Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

References

• Jacob F, Monod J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961;3:318–356.
   PubMed Web of Science ®Google Scholar
• Gertz J, Siggia ED, Cohen BA. Analysis of combinatorial cis-regulation in synthetic and genomic promoters. Nature. 2009;457:215–218.
   PubMed Web of Science ®Google Scholar
• Berg OG, Winter RB, Von Hippel PH. Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry. 1981;20:6929–6948.
   PubMed Web of Science ®Google Scholar
• Mahmutovic A, Berg OG, Elf J. What matters for lac repressor search in vivo–sliding, hopping, intersegment transfer, crowding on DNA or recognition? Nucleic Acids Res. 2015;43:3454–3464.
   PubMed Web of Science ®Google Scholar
• Halford SE, Marko JF. How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res. 2004;32:3040–3052.
   PubMed Web of Science ®Google Scholar
• Gowers DM, Halford SE. Protein motion from non-specific to specific DNA by three-dimensional routes aided by supercoiling. EMBO J. 2003;22:1410–1418.
   PubMed Web of Science ®Google Scholar
• Whiteside ST, Goodbourn S. Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation. J Cell Sci. 1993;104(Pt 4):949–955.
   PubMedGoogle Scholar
• Harbison CT, Gordon DB, Lee TI, et al. Transcriptional regulatory code of a eukaryotic genome. Nature. 2004;431:99–104.
   PubMed Web of Science ®Google Scholar
• Schmidt HG, Sewitz S, Andrews SS, et al. An integrated model of transcription factor diffusion shows the importance of intersegmental transfer and quaternary protein structure for target site finding. PLOS one. 2014;9:e108575.
   Google Scholar
• Li G-W, Xie XS. Central dogma at the single-molecule level in living cells. Nature. 2011;475:308–315.
   PubMed Web of Science ®Google Scholar
• Gebhardt JCM, Suter DM, Roy R, et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat Methods. 2013;10:421–426.
   PubMed Web of Science ®Google Scholar
• Normanno D, Boudarène L, Dugast-Darzacq C, et al. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher. Nat Commun. 2015;6:7357.
   PubMed Web of Science ®Google Scholar
• Mazza D, Abernathy A, Golob N, et al. A benchmark for chromatin binding measurements in live cells. Nucleic Acids Res. 2012;40:e119.
   PubMed Web of Science ®Google Scholar
• Liu Z, Legant WR, Chen B-C, et al. 3D imaging of Sox2 enhancer clusters in embryonic stem cells. Elife. 2014;3:e04236.
   PubMed Web of Science ®Google Scholar
• Chen J, Zhang Z, Li L, et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell. 2014;156:1274–1285.
   PubMed Web of Science ®Google Scholar
• Zhang Z, English BP, Grimm JB, et al. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis. Genes Dev. 2016;30:2106–2118.
   PubMed Web of Science ®Google Scholar
• Hammar P, Leroy P, Mahmutovic A, et al. The lac repressor displays facilitated diffusion in living cells. Science. 2012;336:1595–1598.
   PubMed Web of Science ®Google Scholar
• Nehlin JO, Carlberg M, Ronne H. Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J. 1991;10:3373–3377.
   PubMed Web of Science ®Google Scholar
• Frolova E. Binding of the glucose-dependent Mig1p repressor to the GAL1 and GAL4 promoters in vivo: regulationby glucose and chromatin structure. Nucleic Acids Res. 1999;27:1350–1358.
   PubMed Web of Science ®Google Scholar
• De Vit MJ, Waddle JA, Johnston M. Regulated nuclear translocation of the Mig1 glucose repressor. Mol Biol Cell. 1997;8:1603–1618.
   PubMed Web of Science ®Google Scholar
• Bendrioua L, Smedh M, Almquist J, et al. Yeast AMP-activated protein kinase monitors glucose concentration changes and absolute glucose levels. J Biol Chem. 2014;289:12863–12875.
   PubMed Web of Science ®Google Scholar
• Shashkova S, Wollman AJM, Leake MC, et al. The yeast Mig1 transcriptional repressor is dephosphorylated by glucose-dependent and -independent mechanisms. FEMS Microbiol Lett. 2017;364. DOI:10.1093/femsle/fnx133.
   PubMed Web of Science ®Google Scholar
• Wollman A. J. M. J., Shashkova S, Hedlund EG, et al. Transcription factor clusters regulate genes in eukaryotic cells. Elife. 2017;6:e27451.
   Google Scholar
• Leake MC. The physics of life: one molecule at a time. Philos Trans R Soc Lond B Biol Sci. 2013;368:20120248.
   PubMed Web of Science ®Google Scholar
• Miller H, Zhou Z, Shepherd J, et al. Single-molecule techniques in biophysics: a review of the progress in methods and applications. Rep Prog Phys. 2018;81:24601.
   Web of Science ®Google Scholar
• Shashkova S, Leake MC. Single-molecule fluorescence microscopy review: shedding new light on old problems. Biosci Rep. 2017;37. DOI:10.1042/BSR20170031.
   PubMed Web of Science ®Google Scholar
• Huang B, Wu H, Bhaya D, et al. Counting Low-Copy Number Proteins in a Single Cell. Science. 2007;315:81–84.
   PubMed Web of Science ®Google Scholar
• Wu M, Singh AK. Single-cell protein analysis. Curr Opin Biotechnol. 2012;23:83–88.
   PubMed Web of Science ®Google Scholar
• Bryan SJ, Burroughs NJ, Shevela D, et al. Localisation and interactions of the Vipp1 protein in cyanobacteria. Mol Microbiol. 2014;94:1179–1195.
   Web of Science ®Google Scholar
• Nenninger A, Mastroianni G, Robson A, et al. Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli. Mol Microbiol. 2014;92:1142–1153.
   PubMed Web of Science ®Google Scholar
• Lenn T, Leake MC. Single-molecule studies of the dynamics and interactions of bacterial OXPHOS complexes. Biochim Biophys Acta Bioenerg. 2016;1857:224–231.
   PubMed Web of Science ®Google Scholar
• Llorente-Garcia I, Lenn T, Erhardt H, et al. Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation. Biochim Biophys Acta. 2014;1837:811–824.
   PubMed Web of Science ®Google Scholar
• Lenn YT, Leake MC, Mullineaux CW. Are Escherichia coli OXPHOS complexes concentrated in specialized zones within the plasma membrane? Biochem Soc Trans. 2008;36:1032–1036.
   PubMed Web of Science ®Google Scholar
• Lenn T, Leake MC, Mullineaux CW. Clustering and dynamics of cytochrome bd-I complexes in the Escherichia coli plasma membrane in vivo. Mol Microbiol. 2008;70:1397–1407.
   PubMed Web of Science ®Google Scholar
• Badrinarayanan A, Leake MC. Using fluorescence recovery after photobleaching (FRAP) to study dynamics of the structural maintenance of chromosome (SMC) complex in vivo. Methods Mol Biol. 2016;1431:37–46.
   PubMedGoogle Scholar
• Chiu S-WS-W, Roberts MAJ, Leake MC, et al. Positioning of chemosensory proteins and ftsz through the rhodobacter sphaeroides cell cycle. Mol Microbiol. 2013;90:322–337.
   PubMed Web of Science ®Google Scholar
• Bisson-Filho AW, Hsu Y-P, Squyres GR, et al. Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science. 2017;355:739–743.
   PubMed Web of Science ®Google Scholar
• Lund VA, Wacnik K, Turner RD, et al. Molecular coordination of Staphylococcus aureus cell division. Elife. 2018;7:e32057.
   PubMed Web of Science ®Google Scholar
• Leake MC, Greene NP, Godun RM, et al. Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging. Proc Natl Acad Sci U S A. 2008;105:15376–15381.
   PubMed Web of Science ®Google Scholar
• Reid SWW, Leake MC, Chandler JH, et al. The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11. Proc Natl Acad Sci U S A. 2006;103:8066–8071.
   PubMed Web of Science ®Google Scholar
• Sowa Y, Rowe AD, Leake MC, et al. Direct observation of steps in rotation of the bacterial flagellar motor. Nature. 2005;437:916–919.
   PubMed Web of Science ®Google Scholar
• Pilizota T, Brown MT, Leake MC, et al. A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor. Proc Natl Acad Sci U S A. 2009;106:11582–11587.
   PubMed Web of Science ®Google Scholar
• Lo C-J, Leake MC, Pilizota T, et al. Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load. Biophys J. 2007;93:294–302.
   PubMed Web of Science ®Google Scholar
• Reyes-Lamothe R, Sherratt DJ, Leake MC. Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Science. 2010;328:498–501.
   PubMed Web of Science ®Google Scholar
• Badrinarayanan A, Reyes-Lamothe R, Uphoff S, et al. In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science. 2012;338:528–531.
   PubMed Web of Science ®Google Scholar
• Wollman AJM, Syeda AH, McGlynn P, et al. Single-molecule observation of DNA replication repair pathways in E. coli. Adv Exp Med Biol. 2016;915:5–16.
   PubMed Web of Science ®Google Scholar
• Leake MC. The Biophysics of Infection. Adv Exp Med Biol. 2016;915:1–3.
   PubMed Web of Science ®Google Scholar
• Miller H, Wollman AJM, Leake MC. Designing a single-molecule biophysics tool for characterising DNA damage for techniques that kill infectious pathogens through DNA damage effects. Adv Exp Med Biol. 2016;915:115–127.
   PubMed Web of Science ®Google Scholar
• Wollman AJM, Miller H, Foster S, et al. An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy. Phys Biol. 2016;5:55002.
   Google Scholar
• Lin Y, Sohn CH, Dalal CK, et al. Combinatorial gene regulation by modulation of relative pulse timing. Nature. 2015;527:54–58.
   PubMed Web of Science ®Google Scholar
• Plank M, Wadhams GH, Leake MC. Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes. Integr Biol (Camb). 2009;1:602–612.
   PubMed Web of Science ®Google Scholar
• M. C. Leake, Chandler, JH, Wadhams, GH, et al. Stoichiometry and turnover in single, functioning membrane protein complexes. Nature. 2006;443:355–358.
   PubMed Web of Science ®Google Scholar
• Shashkova S, Wollman A, Hohmann S, et al. Characterising maturation of GFP and mCherry of genomically integrated fusions in saccharomyces cerevisiae. Bio Protoc. 2018;7:e2710.
   Google Scholar
• Miller H, Cosgrove J, Wollman A, et al. High-speed single-molecule trackiung of CXCL13 in the B-Follicle. Front Immunol. 2018.
   Web of Science ®Google Scholar
• Tokunaga M, Imamoto N, Sakata-Sogawa K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods. 2008;5:159–161.
   PubMed Web of Science ®Google Scholar
• Chen B-C, Liu Y, Duzhko VV, et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science. 2014;346:1257998.
   PubMed Web of Science ®Google Scholar
• Abrahamsson S, Chen J, Hajj B, et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat Methods. 2013;10:60–63.
   PubMed Web of Science ®Google Scholar
• B. Yang, Wang, Y, Feng, S, et al. High numerical aperture epi-illumination selective plane illumination microscopy. bioRxiv. 2018;273359. Available from: https://doi.org/10.1101/273359
   Google Scholar
• Dean KM, Roudot P, Reis C, et al. Diagonally scanned light-sheet microscopy for fast volumetric imaging of adherent cells. Biophys J. 2016;110:1456–1465.
   PubMed Web of Science ®Google Scholar
• McKinney SA, Murphy CS, Hazelwood KL, et al. A bright and photostable photoconvertible fluorescent protein. Nat Methods. 2009;6:131–133.
   PubMed Web of Science ®Google Scholar
• Duan Z, Andronescu M, Schutz K, et al. A three-dimensional model of the yeast genome. Nature. 2010;465:363–367.
   PubMed Web of Science ®Google Scholar
• Miller H, Zhou Z, Wollman AJM, et al. Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes. Methods. 2015;88:81–88.
   PubMed Web of Science ®Google Scholar
• Wollman AJM, Leake MC. Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss. 2015;184:401–424.
   PubMed Web of Science ®Google Scholar
• Izeddin I, Récamier V, Bosanac L, et al. Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus. Elife. 2014;3:e02230.
   Web of Science ®Google Scholar
• Adam SA. The nuclear pore complex. Genome Biol. 2001;2:reviews0007.1- reviews0007.7.
   PubMed Web of Science ®Google Scholar
• Strambio-de-Castillia C, Niepel M, Rout MP. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat Rev Mol Cell Biol. 2010;11:490–501.
   PubMed Web of Science ®Google Scholar
• Yang W, Gelles J, Musser SM. Imaging of single-molecule translocation through nuclear pore complexes. Proc Natl Acad Sci U S A. 2004;101:12887–12892.
   PubMed Web of Science ®Google Scholar
• Lowe AR, Siegel JJ, Kalab P, et al. Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking. Nature. 2010;467:600–603.
   PubMed Web of Science ®Google Scholar
• Treitel MA, Carlson M. Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein. Proc Natl Acad Sci U S A. 1995;92:3132–3136.
   PubMed Web of Science ®Google Scholar
• Phillip Y, Schreiber G. Formation of protein complexes in crowded environments–from in vitro to in vivo. FEBS Lett. 2013;587:1046–1052.
   PubMed Web of Science ®Google Scholar
• Sode K, Ochiai S, Kobayashi N, et al. Effect of reparation of repeat sequences in the human alpha-synuclein on fibrillation ability. Int J Biol Sci. 2007;3:1–7.
   Web of Science ®Google Scholar
• Avitabile C, D'Andrea LD, Romanelli A. Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells. Sci Rep. 2014;4:337–360.
   Web of Science ®Google Scholar
• Cisse II, Izeddin I, Causse SZ, et al. Real-time dynamics of RNA polymerase II clustering in live human cells. Science. 2013;341:664–667.
   PubMed Web of Science ®Google Scholar
• Cho W-K, Jayanth N, English BP, et al. RNA polymerase II cluster dynamics predict mRNA output in living cells. Elife. 2016;5:e13617.
   PubMed Web of Science ®Google Scholar
• Qian J, Alföldi J, Pyntikova T, et al. B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity. Cell. 2014;159:1524–1537.
   PubMed Web of Science ®Google Scholar
• Mir M, Reimer A, Haines JE, et al. Dense bicoid hubs accentuate binding along the morphogen gradient. Genes & Dev. 2017;31:1784–1794.
   PubMed Web of Science ®Google Scholar
• Liu J, Perumal NB, Oldfield CJ, et al. Intrinsic disorder in transcription factors. Biochemistry. 2006;45:6873–6888.
   PubMed Web of Science ®Google Scholar
• Uversky VN, Patel VB. Intrinsically disordered proteins and their (disordered) proteomes in neurodegenerative disorders. Front Aging Neurosci. 2015;7:18.
   PubMed Web of Science ®Google Scholar
• Leake MC, Wilson D, Gautel M, et al. The elasticity of single titin molecules using a two-bead optical tweezers assay. Biophys J. 2004;87:1112–1135.
   PubMed Web of Science ®Google Scholar
• Leake MC, Wilson D, Bullard B, et al. The elasticity of single kettin molecules using a two-bead laser-tweezers assay. FEBS Lett. 2003;535:55–60.
   PubMed Web of Science ®Google Scholar