COORDINATE GENE REGULATION IN ANIMAL CELLS

Summary

Principal Investigator: JOHN THOMAS LIS
Affiliation: Cornell University
Country: USA
Abstract: The regulated production of a mature mRNA in eukaryotes requires highly coordinated molecular interactions and biochemical processes that involve the participation of hundreds of polypeptides. Understanding these underlying processes is important in creating novel strategies for intervening in abnormal regulation caused by mutations, epigenetic abnormalities, or infectious agents. The heat shock (HS) genes are a highly regulated set of genes particularly well suited to investigate fundamental features of inducible mRNA production. Here, both well-established and newly developed technologies will be used to discern critical features of promoter architecture and mechanisms of regulation. A critical feature of the promoter regions of heat shock genes and many important highly regulated genes is promoter-proximal RNA Polymerase II pausing (Paused Pol II). In Aim 1 of this application, the establishment and properties of Paused Pol II will be investigated using a variation of our newly developed Global nuclear Run-On and massively-parallel sequencing protocol (GRO- seq) that is designed to provide near-nucleotide resolution mapping of Paused Pol II on a genome-wide scale in Drosophila cells. The precise positioning of all pauses coupled with bioinformatic approaches should greatly aid the identification of general elements and factors used in Paused Pol II regulation. The role of DNA elements and their spatial relationships to each other will be tested using targeted mutagenesis of existing model genes like Hsp70, and new genes uncovered by GRO-seq analyses. We will also define important mechanistic and dynamic properties of Paused Pol II using both our newly developed live cell imaging method and classical pulse-labeling experiments coupled with sensitive and high-resolution biochemical assays of paused RNAs. In Aim 2, we investigate the mechanism of activating promoter-proximal paused Pol II by performing a comprehensive biochemical search for factors that interact with master regulator, HSF, as well as analysis of factors affecting Hsp70 expression that we obtained from directed and genome-wide screens. Existing and newly identified transcription factors will be examined to determine when and where they participate in the activation process in vivo by ChIP assays as well as two-photon microscopy to track in real time the recruitment and dynamics of factors at specific activated loci. In Aim 3, we investigate the mechanisms that bring about rapid large scale loss of nucleosomes over an entire activated locus. Upon HS, nucleosomes are rapidly lost from heat shock loci in a transcription-independent manner. The region of nucleosome loss extends beyond the gene and up to, but not beyond, the locus insulators, scs and scs'. This locus-wide loss of nucleosomes depends on HSF and PARP. The proposed analyses will probe the spatial relationship of HSF and PARP during gene activation and nucleosome loss, testing for interactions between HSF and PARP, and identifying the critical barrier to nucleosome loss in the scs and scs'insulator regions. PUBLIC HEALTH RELEVANCE: The development, health, and viability of an organism depend on a plethora of intra- and extracellular signals that produce a highly orchestrated and regulated gene expression, with much of this regulation occurring at the level of transcription. Some of the approaches in this proposal and the technology being developed are providing direct insights to these molecular mechanisms in the complex but relevant milieu of living cells, while others provide unprecedented genome-wide views of promoter and gene architecture and expression. This information will provide the necessary background for understanding normal and disease states at the level of gene regulation and expression, and for designing strategies for intervening with abnormal expression of genes associated with cancer or disorders originating from mutation or disruption of transcription factor functions.
Funding Period: ----------------1978 - ---------------2013-
more information: NIH RePORT

Top Publications

  1. pmc Drosophila Paf1 modulates chromatin structure at actively transcribed genes
    Karen Adelman
    Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA
    Mol Cell Biol 26:250-60. 2006
  2. ncbi Imaging RNA Polymerase II transcription sites in living cells
    Martin S Buckley
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA Electronic address
    Curr Opin Genet Dev 25:126-30. 2014
  3. pmc Defining the status of RNA polymerase at promoters
    Leighton J Core
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Cell Rep 2:1025-35. 2012
  4. pmc Mechanisms by which transcription factors gain access to target sequence elements in chromatin
    Michael J Guertin
    Department of Molecular Biology and Genetics, Cornell University, United States
    Curr Opin Genet Dev 23:116-23. 2013
  5. pmc Precise maps of RNA polymerase reveal how promoters direct initiation and pausing
    Hojoong Kwak
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Science 339:950-3. 2013
  6. pmc Development of temperature-sensitive mutants of the Drosophila melanogaster P-TEFb (Cyclin T/CDK9) heterodimer using yeast two-hybrid screening
    Soyoun Kim
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Biochem Biophys Res Commun 433:243-8. 2013
  7. pmc Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells
    Charles G Danko
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Mol Cell 50:212-22. 2013
  8. pmc Genome-wide control of RNA polymerase II activity by cohesin
    Cheri A Schaaf
    Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America
    PLoS Genet 9:e1003382. 2013
  9. pmc Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription
    Abbie Saunders
    Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
    Genes Dev 27:1146-58. 2013
  10. pmc A new player in Pol II pausing
    Nicholas J Fuda
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, NY, USA
    EMBO J 32:1796-8. 2013

Scientific Experts

  • JOHN THOMAS LIS
  • Leighton J Core
  • Hojoong Kwak
  • Nicholas J Fuda
  • M Behfar Ardehali
  • Martin S Buckley
  • Jie Yao
  • Steven J Petesch
  • Karen Adelman
  • Irene M Min
  • Michael J Guertin
  • Joshua J Waterfall
  • Katie L Zobeck
  • Watt W Webb
  • Charles G Danko
  • Nasun Hah
  • Soyoun Kim
  • Abbie Saunders
  • John M Pagano
  • Isabel X Wang
  • Colin T Waters
  • Helena Kilpinen
  • Cheri A Schaaf
  • William S Kruesi
  • Leighton Core
  • Adam Siepel
  • W Lee Kraus
  • Warren R Zipfel
  • Suzanne A Hartford
  • H Hans Salamanca
  • M J Guertin
  • Bartlomiej Bartkowiak
  • Michael A Hall
  • Sun Woo Hong
  • Amy C Seila
  • Zhuoyu Ni
  • Brian S White
  • Kylan Szeto
  • Christopher Grunseich
  • Harold G Craighead
  • Alan Bruzel
  • Lee McDaniel
  • Ming Wu
  • Lauren Brady
  • Allison L Richards
  • David Shalloway
  • Abdullah Ozer
  • Rebekka O Sprouse
  • Vivian G Cheung
  • Barbara J Meyer
  • Sarah Thurnheer
  • David W Gohara
  • Luciana Romano-Palumbo
  • Andrea Orioli
  • Moonsoo M Jin
  • Nikolaos I Panousis
  • Sunil K Raghav
  • Ziva Misulovin
  • Nouria Hernandez
  • Xin Luo
  • Robert M Witwicki
  • Deborah Bielser
  • Shuo Ren
  • André L Martins
  • Sebastian M Waszak
  • Dale Dorsett
  • Amanda Koenig
  • Eugenia Migliavacca
  • Andreas R Gschwind
  • David Hacker
  • Gilles Udin
  • Ismaël Padioleau
  • Alex Spector
  • Yanjiao Zhou
  • Emmanouil T Dermitzakis
  • Alisa Yurovsky
  • Bart Deplancke
  • Alexandra Planchon
  • Hilary L Ashe
  • Maria Gutierrez-Arcelus
  • Audrey Watson
  • Tuuli Lappalainen
  • Catherine Sutcliffe
  • Michael Wiederkehr
  • Alexandre Reymond
  • Julien Bryois
  • David C Fargo
  • Daniel A Gilchrist
  • Matthieu Caron
  • John C Schimenti

Detail Information

Publications39

  1. pmc Drosophila Paf1 modulates chromatin structure at actively transcribed genes
    Karen Adelman
    Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA
    Mol Cell Biol 26:250-60. 2006
    ....
  2. ncbi Imaging RNA Polymerase II transcription sites in living cells
    Martin S Buckley
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA Electronic address
    Curr Opin Genet Dev 25:126-30. 2014
    ..In this review, we will focus on recent live-cell imaging studies that are changing our understanding of transcription factories. ..
  3. pmc Defining the status of RNA polymerase at promoters
    Leighton J Core
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Cell Rep 2:1025-35. 2012
    ..Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila...
  4. pmc Mechanisms by which transcription factors gain access to target sequence elements in chromatin
    Michael J Guertin
    Department of Molecular Biology and Genetics, Cornell University, United States
    Curr Opin Genet Dev 23:116-23. 2013
    ..Taken together, these studies support a model for how accessibility originates and then propagates throughout regulatory cascades and development...
  5. pmc Precise maps of RNA polymerase reveal how promoters direct initiation and pausing
    Hojoong Kwak
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Science 339:950-3. 2013
    ..This "complex interaction" model was tested with insertional mutagenesis of the Drosophila Hsp70 core promoter...
  6. pmc Development of temperature-sensitive mutants of the Drosophila melanogaster P-TEFb (Cyclin T/CDK9) heterodimer using yeast two-hybrid screening
    Soyoun Kim
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Biochem Biophys Res Commun 433:243-8. 2013
    ..The use of TS or CS mutants will enable a 'real-time and reversible perturbation' restricted to specific protein-protein interactions, providing a mechanistic insight into the biological process mediated by a target complex...
  7. pmc Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells
    Charles G Danko
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Mol Cell 50:212-22. 2013
    ..Collectively, our results identify previously uncharacterized variation in the rate of transcription and highlight elongation as an important, variable, and regulated rate-limiting step during transcription...
  8. pmc Genome-wide control of RNA polymerase II activity by cohesin
    Cheri A Schaaf
    Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, United States of America
    PLoS Genet 9:e1003382. 2013
    ..The multiple transcriptional roles of cohesin revealed by these studies likely underlie the growth and developmental deficits caused by minor changes in cohesin activity...
  9. pmc Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription
    Abbie Saunders
    Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
    Genes Dev 27:1146-58. 2013
    ..These data are discussed in terms of the efficiency of transcriptional activation required across cell populations during developmental time constraints...
  10. pmc A new player in Pol II pausing
    Nicholas J Fuda
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, NY, USA
    EMBO J 32:1796-8. 2013
    ....
  11. pmc Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation
    William S Kruesi
    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    elife 2:e00808. 2013
    ..DOI:http://dx.doi.org/10.7554/eLife.00808.001. ..
  12. pmc Control of transcriptional elongation
    Hojoong Kwak
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 2703 email
    Annu Rev Genet 47:483-508. 2013
    ..We then discuss Pol II elongation through the bodies of genes and the roles of FACT and SPT6, factors that allow Pol II to move through nucleosomes...
  13. doi Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription
    Helena Kilpinen
    Department of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, Switzerland
    Science 342:744-7. 2013
    ..Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event. ..
  14. pmc Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation
    Martin S Buckley
    Department of Molecular Biology and Genetics
    Genes Dev 28:14-9. 2014
    ..Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation. ..
  15. pmc Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions
    John M Pagano
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America
    PLoS Genet 10:e1004090. 2014
    ..Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes. ..
  16. doi RNA-DNA differences are generated in human cells within seconds after RNA exits polymerase II
    Isabel X Wang
    Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
    Cell Rep 6:906-15. 2014
    ..Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing...
  17. doi Drosophila heat shock system as a general model to investigate transcriptional regulation
    M J Guertin
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
    Cold Spring Harb Symp Quant Biol 75:1-9. 2010
    ..Here, we highlight heat-shock-mediated gene transcription as a model system in which to study common mechanistic features of gene regulation...
  18. ncbi Divergent transcription: a new feature of active promoters
    Amy C Seila
    Koch Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
    Cell Cycle 8:2557-64. 2009
    ..The potential implications of divergent transcription on gene regulation and possible mechanisms that give rise to this phenomenon are discussed...
  19. pmc Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters
    Leighton J Core
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Science 322:1845-8. 2008
    ..These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription...
  20. pmc Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci
    Steven J Petesch
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Cell 134:74-84. 2008
    ..An RNAi screen of 28 transcription and chromatin-related factors reveals that depletion of heat shock factor, GAGA Factor, or Poly(ADP)-Ribose Polymerase or its activity abolishes the loss of nucleosomes upon Hsp70 activation...
  21. pmc Imaging transcription dynamics at endogenous genes in living Drosophila tissues
    Jie Yao
    School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA
    Methods 45:233-41. 2008
    ..This method has provided the experimental capability to visualize the assembly and dynamics of individual transcription factors and regulators and to dissect their functions at their endogenous gene targets in living cells...
  22. pmc Transcription regulation through promoter-proximal pausing of RNA polymerase II
    Leighton J Core
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Science 319:1791-2. 2008
    ..This rate-limiting step occurs after recruitment and initiation of RNA polymerase II at a gene promoter. This stage in early elongation appears to be an important and broadly used target of gene regulation...
  23. ncbi Intranuclear distribution and local dynamics of RNA polymerase II during transcription activation
    Jie Yao
    School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA
    Mol Cell 28:978-90. 2007
    ..Pol II at highly transcribed developmental loci exhibits dynamics resembling combinations of these Hsp70 transcription modes...
  24. pmc P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo
    Zhuoyu Ni
    Department of Molecular Biology and Genetics, 1 School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA
    Mol Cell Biol 28:1161-70. 2008
    ..In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered...
  25. ncbi Imaging Drosophila gene activation and polymerase pausing in vivo
    John T Lis
    Molecular Biology and Genetics, 416 Biotechnology Building, Cornell University, Ithaca, New York 14853, USA
    Nature 450:198-202. 2007
    ....
  26. pmc High-resolution dynamic mapping of histone-DNA interactions in a nucleosome
    Michael A Hall
    Department of Physics Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853, USA
    Nat Struct Mol Biol 16:124-9. 2009
    ..These findings have important implications for how RNA polymerase and other DNA-based enzymes may gain access to DNA associated with a nucleosome...
  27. pmc Spt6 enhances the elongation rate of RNA polymerase II in vivo
    M Behfar Ardehali
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    EMBO J 28:1067-77. 2009
    ..Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development...
  28. pmc Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells
    Irene M Min
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
    Genes Dev 25:742-54. 2011
    ..Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation...
  29. pmc Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome
    Sun Woo Hong
    Department of Chemistry, Global Research Laboratory for RNAi Medicine, BK21 School of Chemical Materials Science, Sungkyunkwan University, Suwon 440 746, Korea
    Proc Natl Acad Sci U S A 106:14276-80. 2009
    ..Therefore, contrary to the current belief, our study points strongly toward a minor role of TFIIH kinase in Pol II transcription, and a more significant role in mRNA capping in budding yeast...
  30. pmc Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells
    Katie L Zobeck
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Mol Cell 40:965-75. 2010
    ..Furthermore, we demonstrate that poly(ADP-ribose) (PAR) polymerase activity is required to maintain the transcription compartment. We propose that PAR polymers locally retain factors in a transcription compartment...
  31. pmc CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1
    Bartlomiej Bartkowiak
    Department of Biochemistry, Duke Center for RNA Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
    Genes Dev 24:2303-16. 2010
    ..Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1...
  32. pmc An RNA aptamer perturbs heat shock transcription factor activity in Drosophila melanogaster
    H Hans Salamanca
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Nucleic Acids Res 39:6729-40. 2011
    ..Additionally, our study demonstrates the utility of the RNA aptamer technology as a promising chemical genetic approach to investigate biological mechanisms, including cancer and for identifying effective drug targets in vivo...
  33. pmc Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription
    M Behfar Ardehali
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Department of Molecular Biology and Biochemistry, Rutgers, State University of New Jersey, Piscataway, NJ, USA
    EMBO J 30:2817-28. 2011
    ..Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation...
  34. pmc Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression
    Suzanne A Hartford
    Department of Biomedical Sciences and Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Proc Natl Acad Sci U S A 108:17702-7. 2011
    ....
  35. pmc Activator-induced spread of poly(ADP-ribose) polymerase promotes nucleosome loss at Hsp70
    Steven J Petesch
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Mol Cell 45:64-74. 2012
    ..This acetylation is critical for the activation and spread of PARP as well as for the rapid nucleosome loss over the Hsp70 loci...
  36. pmc Chromatin landscape dictates HSF binding to target DNA elements
    Michael J Guertin
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America
    PLoS Genet 6:e1001114. 2010
    ..There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in "active" chromatin is a primary determinant of whether HSF can bind following heat shock...
  37. pmc Defining mechanisms that regulate RNA polymerase II transcription in vivo
    Nicholas J Fuda
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
    Nature 461:186-92. 2009
    ....
  38. pmc A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells
    Nasun Hah
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
    Cell 145:622-34. 2011
    ..Collectively, our results provide the most comprehensive measurement of the primary and immediate estrogen effects to date and a resource for understanding rapid signal-dependent transcription in other systems...