 1842–1855.e16.
Bolger, A.M., Lohse, M., Usadel, B., Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 (2014), 2114–2120, 10.1093/bioinformatics/btu170.
Brodsky, S., Jana, T., Mittelman, K., Chapal, M., Kumar, D.K., Carmi, M., Barkai, N., Intrinsically Disordered Regions Direct Transcription Factor In Vivo Binding Specificity. Mol. Cell 79 (2020), 459–471.e4.
Brouwer, I., Lenstra, T.L., Visualizing transcription: key to understanding gene expression dynamics. Curr. Opin. Chem. Biol. 51 (2019), 122–129.
Cho, W.K., Spille, J.H., Hecht, M., Lee, C., Li, C., Grube, V., Cisse, I.I., Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361 (2018), 412–415.
Chong, S., Dugast-Darzacq, C., Liu, Z., Dong, P., Dailey, G.M., Cattoglio, C., Heckert, A., Banala, S., Lavis, L., Darzacq, X., Tjian, R., Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science, 361, 2018, eaar2555.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F., Multiplex genome engineering using CRISPR/Cas systems. Science 339 (2013), 819–823, 10.1126/science.1231143.
Coons, L.A., Burkholder, A.B., Hewitt, S.C., McDonnell, D.P., Korach, K.S., Decoding the Inversion Symmetry Underlying Transcription Factor DNA-Binding Specificity and Functionality in the Genome. iScience 15 (2019), 552–591.
Darling, A.L., Uversky, V.N., Intrinsic Disorder in Proteins with Pathogenic Repeat Expansions. Molecules, 22, 2017, 2027.
Dasgupta, S., Lonard, D.M., O'Malley, B.W., Nuclear receptor coactivators: master regulators of human health and disease. Annu. Rev. Med. 65 (2014), 279–292.
Donovan, B.T., Huynh, A., Ball, D.A., Patel, H.P., Poirier, M.G., Larson, D.R., Ferguson, M.L., Lenstra, T.L., Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting. EMBO J., 38, 2019, e100809.
Everaers, R., Schiessel, H., The physics of chromatin. J. Phys. Condens. Matter, 27, 2015, 060301.
Fan, H.Y., Trotter, K.W., Archer, T.K., Kingston, R.E., Swapping function of two chromatin remodeling complexes. Mol. Cell 17 (2005), 805–815.
Finn, E.H., Misteli, T., Molecular basis and biological function of variability in spatial genome organization. Science, 365, 2019, eaaw9498.
Garcia, D.A., Fettweis, G., Presman, D.M., Paakinaho, V., Jarzynski, C., Upadhyaya, A., Hager, G.L., Power-law behaviour of transcription factor dynamics at the single-molecule level implies a continuum affinity model. bioRxiv, 2020, 10.1101/637355.
Goldstein, I., Hager, G.L., Dynamic enhancer function in the chromatin context. Wiley Interdiscip. Rev. Syst. Biol. Med., 10, 2018, e1390.
Grimm, J.B., English, B.P., Chen, J., Slaughter, J.P., Zhang, Z., Revyakin, A., Patel, R., Macklin, J.J., Normanno, D., Singer, R.H., et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12 (2015), 244–250 3, 250.
Grünwald, D., Spottke, B., Buschmann, V., Kubitscheck, U., Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells. Mol. Biol. Cell 17 (2006), 5017–5027.
Innis, J.W., Mortlock, D., Chen, Z., Ludwig, M., Williams, M.E., Williams, T.M., Doyle, C.D., Shao, Z., Glynn, M., Mikulic, D., et al. Polyalanine expansion in HOXA13: three new affected families and the molecular consequences in a mouse model. Hum. Mol. Genet. 13 (2004), 2841–2851.
Izeddin, I., Récamier, V., Bosanac, L., Cissé, I.I., Boudarene, L., Dugast-Darzacq, C., Proux, F., Bénichou, O., Voituriez, R., Bensaude, O., et al. Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus. eLife, 3, 2014, e02230.
James, G., Witten, D., Hastie, T., Tibshirani, R., An Introduction to Statistical Learning: With Applications in R. 2017, Springer.
Jaynes, E.T., Bretthorst, G.L., Probability Theory: The Logic of Science. 2019, Cambridge University Press.
Jewell, C.M., Scoltock, A.B., Hamel, B.L., Yudt, M.R., Cidlowski, J.A., Complex human glucocorticoid receptor dim mutations define glucocorticoid induced apoptotic resistance in bone cells. Mol. Endocrinol. 26 (2012), 244–256.
John, S., Sabo, P.J., Johnson, T.A., Sung, M.H., Biddie, S.C., Lightman, S.L., Voss, T.C., Davis, S.R., Meltzer, P.S., Stamatoyannopoulos, J.A., Hager, G.L., Interaction of the glucocorticoid receptor with the chromatin landscape. Mol. Cell 29 (2008), 611–624.
Johnson, T.A., Chereji, R.V., Stavreva, D.A., Morris, S.A., Hager, G.L., Clark, D.J., Conventional and pioneer modes of glucocorticoid receptor interaction with enhancer chromatin in vivo. Nucleic Acids Res. 46 (2018), 203–214.
Kent, S., Brown, K., Yang, C.H., Alsaihati, N., Tian, C., Wang, H., Ren, X., Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging. Cell Rep., 33, 2020, 108248.
Khan, S.H., Awasthi, S., Guo, C., Goswami, D., Ling, J., Griffin, P.R., Simons, S.S. Jr., Kumar, R., Binding of the N-terminal region of coactivator TIF2 to the intrinsically disordered AF1 domain of the glucocorticoid receptor is accompanied by conformational reorganizations. J. Biol. Chem. 287 (2012), 44546–44560.
Koo, P.K., Mochrie, S.G., Systems-level approach to uncovering diffusive states and their transitions from single-particle trajectories. Phys. Rev. E, 94, 2016, 052412.
Koo, P.K., Weitzman, M., Sabanaygam, C.R., van Golen, K.L., Mochrie, S.G., Extracting Diffusive States of Rho GTPase in Live Cells: Towards In Vivo Biochemistry. PLoS Comput. Biol., 11, 2015, e1004297.
Koslover, E.F., Fuller, C.J., Straight, A.F., Spakowitz, A.J., Local geometry and elasticity in compact chromatin structure. Biophys. J. 99 (2010), 3941–3950.
Kumar, R., Litwack, G., Structural and functional relationships of the steroid hormone receptors’ N-terminal transactivation domain. Steroids 74 (2009), 877–883.
Langmead, B., Salzberg, S.L., Fast gapped-read alignment with Bowtie 2. Nat. Methods 9 (2012), 357–359.
Lazar, M.A., Maturing of the nuclear receptor family. J. Clin. Invest. 127 (2017), 1123–1125.
Lerner, J., Gomez-Garcia, P.A., McCarthy, R.L., Liu, Z., Lakadamyali, M., Zaret, K.S., Two-Parameter Mobility Assessments Discriminate Diverse Regulatory Factor Behaviors in Chromatin. Mol. Cell 79 (2020), 677–688.e6.
Levi, V., Gratton, E., Exploring dynamics in living cells by tracking single particles. Cell Biochem. Biophys. 48 (2007), 1–15.
Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., The sequence alignment/map format and SAMtools. Bioinformatics 25 (2009), 2078–2079, 10.1093/bioinformatics/btp352.
Lim, H.W., Uhlenhaut, N.H., Rauch, A., Weiner, J., Hübner, S., Hübner, N., Won, K.J., Lazar, M.A., Tuckermann, J., Steger, D.J., Genomic redistribution of GR monomers and dimers mediates transcriptional response to exogenous glucocorticoid in vivo. Genome Res. 25 (2015), 836–844.
Liu, Z., Tjian, R., Visualizing transcription factor dynamics in living cells. J. Cell Biol. 217 (2018), 1181–1191.
Lu, H., Yu, D., Hansen, A.S., Ganguly, S., Liu, R., Heckert, A., Darzacq, X., Zhou, Q., Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558 (2018), 318–323.
Lu, Y., Wu, T., Gutman, O., Lu, H., Zhou, Q., Henis, Y.I., Luo, K., Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression. Nat. Cell Biol. 22 (2020), 453–464.
Luisi, B.F., Xu, W.X., Otwinowski, Z., Freedman, L.P., Yamamoto, K.R., Sigler, P.B., Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352 (1991), 497–505.
Mazza, D., Abernathy, A., Golob, N., Morisaki, T., McNally, J.G., A benchmark for chromatin binding measurements in live cells. Nucleic Acids Res., 40, 2012, e119.
Mazza, D., Ganguly, S., McNally, J.G., Monitoring dynamic binding of chromatin proteins in vivo by single-molecule tracking. Methods Mol. Biol. 1042 (2013), 117–137.
Metzler, R., Jeon, J.H., Cherstvy, A.G., Barkai, E., Anomalous diffusion models and their properties: non-stationarity, non-ergodicity, and ageing at the centenary of single particle tracking. Phys. Chem. Chem. Phys. 16 (2014), 24128–24164.
Mikuni, S., Tamura, M., Kinjo, M., Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy. FEBS Lett. 581 (2007), 389–393.
Mikuni, S., Yamamoto, J., Horio, T., Kinjo, M., Negative Correlation between the Diffusion Coefficient and Transcriptional Activity of the Glucocorticoid Receptor. Int. J. Mol. Sci., 18, 2017, 1855.
Morisaki, T., Müller, W.G., Golob, N., Mazza, D., McNally, J.G., Single-molecule analysis of transcription factor binding at transcription sites in live cells. Nat. Commun., 5, 2014, 4456.
Muragaki, Y., Mundlos, S., Upton, J., Olsen, B.R., Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13. Science 272 (1996), 548–551.
Nakamura, K., Jeong, S.Y., Uchihara, T., Anno, M., Nagashima, K., Nagashima, T., Ikeda, S., Tsuji, S., Kanazawa, I., SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein. Hum. Mol. Genet. 10 (2001), 1441–1448.
Normanno, D., Boudarène, L., Dugast-Darzacq, C., Chen, J., Richter, C., Proux, F., Bénichou, O., Voituriez, R., Darzacq, X., Dahan, M., Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher. Nat. Commun., 6, 2015, 7357.
Onuki, A., Phase Transition Dynamics. 2007, Cambridge University Press.
Paakinaho, V., Presman, D.M., Ball, D.A., Johnson, T.A., Schiltz, R.L., Levitt, P., Mazza, D., Morisaki, T., Karpova, T.S., Hager, G.L., Single-molecule analysis of steroid receptor and cofactor action in living cells. Nat. Commun., 8, 2017, 15896.
Peng, Z., Mizianty, M.J., Xue, B., Kurgan, L., Uversky, V.N., More than just tails: intrinsic disorder in histone proteins. Mol. Biosyst. 8 (2012), 1886–1901.
Presman, D.M., Hager, G.L., More than meets the dimer: What is the quaternary structure of the glucocorticoid receptor?. Transcription 8 (2017), 32–39.
Presman, D.M., Ogara, M.F., Stortz, M., Alvarez, L.D., Pooley, J.R., Schiltz, R.L., Grøntved, L., Johnson, T.A., Mittelstadt, P.R., Ashwell, J.D., et al. Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor. PLoS Biol., 12, 2014, e1001813.
Presman, D.M., Ganguly, S., Schiltz, R.L., Johnson, T.A., Karpova, T.S., Hager, G.L., DNA binding triggers tetramerization of the glucocorticoid receptor in live cells. Proc. Natl. Acad. Sci. USA 113 (2016), 8236–8241.
Presman, D.M., Ball, D.A., Paakinaho, V., Grimm, J.B., Lavis, L.D., Karpova, T.S., Hager, G.L., Quantifying transcription factor binding dynamics at the single-molecule level in live cells. Methods 123 (2017), 76–88.
Qian, H., Sheetz, M.P., Elson, E.L., Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys. J. 60 (1991), 910–921.
Quinlan, A.R., Hall, I.M., BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26 (2010), 841–842, 10.1093/bioinformatics/btq033.
Ratke, L., Voorhees, P.W., Growth and Coarsening: Ostwald Ripening in Material Processing. 2011, Springer.
Ratman, D., Mylka, V., Bougarne, N., Pawlak, M., Caron, S., Hennuyer, N., Paumelle, R., De Cauwer, L., Thommis, J., Rider, M.H., et al. Chromatin recruitment of activated AMPK drives fasting response genes co-controlled by GR and PPARα. Nucleic Acids Res. 44 (2016), 10539–10553.
Reingruber, J., Holcman, D., Transcription factor search for a DNA promoter in a three-state model. Phys. Rev. E Stat. Nonlin. Soft Matter Phys., 84, 2011, 020901.
Reisser, M., Hettich, J., Kuhn, T., Popp, A.P., Große-Berkenbusch, A., Gebhardt, J.C.M., Inferring quantity and qualities of superimposed reaction rates from single molecule survival time distributions. Sci. Rep., 10, 2020, 1758.
Reiter, F., Wienerroither, S., Stark, A., Combinatorial function of transcription factors and cofactors. Curr. Opin. Genet. Dev. 43 (2017), 73–81.
Rogatsky, I., Wang, J.C., Derynck, M.K., Nonaka, D.F., Khodabakhsh, D.B., Haqq, C.M., Darimont, B.D., Garabedian, M.J., Yamamoto, K.R., Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor. Proc. Natl. Acad. Sci. USA 100 (2003), 13845–13850.
Sabari, B.R., Dall'Agnese, A., Boija, A., Klein, I.A., Coffey, E.L., Shrinivas, K., Abraham, B.J., Hannett, N.M., Zamudio, A.V., Manteiga, J.C., et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science, 361, 2018, eaar3958.
Schwarz, G., Estimating the dimension of a model. Ann. Stat. 6 (1978), 461–464.
Shakya, A., Park, S., Rana, N., King, J.T., Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization. Biophys. J. 118 (2020), 753–764.
Shin, Y., Brangwynne, C.P., Liquid phase condensation in cell physiology and disease. Science, 357, 2017, eaaf4382.
Simons, S.S. Jr., Kumar, R., Variable steroid receptor responses: Intrinsically disordered AF1 is the key. Mol. Cell. Endocrinol. 376 (2013), 81–84.
Simons, S.S. Jr., Edwards, D.P., Kumar, R., Minireview: dynamic structures of nuclear hormone receptors: new promises and challenges. Mol. Endocrinol. 28 (2014), 173–182.
Stasevich, T.J., Mueller, F., Michelman-Ribeiro, A., Rosales, T., Knutson, J.R., McNally, J.G., Cross-validating FRAP and FCS to quantify the impact of photobleaching on in vivo binding estimates. Biophys. J. 99 (2010), 3093–3101.
Stavreva, D.A., Coulon, A., Baek, S., Sung, M.H., John, S., Stixova, L., Tesikova, M., Hakim, O., Miranda, T., Hawkins, M., et al. Dynamics of chromatin accessibility and long-range interactions in response to glucocorticoid pulsing. Genome Res. 25 (2015), 845–857.
Stavreva, D.A., Garcia, D.A., Fettweis, G., Gudla, P.R., Zaki, G.F., Soni, V., McGowan, A., Williams, G., Huynh, A., Palangat, M., et al. Transcriptional Bursting and Co-bursting Regulation by Steroid Hormone Release Pattern and Transcription Factor Mobility. Mol. Cell 75 (2019), 1161–1177.e11.
Stortz, M., Pecci, A., Presman, D.M., Levi, V., Unraveling the molecular interactions involved in phase separation of glucocorticoid receptor. BMC Biol., 18, 2020, 59.
