Transcription Dynamics in Living Cells.

The transcription cycle can be roughly divided into three stages: initiation, elongation, and termination. Understanding the molecular events that regulate all these stages requires a dynamic view of the underlying processes. The development of techniques to visualize and quantify transcription in single living cells has been essential in revealing the transcription kinetics. They have revealed that (a) transcription is heterogeneous between cells and (b) transcription can be discontinuous within a cell. In this review, we discuss the progress in our quantitative understanding of transcription dynamics in living cells, focusing on all parts of the transcription cycle. We present the techniques allowing for single-cell transcription measurements, review evidence from different organisms, and discuss how these experiments have broadened our mechanistic understanding of transcription regulation.

[1]  Michael R. Green,et al.  Dissecting the Regulatory Circuitry of a Eukaryotic Genome , 1998, Cell.

[2]  Jeffry D Sander,et al.  Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins , 2013, Nature Biotechnology.

[3]  Paul J. Choi,et al.  Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells , 2010, Science.

[4]  P. Pasceri,et al.  Rapid Transcriptional Pulsing Dynamics of High Expressing Retroviral Transgenes in Embryonic Stem Cells , 2012, PloS one.

[5]  E. O’Shea,et al.  Noise in protein expression scales with natural protein abundance , 2006, Nature Genetics.

[6]  N. Krogan,et al.  The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II , 2004, Nature.

[7]  Robert H Singer,et al.  Structural basis for the coevolution of a viral RNA–protein complex , 2008, Nature Structural &Molecular Biology.

[8]  J. Manley,et al.  Evolutionarily conserved interaction between CstF-64 and PC4 links transcription, polyadenylation, and termination. , 2001, Molecular cell.

[9]  Leighton J. Core,et al.  Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing , 2013, Science.

[10]  Diana P Bratu,et al.  Visualizing the distribution and transport of mRNAs in living cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Melissa S. Jurica,et al.  Linking Stochastic Fluctuations in Chromatin Structure and Gene Expression , 2013, PLoS biology.

[12]  Peter M. A. Sloot,et al.  Promoter Sequence Determines the Relationship between Expression Level and Noise , 2013, PLoS biology.

[13]  D. Larson,et al.  Single-RNA counting reveals alternative modes of gene expression in yeast , 2008, Nature Structural &Molecular Biology.

[14]  F. Rigo,et al.  Poly(A) Signal-Dependent Transcription Termination Occurs through a Conformational Change Mechanism that Does Not Require Cleavage at the Poly(A) Site. , 2015, Molecular cell.

[15]  M. Elowitz,et al.  Functional Roles of Pulsing in Genetic Circuits , 2013, Science.

[16]  S. Marquardt,et al.  Kinetic competition between RNA Polymerase II and Sen1-dependent transcription termination. , 2013, Molecular cell.

[17]  Adam P. Arkin,et al.  HIV Promoter Integration Site Primarily Modulates Transcriptional Burst Size Rather Than Frequency , 2010, PLoS Comput. Biol..

[18]  Michael B. Elowitz,et al.  Combinatorial gene regulation by modulation of relative pulse timing , 2015, Nature.

[19]  A. Kornblihtt,et al.  The transcriptional cycle of HIV-1 in real-time and live cells , 2007, The Journal of Cell Biology.

[20]  J. Lis,et al.  Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons , 2014, eLife.

[21]  D. Tranchina,et al.  Stochastic mRNA Synthesis in Mammalian Cells , 2006, PLoS biology.

[22]  Felix Naef,et al.  Stimulus-induced modulation of transcriptional bursting in a single mammalian gene , 2013, Proceedings of the National Academy of Sciences.

[23]  Arlen W. Johnson,et al.  The role of Rat1 in coupling mRNA 3'-end processing to transcription termination: implications for a unified allosteric-torpedo model. , 2006, Genes & development.

[24]  R. Segev,et al.  GENERAL PROPERTIES OF THE TRANSCRIPTIONAL TIME-SERIES IN ESCHERICHIA COLI , 2011, Nature Genetics.

[25]  Hiroshi Kimura,et al.  The transcription cycle of RNA polymerase II in living cells , 2002, The Journal of cell biology.

[26]  W. Keller,et al.  The role of the putative 3' end processing endonuclease Ysh1p in mRNA and snoRNA synthesis. , 2008, RNA.

[27]  Jayasha Shandilya,et al.  The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling. , 2012, Biochimica et biophysica acta.

[28]  Liat Rosenfeld,et al.  Single-allele analysis of transcription kinetics in living mammalian cells , 2010, Nature Methods.

[29]  D. Larson,et al.  Complexity of RNA polymerase II elongation dynamics. , 2012, Biochimica et biophysica acta.

[30]  E. Cox,et al.  Real-Time Kinetics of Gene Activity in Individual Bacteria , 2005, Cell.

[31]  R. Singer,et al.  Quantifying Protein-mRNA Interactions in Single Live Cells , 2015, Cell.

[32]  Dan S. Tawfik,et al.  Noise–mean relationship in mutated promoters , 2012, Genome research.

[33]  Nacho Molina,et al.  Mammalian Genes Are Transcribed with Widely Different Bursting Kinetics , 2011, Science.

[34]  Myles Brown,et al.  Cofactor Dynamics and Sufficiency in Estrogen Receptor–Regulated Transcription , 2000, Cell.

[35]  Shasha Chong,et al.  Mechanism of Transcriptional Bursting in Bacteria , 2014, Cell.

[36]  R. Singer,et al.  Transcriptional Pulsing of a Developmental Gene , 2006, Current Biology.

[37]  J. Raser,et al.  Control of Stochasticity in Eukaryotic Gene Expression , 2004, Science.

[38]  Thomas Ried,et al.  From Silencing to Gene Expression Real-Time Analysis in Single Cells , 2004, Cell.

[39]  D. Larson,et al.  Direct observation of frequency modulated transcription in single cells using light activation , 2013, eLife.

[40]  Sungtae Kim,et al.  1,25‐Dihydroxyvitamin D3 Stimulates Cyclic Vitamin D Receptor/Retinoid X Receptor DNA‐Binding, Co‐activator Recruitment, and Histone Acetylation in Intact Osteoblasts , 2004, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[41]  Ignacio Izeddin,et al.  Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells , 2013, Science.

[42]  Hiroshi Kimura,et al.  Regulation of RNA polymerase II activation by histone acetylation in single living cells , 2014, Nature.

[43]  Mads Kærn,et al.  Chromosomal position effects are linked to sir2-mediated variation in transcriptional burst size. , 2011, Biophysical journal.

[44]  D. Sherrington.  Stochastic Processes in Physics and Chemistry , 1983 .

[45]  D. Brow,et al.  Ssu72 Protein Mediates Both Poly(A)-Coupled and Poly(A)-Independent Termination of RNA Polymerase II Transcription , 2003, Molecular and Cellular Biology.

[46]  André L. Martins,et al.  Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells. , 2013, Molecular cell.

[47]  O. Miller,et al.  Visualization of Nucleolar Genes , 1969, Science.

[48]  Michael L. Simpson,et al.  Transcriptional burst frequency and burst size are equally modulated across the human genome , 2012, Proceedings of the National Academy of Sciences.

[49]  O. Jänne,et al.  Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex* , 2002, The Journal of Biological Chemistry.

[50]  Maxime Dahan,et al.  Intra-nuclear mobility and target search mechanisms of transcription factors: a single-molecule perspective on gene expression. , 2012, Biochimica et biophysica acta.

[51]  R. Singer,et al.  Localization of ASH1 mRNA particles in living yeast. , 1998, Molecular cell.

[52]  J. Peccoud,et al.  Markovian Modeling of Gene-Product Synthesis , 1995 .

[53]  A. Oudenaarden,et al.  Cellular Decision Making and Biological Noise: From Microbes to Mammals , 2011, Cell.

[54]  J. Derisi,et al.  Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise , 2006, Nature.

[55]  Nick S. Jones,et al.  Connecting Variability in Global Transcription Rate to Mitochondrial Variability , 2010, PLoS biology.

[56]  Robert H Singer,et al.  Single-molecule analysis of gene expression using two-color RNA labeling in live yeast , 2012, Nature Methods.

[57]  Hernan G. Garcia,et al.  Quantitative Imaging of Transcription in Living Drosophila Embryos Links Polymerase Activity to Patterning , 2013, Current Biology.

[58]  Tetsushi Sakuma,et al.  Stochastic promoter activation affects Nanog expression variability in mouse embryonic stem cells , 2014, Scientific Reports.

[59]  N. Kampen,et al.  Stochastic processes in physics and chemistry , 1981 .

[60]  K. Nasmyth,et al.  Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter. , 1999, Cell.

[61]  M. Groudine,et al.  Enhancers increase the probability but not the level of gene expression. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[62]  Amir Kazerouninia,et al.  The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity. , 2006, RNA.

[63]  Davide Mazza,et al.  FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know? , 2010, Current opinion in cell biology.

[64]  J. McNally,et al.  Fast transcription rates of RNA polymerase II in human cells , 2011, EMBO reports.

[65]  Johan Paulsson,et al.  Non-genetic heterogeneity from stochastic partitioning at cell division , 2011, Nature Genetics.

[66]  J. Widom,et al.  Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity , 2014, Proceedings of the National Academy of Sciences.

[67]  Aleksandra M. Walczak,et al.  Live Imaging of Bicoid-Dependent Transcription in Drosophila Embryos , 2013, Current Biology.

[68]  R. Tjian,et al.  Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation , 2015, eLife.

[69]  Iris Müller,et al.  Methylation of H3K4 Is Required for Inheritance of Active Transcriptional States , 2010, Current Biology.

[70]  J. Weissman,et al.  Nascent transcript sequencing visualizes transcription at nucleotide resolution , 2011, Nature.

[71]  N. Proudfoot,et al.  Human 5′ → 3′ exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites , 2004, Nature.

[72]  X. Darzacq,et al.  In vivo dynamics of RNA polymerase II transcription , 2007, Nature Structural &Molecular Biology.

[73]  A. Coulon,et al.  Eukaryotic transcriptional dynamics: from single molecules to cell populations , 2013, Nature Reviews Genetics.

[74]  Mads Kærn,et al.  Noise in eukaryotic gene expression , 2003, Nature.

[75]  Yoav Freund,et al.  Visualization of Individual Scr mRNAs during Drosophila Embryogenesis Yields Evidence for Transcriptional Bursting , 2009, Current Biology.

[76]  M. Ko,et al.  A stochastic model for gene induction. , 1991, Journal of theoretical biology.

[77]  S. Taylor,et al.  A refractory phase in cyclic AMP-responsive transcription requires down regulation of protein kinase A , 1995, Molecular and cellular biology.

[78]  L. Minvielle-Sebastia,et al.  Coupling termination of transcription to messenger RNA maturation in yeast. , 1998, Science.

[79]  N. Proudfoot.  Ending the message: poly(A) signals then and now. , 2011, Genes & development.

[80]  N. Krogan,et al.  Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes , 2004, The EMBO journal.

[81]  Hye Yoon Park,et al.  Visualization of Dynamics of Single Endogenous mRNA Labeled in Live Mouse , 2014, Science.

[82]  S. McKnight,et al.  Post-replicative nonribosomal transcription units in D. melanogaster embryos , 1979, Cell.

[83]  C. J. Zopf,et al.  Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression , 2013, PLoS Comput. Biol..

[84]  F S Fay,et al.  Visualization of single RNA transcripts in situ. , 1998, Science.

[85]  S. Itzkovitz,et al.  Bursty gene expression in the intact mammalian liver. , 2015, Molecular cell.

[86]  Steven Hahn,et al.  A transcription reinitiation intermediate that is stabilized by activator , 2000, Nature.

[87]  N. Cohen,et al.  Fluctuations, pauses, and backtracking in DNA transcription. , 2008, Biophysical journal.

[88]  A. Hinnen,et al.  Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements. , 1986, The EMBO journal.

[89]  Naama Barkai,et al.  Expression noise and acetylation profiles distinguish HDAC functions. , 2012, Molecular cell.

[90]  S. McKnight,et al.  Electron microscopic analysis of chromatin replication in the cellular blastoderm drosophila melanogaster embryo , 1977, Cell.

[91]  Leighton J. Core,et al.  Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters , 2008, Science.

[92]  B. Cairns.  The logic of chromatin architecture and remodelling at promoters , 2009, Nature.

[93]  Heike Brand,et al.  Estrogen Receptor-α Directs Ordered, Cyclical, and Combinatorial Recruitment of Cofactors on a Natural Target Promoter , 2003, Cell.

[94]  Paul Wach,et al.  Evidence for a common mode of transcription factor interaction with chromatin as revealed by improved quantitative fluorescence recovery after photobleaching. , 2008, Biophysical journal.

[95]  X. Xie,et al.  Probing Gene Expression in Live Cells, One Protein Molecule at a Time , 2006, Science.

[96]  J. McNally,et al.  The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. , 2000, Science.

[97]  Brian Munsky,et al.  Transcription Factors Modulate c-Fos Transcriptional Bursts , 2014, Cell reports.

[98]  Michael A. Cortazar,et al.  Effects of Transcription Elongation Rate and Xrn2 Exonuclease Activity on RNA Polymerase II Termination Suggest Widespread Kinetic Competition. , 2015, Molecular cell.

[99]  Jennifer A. Doudna,et al.  Programmable RNA Tracking in Live Cells with CRISPR/Cas9 , 2016, Cell.

[100]  Farren J. Isaacs,et al.  Phenotypic consequences of promoter-mediated transcriptional noise. , 2006, Molecular cell.

[101]  Hernan G. Garcia,et al.  Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos , 2014, Proceedings of the National Academy of Sciences.

[102]  Hye Yoon Park,et al.  A transgenic mouse for in vivo detection of endogenous labeled mRNA , 2010, Nature Methods.

[103]  D. K. Hawley,et al.  Functional steps in transcription initiation and reinitiation from the major late promoter in a HeLa nuclear extract. , 1987, The Journal of biological chemistry.

[104]  Kirsten L. Frieda,et al.  A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell , 2008, Science.

[105]  A. Coulon,et al.  Kinetic competition during the transcription cycle results in stochastic RNA processing , 2014, eLife.

[106]  Christopher B. Burge,et al.  c-Myc Regulates Transcriptional Pause Release , 2010, Cell.

[107]  Shawn C. Little,et al.  Precise Developmental Gene Expression Arises from Globally Stochastic Transcriptional Activity , 2013, Cell.

[108]  Watt W Webb,et al.  Intranuclear distribution and local dynamics of RNA polymerase II during transcription activation. , 2007, Molecular cell.

[109]  W. Gu,et al.  Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[110]  Philip D. Campbell,et al.  Dynamic visualization of transcription and RNA subcellular localization in zebrafish , 2015, Development.

[111]  Bin Wu,et al.  Real-Time Observation of Transcription Initiation and Elongation on an Endogenous Yeast Gene , 2011, Science.

[112]  M. L. Simpson,et al.  Transcriptional bursting from the HIV-1 promoter is a significant source of stochastic noise in HIV-1 gene expression. , 2010, Biophysical journal.

[113]  Craig D. Kaplan,et al.  From Structure to Systems: High-Resolution, Quantitative Genetic Analysis of RNA Polymerase II , 2013, Cell.

[114]  M. Ko,et al.  The dose dependence of glucocorticoid‐inducible gene expression results from changes in the number of transcriptionally active templates. , 1990, The EMBO journal.

[115]  W. Webb,et al.  Dynamics of heat shock factor association with native gene loci in living cells , 2006, Nature.

[116]  P. Cramer,et al.  Torpedo Nuclease Rat1 Is Insufficient to Terminate RNA Polymerase II in Vitro* , 2009, The Journal of Biological Chemistry.

[117]  James G McNally,et al.  Dynamic behavior of transcription factors on a natural promoter in living cells , 2002, EMBO reports.

[118]  Julian R. E. Davis,et al.  Dynamic Analysis of Stochastic Transcription Cycles , 2011, PLoS biology.

[119]  K. Docherty,et al.  Transcription factor cycling on the insulin promoter , 2006, FEBS letters.

[120]  Craig D. Kaplan,et al.  Dissection of Pol II Trigger Loop Function and Pol II Activity–Dependent Control of Start Site Selection In Vivo , 2012, PLoS genetics.

[121]  M. Elowitz,et al.  Frequency-modulated nuclear localization bursts coordinate gene regulation , 2008, Nature.

[122]  D. Bentley.  Coupling mRNA processing with transcription in time and space , 2014, Nature Reviews Genetics.

[123]  Robert Tjian,et al.  Imaging transcription in living cells. , 2009, Annual review of biophysics.

[124]  A. Raj,et al.  Single mammalian cells compensate for differences in cellular volume and DNA copy number through independent global transcriptional mechanisms. , 2015, Molecular cell.

[125]  G. Beslon,et al.  Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts , 2013, BMC Biology.

[126]  D. Granner,et al.  Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin. , 1984, The Journal of biological chemistry.

[127]  Erin K O'Shea,et al.  Signal-dependent dynamics of transcription factor translocation controls gene expression , 2011, Nature Structural &Molecular Biology.

[128]  T. Lenstra,et al.  Single-Molecule Imaging Reveals a Switch between Spurious and Functional ncRNA Transcription. , 2015, Molecular cell.

[129]  S. Jaffrey,et al.  RNA Mimics of Green Fluorescent Protein , 2011, Science.

[130]  Corentin Spriet,et al.  Concurrent Fast and Slow Cycling of a Transcriptional Activator at an Endogenous Promoter , 2008, Science.

[131]  J. Monod,et al.  Genetic regulatory mechanisms in the synthesis of proteins. , 1961, Journal of Molecular Biology.

[132]  Christopher R. Brown,et al.  Nucleosomal promoter variation generates gene expression noise , 2014, Proceedings of the National Academy of Sciences.

[133]  A. Kornblihtt,et al.  A slow RNA polymerase II affects alternative splicing in vivo. , 2003, Molecular cell.

[134]  T. Curran,et al.  Mapping patterns of c-fos expression in the central nervous system after seizure. , 1987, Science.

[135]  Geoffrey J Barton,et al.  Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation , 2012, Proceedings of the National Academy of Sciences.

[136]  Michael B. Elowitz,et al.  Dynamic Heterogeneity and DNA Methylation in Embryonic Stem Cells , 2014, Molecular cell.

[137]  E. Airoldi,et al.  Accounting for Experimental Noise Reveals That mRNA Levels, Amplified by Post-Transcriptional Processes, Largely Determine Steady-State Protein Levels in Yeast , 2014, bioRxiv.

[138]  Iris Müller,et al.  Digital nature of the immediate-early transcriptional response , 2010, Development.

[139]  R. Padgett,et al.  Rates of in situ transcription and splicing in large human genes , 2009, Nature Structural &Molecular Biology.

[140]  N. Friedman,et al.  Stochastic protein expression in individual cells at the single molecule level , 2006, Nature.