Live-cell imaging uncovers the relationship between histone acetylation, transcription initiation, and nucleosome mobility

Post-translational protein modifications play an important role in the regulation of gene dynamics. Certain modifications, such as histone acetylation and RNA polymerase II phosphorylation, are associated with transcriptionally active chromatin. However, the spatial and temporal relationship between chromatin and post-translational protein modifications, and how these dynamics facilitate selective gene expression, remain poorly understood. In this study, we address this problem by developing a general methodology for quantifying in live cells the dynamics of chromatin across multiple time and length scales in the context of residue-specific protein modifications. By combining Fab-based labeling of endogenous protein modifications with single-molecule imaging, we track the dynamics of chromatin enriched with histone H3 Lysine-27 acetylation (H3K27ac) and RNA polymerase II Serine-5 phosphorylation (RNAP2-Ser5ph). Our analysis reveals chromatin enriched with H3K27ac is separated from chromatin enriched with RNAP2-Ser5ph. Furthermore, in these separated sites, we show the presence of the two modifications are inversely correlated with one another on the minutes timescale. We then track single nucleosomes in both types of sites on the sub-second timescale and again find evidence for distinct and opposing changes in their diffusive behavior. While nucleosomes diffuse ∼15% faster in chromatin enriched with H3K27ac, they diffuse ∼15% slower in chromatin enriched with RNAP2-Ser5ph. Taken together, these results argue that high levels of H3K27ac and RNAP2-Ser5ph are not often present together at the same place and time, but rather each modification marks distinct sites that are transcriptionally poised or active, respectively.

[1]  K. Maeshima,et al.  Chromatin behavior in living cells: Lessons from single‐nucleosome imaging and tracking , 2022, BioEssays : news and reviews in molecular, cellular and developmental biology.

[2]  Romain F. Laine,et al.  TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines , 2022, Nature Methods.

[3]  K. Helin,et al.  Histone editing elucidates the functional roles of H3K27 methylation and acetylation in mammals , 2022, Nature Genetics.

[4]  H. Kimura,et al.  Imaging transcription elongation dynamics by new technologies unveils the organization of initiation and elongation in transcription factories. , 2022, Current opinion in cell biology.

[5]  M. Lakadamyali.  Single nucleosome tracking to study chromatin plasticity , 2022, Current Opinion in Cell Biology.

[6]  H. Kimura,et al.  Live imaging of transcription sites using an elongating RNA polymerase II–specific probe , 2021, The Journal of cell biology.

[7]  L. Mirny,et al.  Spatial organization of transcribed eukaryotic genes , 2020, Nature Cell Biology.

[8]  M. Hendzel,et al.  The solid and liquid states of chromatin , 2021, Epigenetics & chromatin.

[9]  H. Kimura,et al.  Live-cell imaging probes to track chromatin modification dynamics , 2021, Microscopy.

[10]  T. Stasevich,et al.  Bead Loading Proteins and Nucleic Acids into Adherent Human Cells. , 2021, Journal of visualized experiments : JoVE.

[11]  Òscar Garibo i Orts,et al.  Objective comparison of methods to decode anomalous diffusion , 2021, Nature Communications.

[12]  J. Lippincott-Schwartz,et al.  A General Method to Improve Fluorophores Using Deuterated Auxochromes , 2021, JACS Au.

[13]  S. Henikoff,et al.  The Yin and Yang of Histone Marks in Transcription. , 2021, Annual review of genomics and human genetics.

[14]  J. Forman-Kay,et al.  Phosphorylation-dependent regulation of messenger RNA transcription, processing and translation within biomolecular condensates. , 2021, Current opinion in cell biology.

[15]  T. Schlick,et al.  Mesoscale Modeling and Single-Nucleosome Tracking Reveal Remodeling of Clutch Folding and Dynamics in Stem Cell Differentiation , 2021, Cell reports.

[16]  B. Munsky,et al.  Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene , 2020, Nature Communications.

[17]  M. Levine,et al.  Enhancer-promoter communication: hubs or loops? , 2020, Current opinion in genetics & development.

[18]  Ashutosh Kumar Singh,et al.  Direct observation of RAG recombinase recruitment to chromatin and the IgH locus in live pro-B cells , 2020, bioRxiv.

[19]  J. Lerner,et al.  Two-Parameter Mobility Assessments Discriminate Diverse Regulatory Factor Behaviors in Chromatin. , 2020, Molecular cell.

[20]  Zhuqiang Zhang,et al.  Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells , 2020, Genome Biology.

[21]  M. Weiss,et al.  Elucidating the Origin of Heterogeneous Anomalous Diffusion in the Cytoplasm of Mammalian Cells. , 2019, Physical review letters.

[22]  C. Brangwynne,et al.  Nucleated transcriptional condensates amplify gene expression , 2019, Nature Cell Biology.

[23]  Joel Nothman,et al.  SciPy 1.0-Fundamental Algorithms for Scientific Computing in Python , 2019, ArXiv.

[24]  M. Weiss.  Resampling single-particle tracking data eliminates localization errors and reveals proper diffusion anomalies. , 2019, Physical review. E.

[25]  M. Cosma,et al.  Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo , 2019, Nucleic acids research.

[26]  Leighton J. Core,et al.  Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation , 2019, Genes & development.

[27]  Giacomo Cavalli,et al.  Principles of genome folding into topologically associating domains , 2019, Science Advances.

[28]  L. S. Churchman,et al.  The Long and the Short of the RNA Polymerase C-Terminal Domain and Phase Separation. , 2019, Molecular cell.

[29]  D. Grunwald,et al.  Cell cycle– and genomic distance–dependent dynamics of a discrete chromosomal region , 2019, The Journal of cell biology.

[30]  H. Kimura,et al.  Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II , 2019, The Journal of cell biology.

[31]  Nacho Molina,et al.  Visualization of Endogenous Transcription Factors in Single Cells Using an Antibody Electroporation-Based Imaging Approach. , 2019, Methods in molecular biology.

[32]  Nicholas A. Sinnott-Armstrong,et al.  Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells , 2018, Science.

[33]  Charles H. Li,et al.  Mediator and RNA polymerase II clusters associate in transcription-dependent condensates , 2018, Science.

[34]  R. Tjian,et al.  Imaging dynamic and selective low-complexity domain interactions that control gene transcription , 2018, Science.

[35]  Maxime Dahan,et al.  Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells , 2018, Nature Materials.

[36]  Thomas Gregor,et al.  Dynamic interplay between enhancer-promoter topology and gene activity , 2018, Nature Genetics.

[37]  Thomas Germier,et al.  Real-time imaging of specific genomic loci in eukaryotic cells using the ANCHOR DNA labelling system. , 2018, Methods.

[38]  Nacho Molina,et al.  Imaging of native transcription factors and histone phosphorylation at high resolution in live cells , 2018, The Journal of cell biology.

[39]  T. Meyer,et al.  Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements , 2018, Science.

[40]  Christopher A. Lavender,et al.  Widespread transcriptional pausing and elongation control at enhancers , 2018, Genes & development.

[41]  J. Ellenberg,et al.  Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement , 2017, Biophysical journal.

[42]  M. Tomita,et al.  Dynamic organization of chromatin domains revealed by super-resolution live-cell imaging , 2017 .

[43]  Huimin Zhao,et al.  CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci , 2017, bioRxiv.

[44]  Nicholas A Moringo,et al.  Single Particle Tracking: From Theory to Biophysical Applications. , 2017, Chemical reviews.

[45]  Timothy J Stasevich,et al.  Imaging Translational and Post-Translational Gene Regulatory Dynamics in Living Cells with Antibody-Based Probes. , 2017, Trends in genetics : TIG.

[46]  L. S. Churchman,et al.  The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain , 2017, Nature Reviews Molecular Cell Biology.

[47]  Johannes Schindelin,et al.  TrackMate: An open and extensible platform for single-particle tracking. , 2017, Methods.

[48]  Huimin Chen,et al.  Transcription Dynamics in Living Cells. , 2016, Annual review of biophysics.

[49]  J. Grimm,et al.  RNA Polymerase II cluster dynamics predict mRNA output in living cells , 2016, eLife.

[50]  Shaojie Zhang,et al.  Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow , 2016, Nature Biotechnology.

[51]  M. Garcia-Parajo,et al.  A review of progress in single particle tracking: from methods to biophysical insights , 2015, Reports on progress in physics. Physical Society.

[52]  H. Kimura,et al.  Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo , 2015, Histochemistry and Cell Biology.

[53]  Hiroshi Ochiai,et al.  Simultaneous live imaging of the transcription and nuclear position of specific genes , 2015, Nucleic acids research.

[54]  J. J. Macklin,et al.  A general method to improve fluorophores for live-cell and single-molecule microscopy , 2014, Nature Methods.

[55]  D. Krapf.  Mechanisms underlying anomalous diffusion in the plasma membrane. , 2015, Current topics in membranes.

[56]  Henry Pinkard,et al.  Advanced methods of microscope control using μManager software. , 2014, Journal of biological methods.

[57]  Andrey G. Cherstvy,et al.  Anomalous diffusion models and their properties: non-stationarity, non-ergodicity, and ageing at the centenary of single particle tracking. , 2014, Physical chemistry chemical physics : PCCP.

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

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

[60]  Ming-Ming Zhou,et al.  Writers and readers of histone acetylation: structure, mechanism, and inhibition. , 2014, Cold Spring Harbor perspectives in biology.

[61]  S. Manley,et al.  Reduced dyes enhance single-molecule localization density for live superresolution imaging. , 2014, Chemphyschem : a European journal of chemical physics and physical chemistry.

[62]  Wei Zhang,et al.  Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System , 2014, Cell.

[63]  Hiroshi Kimura,et al.  Histone modifications for human epigenome analysis , 2013, Journal of Human Genetics.

[64]  D. Eick,et al.  Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription. , 2013, Biochimica et biophysica acta.

[65]  Takeharu Nagai,et al.  Local nucleosome dynamics facilitate chromatin accessibility in living mammalian cells. , 2012, Cell reports.

[66]  S. V. van Heeringen,et al.  Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis , 2012, Genome research.

[67]  J. McNally,et al.  A benchmark for chromatin binding measurements in live cells , 2012, Nucleic acids research.

[68]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[69]  M. Petter,et al.  The Role of Bromodomain Proteins in Regulating Gene Expression , 2012, Genes.

[70]  Yuan Gao,et al.  Genome-wide ChIP-seq mapping and analysis reveal butyrate-induced acetylation of H3K9 and H3K27 correlated with transcription activity in bovine cells , 2012, Functional & Integrative Genomics.

[71]  Robert W. Li,et al.  Genome-wide ChIP-seq mapping and analysis reveal butyrate-induced acetylation of H3K9 and H3K27 correlated with transcription activity in bovine cells , 2012, Functional & Integrative Genomics.

[72]  Hiroshi Kimura,et al.  Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling , 2011, Nucleic acids research.

[73]  Karolin Luger,et al.  Nucleosome structure(s) and stability: variations on a theme. , 2011, Annual review of biophysics.

[74]  L. Mahadevan,et al.  Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP , 2011, Proceedings of the National Academy of Sciences.

[75]  Aubrey V. Weigel,et al.  Ergodic and nonergodic processes coexist in the plasma membrane as observed by single-molecule tracking , 2011, Proceedings of the National Academy of Sciences.

[76]  Andrew J. Bannister,et al.  Regulation of chromatin by histone modifications , 2011, Cell Research.

[77]  Ryan A. Flynn,et al.  A unique chromatin signature uncovers early developmental enhancers in humans , 2011, Nature.

[78]  Li-Rong Yu,et al.  Distinct roles of GCN5/PCAF‐mediated H3K9ac and CBP/p300‐mediated H3K18/27ac in nuclear receptor transactivation , 2011, The EMBO journal.

[79]  R. Young,et al.  Histone H3K27ac separates active from poised enhancers and predicts developmental state , 2010, Proceedings of the National Academy of Sciences.

[80]  Peter R Cook,et al.  A model for all genomes: the role of transcription factories. , 2010, Journal of molecular biology.

[81]  S. Buratowski.  Progression through the RNA polymerase II CTD cycle. , 2009, Molecular cell.

[82]  John T. Lis,et al.  Defining mechanisms that regulate RNA polymerase II transcription in vivo , 2009, Nature.

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

[84]  M. Tokunaga,et al.  Highly inclined thin illumination enables clear single-molecule imaging in cells , 2008, Nature Methods.

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

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

[87]  L. Mahadevan,et al.  Enhanced histone acetylation and transcription: a dynamic perspective. , 2006, Molecular cell.

[88]  Carlos Ortiz-de-Solorzano,et al.  Consistent and Elastic Registration of Histological Sections Using Vector-Spline Regularization , 2006, CVAMIA.

[89]  E. Seto,et al.  Acetylation and deacetylation of non-histone proteins. , 2005, Gene.

[90]  P. Doyle,et al.  Static and dynamic errors in particle tracking microrheology. , 2005, Biophysical journal.

[91]  Peter R. Cook,et al.  Principles of Nuclear Structure and Function , 2001 .

[92]  C. Allis,et al.  The language of covalent histone modifications , 2000, Nature.

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

[94]  T. Richmond,et al.  Crystal structure of the nucleosome core particle at 2.8 Å resolution , 1997, Nature.

[95]  Joel S. Demski,et al.  A Dynamic Perspective , 1997 .

[96]  Andrew W. Murray,et al.  GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion , 1996, Current Biology.

[97]  P. Chiaravelli.  Variations on a theme. , 1990, Journal of the American Dental Association.

[98]  A. Mirsky,et al.  ACETYLATION AND METHYLATION OF HISTONES AND THEIR POSSIBLE ROLE IN THE REGULATION OF RNA SYNTHESIS. , 1964, Proceedings of the National Academy of Sciences of the United States of America.