Group leader Pernette Verschure

tel.: 020-5255151
e-mail: p.j.verschure@uva.nl

Research

Research objectives
Epigenetic gene control
Epigenetic gene control is a major mechanism for cell-type specific commitment in multicellular organisms. Epigenetic regulatory mechanisms, such as histone modifications, have been directly linked with transcriptional regulation and domain-wide changes in chromatin structure. Current understanding of this complex process is predominantly qualitative. The mechanistic interaction of individual epigenetic factors that determine the transcription rate of single genes, is largely unknown. Our aim is to understand epigenetic gene regulation using a combined approach of quantitative cell biological experiments and mathematical modeling. To this end we monitor epigenetic gene control in in vivo cell systems, i.e. engineered chromatin constructs in living mammalian cells.

Nuclear organization
We focus on epigenetic gene control and the organization of chromosomes and chromatin within the cell nucleus (12-30). Important insights of functional chromosome organization in the interphase cell nucleus were obtained analyzing intact nuclear organization using confocal microscopy and 3D image analysis-processing tools. Interphase chromosomes were found to represent open structures consisting of condensed chromatin fibers. Specific genomic loci were found to occur at condensed chromatin domain surfaces (12, 13, 16, 17), Figure 1. Condensed chromatin was shown to be accessible for large macromolecules, demonstrating that other mechanisms than inaccessibility determine gene silencing in heterochromatin (18). Using a dominant negative approach, we established that pericentromeric heterochromatin is maintained without accumulation of HP1 (28).

Fig.1
Distribution of transcription sites in relation to the chromosome territories of the two X chromosomes in female primary fibroblasts. Labeling of nascent RNA by incorporation of BrUTP (green) in permeabilized cells (run-on-transcription) is combined with FISH labeling of X chromosome territories (red). Transcription sites occur as defined spots throughout one of the two X chromosome territories (most likely Xactive), whereas almost no transcription sites are observed in the other X chromosome territory (most likely Xinactive).
Chromosome territories have a distinct substructure, showing strongly labeled structures that are surrounded by less intensely labeled structures. The intensely labeled structures have a diameter in the range of 300 – 450 nm. Nascent RNA preferentially accumulates between the intensely  labeled structures, in the areas with little or no FISH label.

Functional relationship between gene expression, chromatin structure and epigenetic gene control
Recently, (ALW-NWO-PULS and VIDI-ALW-NWO grant), relationships between dynamic chromatin behaviour and epigenetic gene silencing were investigated using a lac-operator-repressor targeting system in living mammalian cells (21, 23, 26, 27). We showed that HP1 targeting to amplified chromosome regions causes local heterochromatinization, Figure 2. A novel in vivo engineered cell system is created extending the initial studies, to systematically measure causal relationships of epigenetic gene control mechanisms*. In addition, we create epigenetic gene regulatory circuits with virtually any desired property including multistability or oscillation. We measure system responses depending on the system’s input conditions (targeting of epigenetic regulatory proteins).
Kinetic models are used to analyze the quantitative kinetic data. The models are used to quantify and explain the reduction or increase of transcription rate, as function of the epigenetic status of the domain. Stochastic kinetic models are used to explain cell-cell variability (“noise”). The combined experimental-modelling approach enables us to obtain insight into the mechanistic behaviour of the system and to assist in design of new engineered cell systems and to compare synthetic variants that rely on different epigenetic components. This research is proof-of-principle for the creation of more complex synthetic epigenetic gene control systems and opens an unexplored field of research with great potential for medicine.

Direct HP1 targeting induces local compaction of the chromatin structure and enhanced H3K9 tri-methylation. The amplified lac operator chromosome region (green signal) and the immunolabeled histone H3 K9 tri-methylation pattern (red signal) are shown in the interphase cell nucleus. Control cell (AI, II, II) with a decondensed fiber-like chromatin structure of the chromosomal array (transfection with EGFP-lac repressor fusion construct) versus a cell after HP1 targeting (transfection with EGFP-LacR-HP-1a construct), (BI, II, III) showing local condensation of the chromatin structure and enhanced H3K9 tri-methylation.

*A patent is approved to use our in vivo cell system to test the regulatory action of compounds interfering with epigenetic dysregulation.

Huntington’s disease
We focus on the involvement of epigenetic gene control in the neurodegenerative disease, Huntington’s disease (HD) in conjunction with, a computational-bioinfmatics based analysis (Dr. Breit, UvA). The pathogenesis of HD is complex and multi-factorial. Our ability to eventually correct pathological malfunctions by interfering with gene dysregulation clearly depends on understanding the rules that dictate molecular mechanisms of chromatin organization.

Projects

Project 1 Regulation of eukaryotic gene expression at the level of chromatin domains
Maartje Brink (PhD student), Rabab Charafeddine (Master student), Ineke van der Kraan (technician)

General aim
To understand the molecular mechanisms that regulate gene expression at the level of chromatin domains.

Experimental set-up
A novel in vivo engineered cell system is developed to systematically analyse effects of targeting histone-modifying enzymes and proteins inducing histone-modifying enzymes on (i) changes in condensation of the defined chromatin domain, as visualized in living cells, (ii) spreading of the functional state in the defined domain in cis, and (iii) gene expression levels of a reporter gene in the defined domain, allowing systematic and quantitative measurements of the change in transcription rate as function of the epigenetic status of the domain.

Project 2 Regulation of eukaryotic epigenetic gene control at the level of chromatin domains: Real-time kinetics of transcription activity in single cells
Diewertje Piebes (technician),  vacancy PhD student

General aim
To understand epigenetic gene regulation using our in vivo cell system* that enables measurements of mRNA synthesis kinetics in real-time in single living cells in an induced variable epigenetic context.

Experimental set-up
A novel in vivo engineered cell system is used, allowing systematic and quantitative measurements of the change in transcription rate as function of the epigenetic status of the domain. Real-time transcription rate of the engineered cell system is measured in single cells. Transcripts containing MS2 viral-RNA are detected by constitutively expressed fluorescently tagged MS2 protein. Kinetic models are used to analyze the quantitative kinetic data. The models are used to quantify and explain the reduction or increase of transcription rate, as function of the epigenetic status of the domain. Stochastic kinetic models are used to explain cell-cell variability (“noise”). The combined experimental-modelling approach enables us to obtain insight into the mechanistic behaviour of the system. This interdisciplinary approach represents a yet unexplored path in the field of epigenetic gene control.

Project 3 An engineered synthetic system to understand the complex behaviour of epigenetic gene control systems, vacancy PhD student

General aim
To understand epigenetic gene regulation using synthetic engineered gene circuits consisting of mammalian cells carrying episomal vectors that have the potential to regulate (epi-) genetic control in small gene networks.

Experimental set-up
Epigenetic gene regulatory circuits with virtually any desired property including multistability or oscillations are constructed. We measure system responses depending on the system’s input conditions (targeting of epigenetic regulatory proteins). The experimentally measured data are compared with computational simulations of simple mathematical models of the system to assist in episome vector design and to compare synthetic variants that rely on different epigenetic components

Project 4 Dysregulation of gene expression in Huntington’s disease, undergraduate student

General aim
To understand the upstream molecular processes of transcriptional dysregulation during the early phases of HD

Experimental set-up
We determine in vivo during the onset of HD the presence of factors of the transcription machinery as well as regulatory proteins that influence the action of the transcription machinery at specific loci. To this end we use Chromatin Immunoprecipitation (ChIP) to measure either the genome-wide or the gene specific distribution of regulatory proteins and/or enzymes during the early phases of HD. The presence of regulatory proteins and enzymes at the coding region of the genes as measured by either microarray or parallel sequence analysis (ChIP- on CHIP or ChIPseq) are compared with the steady-state transcription levels as measured with RT-qPCR. We use a HD cell model system in a rat neuroprecursor cell line (PC12 rat phaeochromocytoma cells). These cells contain the N-terminal fraction of the Htt exon 1 gene containing either 23 or 74 CAG repeats tagged with EGFP in a tetracycline response expression construct.

Publications

1. CMarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S, Fu X-D, Van Driel R, Fakan F (1999): Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. Mol. Biol. Cell. 10: 211-223.
   
2. Verschure PJ, Van der Kraan I, Manders EMM, Van Driel R (1999): Relationship between chromosome territories and the spatial distribution of transcription sites. J. Cell Biol.  4:13-24.
   
3. CMarko D, Verschure PJ, Rothblum LI, Hernandez-Verdun D, Amalric F, Van Driel R, Fakan S (2000): Ultrastructural analysis of nucleolar transcription in the cells microinjected with 5-bromo-UTP. Histochem. Cell Biol. 113:181-187.
   
4. Mone MJ, Volker M, Nikaido O, Mullenders LHF, Van Zeeland AA, Verschure PJ, Manders EMM and Van Driel R (2001): Local induced DNA damage in cell nuclei results in local transcription inhibition. EMBO Rep. 21: 1013-1017.
   
5. Verschure PJ, Van der Kraan I, Enserink, JM, Mone MJ, Manders EMM, Van Driel R (2002): Large-scale chromatin organization and gene expression in the mammalian interphase nucleus. J. Histochem. Cytochem. 50: 1303-1312.
   
6. CMarko D, Verschure PJ, Otte AP, Van Driel R, Fakan F (2003): Polycomb group gene silencing proteins are concentrated in the perichromatin compartment of the mammalian nucleus. J. Cell Science 116: 335-343.
   
7. Verschure PJ, Van der Kraan I, Manders EMM, Houtsmuller AB, Van Driel R (2003): Chromatin domains in the mammalian cell nucleus are accessible for large macromolecules. EMBO Rep. 4: 861-866.
   
8. Van Driel R, Fransz PF, Verschure PJ (2003): The eukaryotic genome: a system regulated at different hierarchical levels. J. Cell Sci 116: 4067-4075.
   
9. Verschure PJ (2004): Positioning the genome within the nucleus. Biol. Cell 96: 569-577.
   
10. Verschure PJ, Van der Kraan I, de Leeuw, W, Van der Vlag J, Carpenter AE, Belmont AS, Van Driel R. (2005): In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Mol. Cell. Biol.  25: 4552-4564.
    
11. Visser AE, Van Driel R, PJ Verschure: Functional organization of chromosomes in the interphase nucleus. In: Visions of the nucleus – Eukaryotic DNA [2004] P Hemmerich & S Diekmann, eds, American Scientific Publishers, 25650 North Lewis Way, Stevenson Ranch, CA 91381, USA
    
12. Cremazy FGE, Manders EMM, Bastaens PIH, Kramer G, Hager GL, Van Munster EB, Verschure PJ, Gadella TWJ, Van Driel R (2005): Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM. Exp. Cell Res. 309: 390-396.
    
13. Brink MC, Y der Velden, W de Leeuw, J Mateos-Langerak, AS Belmont, R Van Driel, Verschure PJ (2006): Truncated HP-1 lacking a functional chromodomain induces heterochromatinization upon in vivo targeting. Histochem. Cell Biol. 125:53-61.
    
14. Visser AE, PJ Verschure, WM Gommans, HJ Haisma, MG Rots (2006): Step into the groove: engineered transcription factors as modulators of gene expression. Adv Genet. 56:131-61.
    
15. Verschure PJ, Visser AE, MG Rots (2006): Step out of the groove: epigenetic gene control systems and engineered transcription factors. Adv Genet. 56:163-204.
    
16. Verschure PJ (2006): Chromosome organization and gene control: It is difficult to see the picture when you are inside the frame. J. Cell. Biochem. 99(1):23-34.
    
17. De Leeuw, W, Verschure PJ, van Liere R (2006): Visualization and analysis of large data collections: a case study applied to confocal microscopy data. IEEE Trans Vis Comput Graph. 12(5):1251-8.
    
18. Mateos-Langerak J, Brink MC, Luijsterburg MS, van der Kraan I, Van Driel R, Verschure PJ (2007): Pericentromeric Heterochromatin Domains Are Maintained Without Accumulation of HP1. Mol Biol Cell. 18:1464-1471.
    
19. Van Royen, ME, Cunha, SM, Brink, MC, Mattern, KA, Nigg, AL, Dubbink, HJ, Verschure, PJ, Trapman, J., Houtsmuller, AB (2007): Compartmentalization of androgen receptor protein-protein interactions in living cells. J. Cell Biol. 177:63-72.

Patent

The approval of a patent of the cell system is completed ’A method for regulating eukaryotic gene expression at the level of chromatin’: PCT/NL2008/050150 (Octrooiaanvrage EU 07104423.4 and VS 60/907,077).

Techniques 

1. DNA manipulation techniques
2. mammalian cell culture techniques
3. creation of clonal cell lines including integration via homologous recombination
4. immunofluorescence labeling
5. 3D fluorescence in situ hybridization
6. state-of-the-art microscopical analysis (CLSM, LM, FRAP/FLIP)
7. image analysis and processing (image deconvolution, quantitative 3D image analysis)
8. RT-QPCR
9. ChIP

Educational contributions

Bachelor level
Course on Biomolecular networks
Course on Gene Regulation
Course on Systems Biology 1
Literature-projects
Master level
Supervising students and graduate students
Literature-projects
Guest lecturer Groningen University course on Genetics
Guest lecturer Netherlands Institute for Brain Research course on Epigenetics and neurodegenerative diseases

Academic positions

VISITING SCIENTIST

PhD STUDENT	February 1990 - January 1994 Department of Rheumatology, University Hospital Nijmegen, the Netherlands, Promoters Prof. Dr. W.B. van den Berg and Prof. Dr. L.A.B. van de Putte Research subject: Insulin-like growth factor-1 regulation of chondrocyte proteoglycan synthesis during experimental arthritis
Study visit 1:	September 1992 Strangeways Research Laboratory, Cambridge, UK Research subject: mRNA detection of growth factors/cytokines and their receptors with in situ hybridisation on cryostat section of joint cartilage
Study visit 2:	March-July 1995 Smith Kline Beecham Pharmaceuticals, Department of Cellular Biochemistry, Philadelphia, USA Research subject: mRNA detection of growth factors with in situ hybridisation techniques on joint cartilage and bone. Participation to establish animal models of osteoarthritis
JUNIOR RESEARCHER	February 1994 - August 1996 Department of Rheumatology, University Hospital Nijmegen, the Netherlands Research subject: Articular cartilage destruction in rheumatoid arthritis and osteoarthritis
VISITING SCIENTIST	February - April 1996 Smith Kline Beecham Pharmaceuticals, Philadelphia, USA Research subject: Bone and cartilage metabolism
POSTDOCTORAL FELLOW	September 1996 - November 1998 E.C. Slater Institute, BioCentrum Amsterdam, University of Amsterdam, the Netherlands, Prof. Dr. R. van Driel European Commission Biomed 2 project Research subject: Higher order chromatin organization and function. 3-D in situ analysis of structure, transcription and replication
PRINCIPAL INVESTIGATOR	December 1998 – December 2003 PULS (Postdoc  Universitaire Loopbaan Stimulering) personal grant ALW-NWO Swammerdam Institute for Life Sciences, University of Amsterdam, the Netherlands, Prof. Dr. R. van Driel, Research project: Visualization of higher order chromatin dynamics in relation to gene expression
RESEARCH VISIT	April 2000 & July 2001Department of Cell and Structural Biology, University of Illinois, Urbana-Campaign, USA, Dr. A.S. Belmont
PRINCIPAL  INVESTIGATOR	December 2003 – November 2007 VIDI personal grant ALW-NWO Swammerdam Institute for Life Sciences, University of Amsterdam, the Netherlands Research project: Regulation of eukaryotic gene expression at the level of chromatin domains
ASSISTANT PROFESSOR	November 2007 – Meervoud personal grant ALW-NWO Nuclear organization group, Swammerdam Institute for Life Sciences, University of Amsterdam, the Netherlands Research project: Regulation of eukaryotic epigenetic gene control at the level of chromatin domains: Real-time kinetics of transcription activity in single cells

Contact
Visiting address:
Dr. Pernette J. Verschure
University of Amsterdam
Swammerdam Institute for Life Sciences
Kruislaan 318
1098 SM Amsterdam
The Netherlands
Phone 31 20 5255151
Fax 31 20 7935
Email: p.j.verschure@uva.nl

Funding <<hier moeten links komen>>
NWO-ALW VIDI
NWO-ALW Meervoud
NISB
HD research

Collaborations <<links naar de collaborators>>

Local collaboration:

  Prof. Dr. D. Gadella and Dr. E.M.M. Manders (Molecular Cytology and Microscopy Centre, SILS, University of Amsterdam, Amsterdam, the Netherlands): Experts in 3-D CLSM living cell analysis, 3D and 4D image analysis and high-precision distance measurements.

Prof. Dr. R. Versteeg (Dept. of Human Genetics, AMC, University of Amsterdam, Amsterdam, the Netherlands): Longstanding experience with construction and integration of large DNA sequences.

  Dr. R. Van Liere and Dr. W. de Leeuw (Dept. of Software Engineering, Center for Mathematics and Computer Science (CWI), Amsterdam, Amsterdam the Netherlands): Experts in virtual reality-aided quantitative image analysis and 3D and 4D image processing with sophisticated mathematical programs.

  Dr. T.M. Breit (Integrative Bioinformatics Unit, Informatics Institute, University of Amsterdam, The Netherlands): Experts in bioinformatics approaches of microarray data sets.

  Dr. E.A. Reits (Protein and aggregation group, Dept. of Cell Biology and Histology, University of Amsterdam, Amsterdam, The Netherlands): Specialists in proteosome distribution and dynamics in normal and neurodegenerative disorders.

  Dr. F.J. Bruggeman and Prof. H.V. Westerhoff (Dept. of Molecular Cell Physiology, Institute of Molecular Cell Biology, Faculty of Earth and Life Sciences, Free University, Amsterdam, The Netherlands): Experts in systems biology approaches using computational models of complex reaction networks.

National collaboration

Dr. A. Houtsmuller (Dept. of Pathology, Josephine Nefkens Institute,            Erasmus University, Rotterdam, the Netherlands): Expert in quantitative analysis of GFP-tagged proteins in living cells using FRAP and FLIP techniques.

  Dr. W. Vermeulen and Prof. Dr. J.H.J. Hoeijmakers (Cell Biology and Genetics, Erasmus MC, Rotterdam, the Netherlands): Extensive experienced in standard molecular and cellular biological techniques to tag, express and characterize GFP proteins.

  Dr. J. van der Vlag (Nefrology Research Laboratory Nijmegen. Centre for Molecular Life Sciences, Division of Nefrology, University Medical Centre, Nijmegen, the Netherlands): Specialists in molecular and cellular biology techniques in eukaryotic cells.

  Dr. M.G. Rots (Therapeutic Gene modulation, Groningen University Institute for Drug Exploration, University of Groningen, The Netherlands): Extensive expertise in transcriptional therapy for therapeutic purposes using among others adenoviral vectors and promoter targeting.

  Dr. E. Zwarthoff  (Dept. of Pathology, Josephine Nefkens Institute,    Erasmus University, Rotterdam, the Netherlands): Expert in genetic events related to tumor cell behaviour

  Dr. B.J.L. Eggen  (Developmental Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands): Expert in ES cell related development  and differentiation aspects.

  Dr. J. Gribnau  (Reproduction and Development, Erasmus MC, Rotterdam, the Netherlands): Experts in X-incactivation and genomic imprinting as followed in living ES cells.

                                               
Interational collaboration

  Prof. Dr. A.S. Belmont (Dept. of Cell and Structural Biology, University of Illinois, Urbana-Champaign, USA): Longstanding experience in the field of nuclear organization and chromatin/chromosome structure. They were the first using a lac operator based system for direct visualization of large-scale chromatin dynamics in living cells showing large-scale chromatin decondensation by targeting of a transcriptional activator.

Dr. S. Fakan (Centre Microscopie electronique, University of Lausanne, Switzerland): Experts in ultrastructural analysis of the interphase nucleus.

  Dr. T. Cremer (Ludwig Maximilians University, Munich, Germany): Specialists in the field of interphase cell nuclear organization.

  Dr. P.B. Singh (Division of Tumor Biology, Dept. of Immunology and Cell Biology, Forschungszentrum Borstel, Borstel, Germany): Experts in the field of heterochromatin induced silencing, specifically describing functional aspects of the heterochromatin associated proteins.

  Dr. J.W. Dobrucki (Cell Biophysics group, Faculty of Biotechnology, Dept. of Biophysics, Jagiellonian University, Krakow, Poland): Specialists in biophysical aspects of DNA damage.

   Dr. A.H.F.M. Peters (Friedrich Miescher Institute for Biomedical Research (FMI), Novartis Research Foundation, Basel, Switzerland): Experts in single cell analysis of maternal and paternal patterning phenomena in early embryos

   Dr. W. Fishle (Max Planck Institute for Biophysical Chemistry, Laboratory of Chromatin Biochemistry, Goettingen, Germany): Experts in Biochemical isolation of chromatin complexes.