Structure and Functional Organisation of the Cell Nucleus

Introduction

The dynamic structure of the interphase nucleus in general and of chromatin and chromosomes in particular are important elements in the control of gene expression and other nuclear functions. Our aim is to understand principles of nuclear organization and chromatin structure. Two closely related main themes are pursued:

Research topics

*  Nuclear organization and gene expression                                                        (group leader: Roel van Driel)

 *  Functional organization of chromosomes in interphase      nuclei of Arabidopsis  (group leader: Paul Fransz)

    Prof. dr Roel van Driel

NUCLEAR ORGANIZATION AND GENE EXPRESSION The interphase nucleus is highly compartmentalized. This is instrumental in the regulation of gene expression. In the past few years we have investigated the relationship between gene expression and nuclear organization. We have analysed the spatial distribution of chromosomes and chromatin in relation to a variety of components of the gene expression machinery. Presently we are extending these studies to the relationship between the large scale folding of chromatin and the epigenetic regulation of gene expression.

    Drs. Martijn J. Moné

NUCLEOTIDE EXCISION REPAIR IN VIVO Nucleotide excision repair (NER) is a versatile DNA repair pathway, repairing a broad range of structurally diverse DNA lesions (e.g. UV damage). A lot of our knowledge on NER originates from in vitro data. Little is known about NER in the context where it all happens: the intact cell nucleus. Combining molecular biological, cell biological and microscopical techniques, we study NER in living cells.

    Dr Bea E. Krenn

MAGNETIC FORCES IN LIVING CELLS Small magnetic beads (300 nm diameter) injected into living cells can be selectively manipulated using a magnetic field. This principle is used to exert forces on biomolecules (DNA, proteins) that are attached to these magnetic beads before micro-injection in the cell nucleus. Goal of our interdisciplinary research project (a collaboration with the Technical University, Twente) is to explore the usefulness of this new technique for fundamental cell research. We want to investigate the properties and dynamic behaviour of single molecules (DNA, RNA, protein) coupled to magnetic (fluorescent) beads.

    Dr Frédéric Cremazy

MECHANISMS UNDERLYING CORTICOSTEROID RECEPTORS ACTION IN LIVING CELLS The glucocorticoid and mineralocorticoid receptors (respectively GR and MR) are hormone-dependant regulators of transcription involved in numerous biological processes: they mediate eukaryotic development, homeostasis, cell differentiation and physiological stress responses on the HPA axis [Gass et al. 2001]. After the activation by steroid hormones and translocation in the nucleus, MR and GR can bind as homodimers or monomers to cis-acting DNA sequences called Hormone Response Elements (HRE) in order to modulate the expression of their target genes [Deroo and Archer 2001]. The corticosteroid effect is a function of the equilibrum between GR and MR action; indeed, gene deletion experiments suggest that these receptors act interdependently. Nevertheless, the basis of this relation and the identity of their common target genes remain unknown. The aim of this project is to understand the molecular mechanisms underlying the MR and GR action by using 4D confocal microscopy: FRET (Fluorescent Resonance Energy Transfer) is a powerful tool to characterize protein-protein interactions in living cells [Bastiaens and Pepperkok 2000]. Using this technique coupled with the GFP technology, we will test the hypothesis that the GR and MR physically interact, possibly forming heterodimers, and in this way, co-regulate specific genomic loci. We will develop a new effective FRET method in order to measure physical interactions between these nuclear receptors and fluorescently-labelled chromatin in the nucleus of living cells. This method will allow us to determine which fractions of MR and GR are bound to the DNA and their dynamic sublocalisation during the transcription process.

    Dr Pernette J. Verschure

FUNCTIONAL ORGANIZATION OF THE CELL NUCLEUS: A KEY TO EUKARYOTIC GENE CONTROL We aim to understand functional aspects of organization of the interphase cell nucleus, its dynamics and underlying molecular mechanisms that regulate it. Therefore, we investigate the spatial relationship between higher order chromosome and chromatin structure and the control of gene expression. To analyze this relationship in situ, we perform immunofluorescence and fluorescence in situ hybridization labeling using confocal microscopy in combination with 3-D image processing procedures as well as virtual reality visualization techniques. Results show that chromosome territories are rather open structures consisting of domains that contain highly compacted chromatin and a considerable volume of interchromatin space. Strikingly, nuclear domains involved in gene transcription but also in epigentic gene silencing are almost exclusively found in the interchromatin areas in between compacted large-scale chromatin structures, depicting a strict relationship between chromatin folding and gene expression (Figure 1). These observations emphasize that compact large-scale chromatin structures on the one hand and nuclear compartments involved in gene expression on the other hand define two distinct structural and functional compartments (Figure 2). At the moment we are investigating the dynamics of chromatin structure of interphase chromosomes when manipulating gene expression with proteins involved in gene silencing, such as heterochromatin protein 1 (HP-1). The manipulated chromatin structure is visualized in living cells by targeting via a lac repressor-GFP fusion protein, to amplified chromosome regions containing large numbers of lac operator repeats.

References:

A.E. Visser, Van Driel, R. and P.J. Verschure 2002. Functional organization of chromosomes in the interphase cell nucleus. Visions of the nucleus - Eukaryotic DNA. Edited by P Hemmerich & S Diekmann. American Scientific Publishers, 25650 North Lewis Way, Stevenson Ranch, CA 91381, USA.

see also links

    Drs. Julio Mateos Langerak

IS THERE A RELATION BETWEEN CHROMATIN STRUCTURE AND GENE EXPRESSION We are trying to find out how large-scale chromatin structure and gene expression are related. Does folding of chromatin influence gene expression? If it does, how does it happen and which mechanisms are involved? To address these questions we are manipulating the activity of proteins that are involved in chromatin organisation. The goal is to see, under the microscope, any effect on structural properties of the chromatin, and correlate these changes with the gene expression pattern.

    Ing. Ineke van der Kraan

FUNCTIONAL ORGANIZATION OF THE CELL NUCLEUS Many important processes take place in the nucleus, Among them replication, transcription and RNA processing. Much attention has been given to the molecular basis of all these processes. We are studying nuclear architecture and aim to understand the functional aspects of organization of the interphase cell nucleus. Techniques used are microinjection, immunochemistry (immunofluorescence and in situ hybridization in combination with 3D image processing procedures). At the moment we are investigating the dynamics of chromatin structure of interphase chromosomes when manipulating gene expression with proteins involved in gene silencing, such as heterochromatin protein 1 (HP1).

    Dr Paul F. Fransz

FUNCTIONAL ORGANIZATION OF CHROMOSOMES IN INTERPHASE NUCLEI OF ARABIDOPSIS The organization and dynamics of euchromatin and heterochromatin in nuclei is studied by microscopical approaches in combination with molecular techniques. This allows us to investigate the relationship between chromosome organization of individual nuclei and gene expression. Using different accessions, mutants and transformants we aim to unravel epigenetic mechanisms that control gene silencing.

    Ir. Federico G. Tessadori

RELATION BETWEEN CHROMATIN STRUCTURE AND EPIGENETIC REGULATION The aim of this project is to get insight into the relation between chromatin structure and epigenetic regulation mechanisms. A cell culturing system is set up to investigate differentiating plant cells (protoplasts, callus). Nuclear phenotypes, including heterochromatin content, DNA methylation pattern, chromosome arrangement and other nuclear parameters are monitored after manipulating the (de)differentiation system (changes in the chemical and hormonal composition of the growth medium affecting the nuclear phenotype) or by using mutant <em>A. thaliana</em> genotypes. The cytological data are compared with gene expression patterns using molecular biology techniques.

    Ing. Roeland Kees Schulkes

FUNCTIONAL ORGANIZATION OF CHROMOSOMES IN INTERPHASE NUCLEI OF ARABIDOPSIS

    Ing. Rechien Bader

EPIGENTIC GENE REGULATION IN PLANTS    Paramutation at the maize b1 gene requires DNA sequences located 100 kbp upstream of the b1 coding region (hereafter called the paramutation sequences). Paramutation at b1 involves changes in DNA methylation and chromatin remodelling at the paramutation sequences. Analyses of the DNA methylation levels of the paramutation sequences using methylation sensitive restriction enzymes revealed a complex pattern of correlations. We would like to unravel the role of DNA methylation in b1 paramutation in more detail and started bisulfite genomic sequencing of the paramutation sequences. To study cause and effect relationships concerning DNA methylation we make use of various available trans-acting mutations affecting paramutation.

Publications

                            2002

Stam, M., Belele, C., Dorweiler, J and Chandler, V.L. (2002) Differential chromatin structure within a tandem array 100 kb upstream of the maize b1 locus is associated with paramutation. Genes and Development 16, 1906-1918.

Stam, M., Belele, C., Ramakrishna, W., Dorweiler, J., Bennetzen, J. and Chandler, V.L. (2002) The regulatory regions required for B’ paramutation and expression are located far upstream of the maize b1 transcribed sequences. Genetics 162, 917-930.

Chandler, V.L., Stam, M. and Sidorenko, L.V. (2002) Long distance cis and trans interactions mediate paramutation. In: Advances in Genetics, Vol. 46. J.C. Dunlap and C.-ting Wu. Academic Press, San Diego, USA, pp. 215-234. 

                            2001

Volker, M., Moné M. J., Karmakar, P., van Hoffen, A., Schul, W., Vermeulen, W., Hoeijmakers, J. H., van Driel, R., van Zeeland, A. A., Mullenders, L. H. (2001) Sequential assembly of the nucleotide excision repair factors in vivo, Mol Cell 8, 213-24

Moné M. J., Volker, M., Nikaido, O., Mullenders, L. H., van Zeeland, A. A., Verschure, P. J., Manders, E. M., van Driel, R. (2001) Local UV-induced DNA damage in cell nuclei results in local transcription inhibition EMBO Rep 2, 1013-7

Kulikova O., Gualtieri G., Geurts R., Kim D.-J, Cook D., Huguet T., de Jong J.H., Fransz P.F. and Bisseling T. (2001) Integration of the FISH-pachytene and genetic maps of Medicago truncatula. Plant J. 27, 49-58

Passarinho P.A., van Hengel A.J., Fransz P.F. and de Vries S.C. (2001) Expression of the Arabidopsis thaliana AtEP3/AtchitIV endochitinase gene. Planta 212, 556-567

Haupt W., Fischer T.C., Winderl S., Fransz P.F. and Torres-Ruiz R.A. (2001) The CENTROMERE1 (CEN1) region of Arabidopsis thaliana: architecture and chromatin and its impact on gene expression, recombination and size estimation of centromeres. Plant J. 27, 285-297

Lysak, M.A., Fransz P.F., Ali, H.B. and Schubert, I. (2001) Chromosome painting in Arabidopsis thaliana. Plant J. 28:689-97

Schubert I, Fransz P.F., Fuchs J. and de Jong J.H. (2001) Chromosome painting in plants. Methods in Cell Science 23:57-69

van Driel R. (2001). Kun je Leven uitrekenen?  Kanker 25, 22-22         

                           2000

Tabata S, Kaneko T, Nakamura Y, Kotani H, Kato T, Asamizu E, Miyajima N, Sasamoto S, Kimura T, Hosouchi T, et al. (130 authors).... and Fransz P. (2000)
Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana. Nature 408, 823-6

Vergunst, A.C., Jansen, L.E., Fransz P.F., de Jong, J.H. and Hooykaas, P.J. (2000). Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event. Chromosoma 109, 287-97

Steimer, A., Amedeo, P., Afsar, K., Fransz P., Scheid, O.M. and Paszkowski, J. (2000). Endogenous targets of transcriptional gene silencing in Arabidopsis. Plant Cell 12, 1165-78

Ali, H.B., Fransz P. and Schubert, I. (2000). Localization of 5S RNA genes on tobacco chromosomes. Chromosome Res 8, 85-7

Fransz P.F., Armstrong, S., de Jong, J.H., Parnell, L.D., van Drunen, C., Dean, C., Zabel, P., Bisseling, T. and Jones, G.H. (2000). Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region. Cell 100, 367-76

Cmarko, D., Verschure P.J., Rothblum, L.I., HernandezVerdun, D., Amalric, F., van Driel R. and Fakan, S. (2000). Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP. Histochem. Cell. Biol. 113, 181-187

De Leeuw, W., Van Liere, R., Verschure P, Visser, A., Manders, E. and van Driel, R. (2000). Visualization of Time Dependent Confocal Microscopy Data. In T. Ertl, B. Hamann, & A. Varshney (Ed.), Proceedings IEEE Visualization 2000,  (pp. 473-476). IEEE Computer Society Press Kipp,

M., Gohring, F., Ostendorp, T., van Drunen, C.M., van Driel R, Przybylski, M. and Fackelmayer, F.O. (2000). SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA. Mol. Cell. Biol. 20, 7480-7489

van der Vlag, J., den Blaauwen, J.L., Sewalt, R.G.A.B., van Driel R. and Otte, A.P. (2000). Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators. J. Biol. Chem. 275, 697-704

Vareli, K., FrangouLazaridis, M., van der Kraan, I., Tsolas, O. and van Driel R. (2000). Nuclear distribution of prothymosin alpha and parathymosin: Evidence that prothymosin alpha is associated with RNA synthesis processing and para-thymosin with early DNA replication. Exp. Cell Res. 257, 152-161