Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy.

Nanoscale spatiotemporal clustering of RNA polymerase II (Pol II) plays an important role in transcription regulation. However, dynamics of individual Pol II clusters in live-cell nuclei has not been measured directly, prohibiting in-depth understanding of their working mechanisms. In this work, we studied the dynamics of Pol II clustering using Bayesian nanoscopy in live mammalian cell nuclei. With 50 nm spatial resolution and 4 s temporal resolution, Bayesian nanoscopy allows direct observation of the assembly and disassembly dynamics of individual Pol II clusters. The results not only provide quantifications of Pol II clusters but also shed light on the understanding of cluster formation and regulation. Our study suggests that transcription factories form on-demand and recruit Pol II molecules in their pre-elongation phase. The assembly and disassembly of individual Pol II clusters take place asynchronously. Overall, the methods developed herein are also applicable to studying a wide realm of real-time nanometer-scale nuclear processes in live cells.

[1]  M. Groudine,et al.  The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation. , 2006, Genes & development.

[2]  H. Handa,et al.  Interplay between positive and negative elongation factors: drawing a new view of DRB , 1998, Genes to cells : devoted to molecular & cellular mechanisms.

[3]  Jennifer A. Mitchell,et al.  Transcription factories are nuclear subcompartments that remain in the absence of transcription. , 2008, Genes & development.

[4]  P. Maini,et al.  Different populations of RNA polymerase II in living mammalian cells , 2005, Chromosome Research.

[5]  P. Cook,et al.  Transcription factories, chromatin loops, and the dysregulation of gene expression in malignancy. , 2013, Seminars in cancer biology.

[6]  Hari Shroff,et al.  Faster fluorescence microscopy: advances in high speed biological imaging. , 2014, Current opinion in chemical biology.

[7]  Benjamien Moeyaert,et al.  Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy. , 2015, ACS nano.

[8]  Jennifer A. Mitchell,et al.  Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells , 2010, Nature Genetics.

[9]  Raymond K. Auerbach,et al.  Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation , 2012, Cell.

[10]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Cook,et al.  RNA polymerase II activity is located on the surface of protein-rich transcription factories , 2008, Journal of Cell Science.

[12]  Cameron S. Osborne,et al.  Myc Dynamically and Preferentially Relocates to a Transcription Factory Occupied by Igh , 2007, PLoS biology.

[13]  Cameron S. Osborne,et al.  Molecular Pathways Molecular Pathways : Transcription Factories and Chromosomal Translocations , 2013 .

[14]  P. Cook,et al.  Transcription factories: genome organization and gene regulation. , 2013, Chemical reviews.

[15]  Zlatko Trajanoski,et al.  Transcription factories , 2012, Front. Gene..

[16]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[17]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[19]  John Bechhoefer,et al.  What is superresolution microscopy , 2014, 1405.1118.

[20]  Rahul Roy,et al.  Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy , 2013, Proceedings of the National Academy of Sciences.

[21]  Yoshihiro Ohta,et al.  Active RNA Polymerases: Mobile or Immobile Molecular Machines? , 2010, PLoS biology.

[22]  Hans-Peter Kriegel,et al.  A Density-Based Algorithm for Discovering Clusters in Large Spatial Databases with Noise , 1996, KDD.

[23]  W. Garrard,et al.  Pronounced cohabitation of active immunoglobulin genes from three different chromosomes in transcription factories during maximal antibody synthesis , 2014, Genes & development.

[24]  Dariusz M Plewczynski,et al.  CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription , 2015, Cell.

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

[26]  E M Manders,et al.  Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. , 1998, Molecular biology of the cell.

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

[28]  B. van Steensel,et al.  Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus , 1993, The Journal of cell biology.

[29]  W. Zipfel,et al.  Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells. , 2010, Molecular cell.

[30]  Cameron S. Osborne,et al.  Active genes dynamically colocalize to shared sites of ongoing transcription , 2004, Nature Genetics.

[31]  Supat Thongjuea,et al.  In vivo live imaging of RNA polymerase II transcription factories in primary cells. , 2013, Genes & development.

[32]  L. Di,et al.  Active Chromatin Hub of the Mouse α-Globin Locus Forms in a Transcription Factory of Clustered Housekeeping Genes , 2006, Molecular and Cellular Biology.

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

[34]  Peter R. Cook,et al.  Similar active genes cluster in specialized transcription factories , 2008, The Journal of cell biology.

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

[36]  Stuart A. Wilson,et al.  The integrity of a lamin-B1-dependent nucleoskeleton is a fundamental determinant of RNA synthesis in human cells , 2008, Journal of Cell Science.

[37]  D. Jackson,et al.  Active RNA polymerases are localized within discrete transcription "factories' in human nuclei. , 1996, Journal of cell science.

[38]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[39]  P. Fraser,et al.  Ultrastructural study of transcription factories in mouse erythroblasts , 2011, Journal of Cell Science.

[40]  Mingshu Zhang,et al.  Development of a reversibly switchable fluorescent protein for super-resolution optical fluctuation imaging (SOFI). , 2015, ACS nano.

[41]  D. Jackson,et al.  Visualization of focal sites of transcription within human nuclei. , 1993, The EMBO journal.

[42]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[43]  Ian M. Carr,et al.  The proteomes of transcription factories containing RNA polymerases I, II or III , 2011, Nature Methods.

[44]  Ying S Hu,et al.  Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells , 2013, Optical Nanoscopy.

[45]  Wesley R. Legant,et al.  3D imaging of Sox2 enhancer clusters in embryonic stem cells , 2014, eLife.

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

[47]  G. Daley,et al.  Human embryonic stem cells , 2004, Bone Marrow Transplantation.

[48]  Dylan T Burnette,et al.  Bayesian localisation microscopy reveals nanoscale podosome dynamics , 2011, Nature Methods.

[49]  Joshua D. Larkin,et al.  Promoter type influences transcriptional topography by targeting genes to distinct nucleoplasmic sites , 2013, Journal of Cell Science.

[50]  F. Iborra,et al.  Association between active genes occurs at nuclear speckles and is modulated by chromatin environment , 2008, The Journal of cell biology.

[51]  Mingshu Zhang,et al.  Bayesian localization microscopy based on intensity distribution of fluorophores , 2015, Protein & Cell.

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

[53]  T. Misteli,et al.  RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. , 1999, Molecular cell.