Group leader Paul Fransz

tel.: 020-525153
e-mail: p.f.fransz@uva.nl

ARABIDOPSIS HETEROCHROMATIN

Our research aim is to unravel structure and formation of heterochromatin and to understand its role in regulating chromosome function. The term heterochromatin is widely used to designate compact chromatin regions, inert chromatin, or chromatin, containing silent genes. We exploit the model plant Arabidopsis thaliana to investigate heterochromatin using microscopical, molecular and genetic tools.

History

Heterochromatin domains. We have shown that Arabidopsis provides an ideal system to study compact chromatin segments in linear chromosomes (Fransz et al., 1998, 2000) and in interphase nuclei (Fransz et al., 2002, Soppe et al., 2002). Each interphase chromosome of Arabidopsis consists of a single heterochromatic domain, or chromocenter, from which euchromatic loops emanate spanning 0.2 – 2 Mbp (Fig. 1). Chromocenters consist mainly of pericentric repeats which are heavily methylated, whereas euchromatin loops contain less methylated DNA and the majority of genes. In addition, chromocenters contain epigenetic markers for silent chromatin, such as methylated histone H3K9. In contrast, euchromatic loops contain acetylated histones as well as methylated histone H3K4. Chromocenter and loops together form a chromosome territory.

Fig. 1. Chromocenter-loop model for the organization of chromosomes in Arabidopsisnuclei.
Heterochromatic segments of an individual chromosome compartmentalize into one chromocenter, while the euchromatic regions forms 0.2-2 Mbp loops around this chromocenter. Chromocenters contain heavily methylated DNA (Me), while euchromatic loops are enriched in acetylated histone H4 (Ac). Major tandem repeats, 45S rDNA (green) and 5S rDNA (red), colocalize with the centromere in the chromocenter. The other colored blocks represent gene-rich regions showing different positions relative to the chromocenter depending on the loop organization.

Relative heterochromatin fraction (RHF). The appearance of heterochromatin domains as discrete chromocenter structures enables to quantify the fraction of compact chromatin. The relative heterochromatin fraction is defined as the fluorescence intensity of all chromocenters relative to the fluorescence intensity of the entire nucleus (Soppe et al., 2002, Tessadori et al., 2004). The RHF value of nuclei of the accession Colombia grown under standard conditions is 10–15%, which matches the percentage of repetitive sequences in the Arabidopsis genome. The method has been applied to establish quantitative differences in heterochromatin during development and between wild type and a genesilencing mutant affected in DNA methylation. For example, a DNA hypomethylation mutant has 30% less heterochromatin compared with the wild type, whereas the F1 shows intermediate levels. However, a minor fraction of mutant cells display wild-type levels of heterochromatin. During leaf development, mesophyll cells differentiate and the nuclei become elliptic and larger.

Plasticity of heterochromatin. Arabidopsis displays highly dynamic changes in heterochromatin compaction which are induced by developmental and environmental cues. In protoplasts all chromocenters except for a few NOR chromocenters have disappeared due to unfolding of the repeat regions (Tessadori et al., 2007a). Even the centromeric repeats showed a spectacular decondensation. Strikingly, the process appeared reversible. Upon culturing, chromocenters reassembled, following a sequential process in which long tandem repeat arrays were the first sequences to condense. Similarly, leaf nuclei undergo a reduction in chromocenter compaction during the floral transition, which is reversed upon the appearance of the first flower buds (Tessadori et al., 2007b). The data suggest that constitutive heterochromatin is not as permanently compact as we always assumed.

Current research

Natural variation in heterochromatin
We investigate natural variation in chromatin compaction by measuring the RHF in different ecotypes. In addition we map QTLs for RHF using the Ler-Cvi recombinant inbred line population. Genes of interest in the QTL region are studied for sequence polymorphism and their effect on nuclear morphology and chromatin compaction. These are key genes in controlling chromatin folding.

former PhD student: Federico Tessadori
PhD student  Penka Pavlova
students: Reuben Smith, Laurens Bossen
collaboration: Martijn van Zanten and Ton Peeters (Utrecht, The Netherlands), Charlie Spillane (Cork, Ireland), Craig Pikaard (St. Louis, USA)

Stress-related chromatin condensation
Based on the fact that large-scale chromatin decondensation occurs under a variety of biotic and abiotic stress conditions, we speculate that stress is a major factor that affects the nuclear organization of chromatin, in particular the decondensation of heterochromatin. We are exploiting a system, in which low light stress induces the reduction of chromatin compaction. The plant response is reversed upon switching on the light. A number of photoreceptors, including cryptochromes and phytochromes, appear to be involved in the transduction of light signals to a change in chromatin organization.

former PhD student: Federico Tessadori
students: Marije Westra
collaboration: Martijn van Zanten and Ton Peeters (Utrecht, The Netherlands)

 Chromocenter organization
The chromocenter-loop studies of Arabidopsis chromosomes revealed that the majority of genes, active and inactive, are outside chromocenters (Fransz et al., 2006). In addition, recondensation of repeat regions into chromocenters follows a sequential pattern depending on the size of the repeat array. Here, we address the question if the chromocenter is organized in ‘subcompartments’. We apply fluorescence in situ hybridization and immunolocalization to investigate the subchromocenter location of individual repeats and epigenetic marks.

PhD student: Penka Pavlova (NWO)
students: Paula van Kleeff
collaboration: Hans de Jong (Wageningen, The Netherlands)

Heterochromatin – euchromatin borders
Apart from the major heterochromatin segments around centromeres, some ecotypes have interstitial heterochromatic segments. For example, Columbia and Wassilevskija contain a heterochromatic knob, hk4S, in the short arm of chromosome 4. This knob has originated from the pericentromere after a paracentric inversion event (Fransz et al., 2000) and accommodates many dispersed transposable elements (TEs). We investigate the genetic and epigenetic consequences of the translocation of a heterochromatin segment to a euchromatin domain.

former Post doc: Gabriella Linc (Marie Curie IF)
collaboration: Ingo Schubert and Hoda Ali (Gatersleben, Germany), Maarten Koornneef and Hans de Jong (Wageningen, The Netherlands), Tom Gerats and Jannie Peters (Nijmegen, The Netherlands),

Publications (1998-2008)

  • Tessadori, F., Chupeau, M.C., Chupeau, Y., Knip, M., Germann, S., Driel, R. van, Fransz, P.F. & Gaudin, V. (2007). Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells. J Cell Sci, 120(7), 1200-1208.
  • Tessadori, F., Schulkes, R.K., Driel, R. van & Fransz, P.F. (2007). Light-regulated large-scale reorganization of chromatin during the floral transition in Arabidopsis. Plant J., 50(5), 848-857.
  • Lindhout B.I., Fransz P., Tessadori F., Meckel T., Hooykaas P.J., van der Zaal B.J. (2007). Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins. Nucl. Ac. Res. 35, e107
  • Fransz P., R. ten Hoopen  and F. Tessadori (2006) Composition and formation of heterochromatin in Arabidopsis thaliana. Chromosome Research 14, 71-82.
  • Lysak M., Fransz P., Schubert I. (2006) Cytogenetic Analyses of Arabidopsis. Methods Mol Biol. 323, p173-186.
  • Mylne J.S., Barrett L., Tessadori F., Mesnage S, Johnson L., Bernatavichute Y, Jacobsen S., Fransz P. and Dean C. (2006) LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1 is required for epigenetic silencing of FLC. Proc. Nat. Acad. Sci. 103, 5012-5017.
  • Libault M., Tessadori F., Germann S., Snijder B., Fransz P., Gaudin V. (2005) The Arabidopsis LHP1 protein is a component of euchromatin. Planta 222, 910-25.
  • Baroux C, Fransz P, and Grossniklaus U. (2004) Nuclear fusions contribute to polyploidization of the gigantic nuclei in the chalazal endosperm of Arabidopsis. Planta 220, 38-46.
  • Van Driel R. and P. Fransz (2004) Nuclear architecture and genome functioning in plants and animals: what can we learn from both? Exp. Cell Res. 296, 86-90.
  • Fransz P. (2004) The interphase nucleus. In: Encyclopedia of Plant and Crop Science. (R.M. Goodman ed.), Marcel Dekker, New York, Basel, p.568-571.
  • Tessadori F, Van Driel R, Fransz P. (2004) Cytogenetics as a tool to study gene regulation. Trends Plant Sci. 9, 147-153.
  • Van Driel R., Fransz P.F. and Verschure P.J. (2003) The eukaryotic genome: a system regulated at different hierarchical levels. J Cell Sci. 116, 4067-4075.
  • Vleghels I, Hontelez J, Ribeiro A, Fransz P, Bisseling T, Franssen H. (2003) Expression of ENOD40 during tomato plant development. Planta 218, 42-49.
  • Fransz P., Soppe W. and Schubert I. (2003) Heterochromatin in interphase nuclei of Arabidopsis. Chrom. Res. Chrom. Res. 11, 227-240.
  • Koornneef M., Fransz P. and De Jong J.H. (2003) Cytogenetic tools for Arabidopsis thaliana. Chrom. Res. Chrom. Res. 11, 183-194.
  • Probst A.V , Mittelsten Scheid O., Fransz P.F. and Paszkowski J. (2003) Transcriptional reactivation can modify or maintain properties and organization of heterochromatin. Plant J. 33, 743–749
  • Fransz P.F. and De Jong J.H. (2002) Chromatin dynamics in plants. Curr. Opin. Plant Biol. 5, 560-567.
  • Fransz P. , De Jong J.H., Lysak M., Ruffini Castiglione M. and Schubert I. (2002) Interphase chromosomes in Arabidopsis are organised as well-defined chromocenters from which euchromatin loops emanate. Proc. Natl. Acad. Sci. 99, 14584 – 14589.
  • Soppe W.J.J., Jasencakova Z., Houben A., Kakutani T., Meister A., Huang H.S., Jacobsen S.E., Schubert I. and Fransz P.F. (2002) DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis EMBO J. 21, 6549-6559.
  • Haupt W., Fischer T.C.,  Winderl S., Fransz P. 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.
  • 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.
  • Lysak M. A., Fransz  P.F, Ali H.B.M.  and Schubert I. (2001) Chromosome painting in Arabidopsis thaliana. The Plant Journal 28, 689-697.
  • Schubert I, Fransz P.F., Fuchs J. and de Jong J.H. (2001) Chromosome painting in plants.Methods in Cell Science23: 57–69.
  • 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.
  • De Jong J.H., X.-B. Zhong, P.F. Fransz, J. Wennekes-van Eden, E. Jacobsen and P. Zabel (2000) High resolution FISH reveals the molecular and chromosomal organisation of repetitive sequences of individual tomato chromosomes. In: Chromosomes today. Vol 13, E. Olmo and C.A. Redi (eds), Birkhäuser Verlag, Basel-Boston-Berlin.
  • Fransz P., Armstrong S., De Jong J.H., Parnell L., Van Drunen C.M., Dean C., Zabel P., Bisseling T. and Jones G. (2000) Integrated Cytogenetic Map of Chromosome Arm 4S of A. thaliana: Structural Organization of Heterochromatic Knob and Centromere Region. Cell 100, 367-376.
  • Steimer A., Amedeo P., Fransz P., Afsar K., Mittelsten Scheid O. and Paszkowski J. (2000) Endogenous targets of transcriptional gene silencing in Arabidopsis thaliana. Plant Cell 12, 1165-1178.
  • Tabata S, Kaneko T, Nakamura Y, Kotani H, Kato T, Asamizu E, Miyajima N, Sasamoto S, Kimura T, Hosouchi T, Kawashima K, Kohara M, Matsumoto M, Matsuno A, Muraki A, Nakayama S, Nakazaki N, Naruo K, Okumura S, Shinpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Sato S, de la Bastide M, Huang E, Spiegel L, Gnoj L, O'Shaughnessy A, Preston R, Habermann K, Murray J, Johnson D, Rohlfing T, Nelson J, Stoneking T, Pepin K, Spieth J, Sekhon M, Armstrong J, Becker M, Belter E, Cordum H, Cordes M, Courtney L, Courtney W, Dante M, Du H, Edwards J, Fryman J, Haakensen B, Lamar E, Latreille P, Leonard S, Meyer R, Mulvaney E, Ozersky P, Riley A, Strowmatt C, Wagner-McPherson C, Wollam A, Yoakum M, Bell M, Dedhia N, Parnell L, Shah R, Rodriguez M, See LH, Vil D, Baker J, Kirchoff K, Toth K, King L, Bahret A, Miller B, Marra M, Martienssen R, McCombie WR, Wilson RK, Murphy G, Bancroft I, Volckaert G, Wambutt R, Dusterhoft A, Stiekema W, Pohl T, Entian KD, Terryn N, Hartley N, Bent E, Johnson S, Langham SA, McCullagh B, Robben J, Grymonprez B, Zimmermann W, Ramsperger U, Wedler H, Balke K, Wedler E, Peters S, van Staveren M, Dirkse W, Mooijman P, Lankhorst RK, Weitzenegger T, Bothe G, Rose M, Hauf J, Berneiser S, Hempel S, Feldpausch M, Lamberth S, Villarroel R, Gielen J, Ardiles W, Bents O, Lemcke K, Kolesov G, Mayer K, Rudd S, Schoof H, Schueller C, Zaccaria P, Mewes HW, Bevan M, Fransz P. (2000) Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana. Nature 408, 823-826.
  • Vergunst A.C, L.E.T Jansen, P.F. Fransz, J.H. de Jong and P.J.J. Hooykaas (2000) Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event. Chromosoma 109, 287-297.
  • The European Union Arabidopsis Genome Sequencing Consortium & The Cold Spring Harbor, Washington University in St Louis and PE Biosystems Arabidopsis Sequencing Consortium (1999) Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana. Nature 402, 769-777
  • Mayer K, Schüller C, Wambutt R, Murphy G, Volckaert G, Pohl T, Düsterhöft A, Stiekema W, Entian KD, Terryn N, Harris B, Ansorge W, Brandt P, Grivell L, Rieger M, Weichselgartner M, de Simone V, Obermaier B, Mache R, Müller M, Kreis M, Delseny M, Puigdomenech P, Watson M, Schmidtheini T, Reichert B, Portatelle D, Perez-Alonso M, Boutry M, Bancroft I, Vos P, Hoheisel J, Zimmermann W, Wedler H, Ridley P, Langham SA, McCullagh B, Bilham L, Robben J, Van der Schueren J, Grymonprez B, Chuang YJ, Vandenbussche F, Braeken M, Weltjens I, Voet M, Bastiaens I, Aert R, Defoor E, Weitzenegger T, Bothe G, Ramsperger U, Hilbert H, Braun M, Holzer E, Brandt A, Peters S, van Staveren M, Dirske W, Mooijman P, Klein Lankhorst R, Rose M, Hauf J, Kötter P, Berneiser S, Hempel S, Feldpausch M, Lamberth S, Van den Daele H, De Keyser A, Buysshaert C, Gielen J, Villarroel R, De Clercq R, Van Montagu M, Rogers J, Cronin A, Quail M, Bray-Allen S, Clark L, Doggett J, Hall S, Kay M, Lennard N, McLay K, Mayes R, Pettett A, Rajandream MA, Lyne M, Benes V, Rechmann S, Borkova D, Blöcker H, Scharfe M, Grimm M, Löhnert TH, Dose S, de Haan M, Maarse A, Schäfer M, Müller-Auer S, Gabel C, Fuchs M, Fartmann B, Granderath K, Dauner D, Herzl A, Neumann S, Argiriou A, Vitale D, Liguori R, Piravandi E, Massenet O, Quigley F, Clabauld G, Mündlein A, Felber R, Schnabl S, Hiller R, Schmidt W, Lecharny A, Aubourg S, Chefdor F, Cooke R, Berger C, Montfort A, Casacuberta E, Gibbons T, Weber N, Vandenbol M, Bargues M, Terol J, Torres A, Perez-Perez A, Purnelle B, Bent E, Johnson S, Tacon D, Jesse T, Heijnen L, Schwarz S, Scholler P, Heber S, Fransz P, Bielke C, Frishman D, Haase D, Lemcke K, Mewes HW, Stocker S, Zaccaria P, Bevan M, Wilson RK, de la Bastide M, Habermann K, Parnell L, Dedhia N, Gnoj L, Schutz K, Huang E, Spiegel L, Sehkon M, Murray J, Sheet P, Cordes M, Abu-Threideh J, Stoneking T, Kalicki J, Graves T, Harmon G, Edwards J, Latreille P, Courtney L, Cloud J, Abbott A, Scott K, Johnson D, Minx P, Bentley D, Fulton B, Miller N, Greco T, Kemp K, Kramer J, Fulton L, Mardis E, Dante M, Pepin K, Hillier L, Nelson J, Spieth J, Ryan E, Andrews S, Geisel C, Layman D, Du H, Ali J, Berghoff A, Jones K, Drone K, Cotton M, Joshu C, Antonoiu B, Zidanic M, Strong C, Sun H, Lamar B, Yordan C, Ma P, Zhong J, Preston R, Vil D, Shekher M, Matero A, Shah R, Swaby IK, O'Shaughnessy A, Rodriguez M, Hoffmann J, Till S, Granat S, Shohdy N, Hasegawa A, Hameed A, Lodhi M, Johnson A, Chen E, Marra M, Martienssen R, McCombie WR.  (1999) Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana. Nature 402, 769-777
  • De Jong J.H., Fransz P.F. and Zabel P. (1999) High resolution FISH in plant - techniques and applications. Trends in Genetics 4, 258-263
  • Fransz P., S. Armstrong, C. Alonso-Blanco, T.C. Fischer, R.A. Torres-Ruiz and G.H. Jones (1998) Cytogenetics for the model system Arabidopsis thaliana. The Plant Journal, 13, 867-876
  • Zhong X.B, Bodeau J., Fransz  P.F., Williamson V.M., Van Kammen A., De Jong J.H. and Zabel P. (1999) FISH to meiotic pachytene chromosomes of tomato locates the root-knot nematode resistance gene Mi-1 and the acid phosphatase gene Aps-1 near the junction of euchromatin and pericentromeric heterochromatin of chromosome arms 6S and 6L, respectively. Theor. Appl. Genet. 98, 365-370
  • Bhatt A.M., Lister C., Page T., Fransz P., Findlay K., Jones G.H., Dickinson H.G. and Dean C. (1999) DIF1, a plant gene essential for meiosis has homology to a double stranded break repair protein. The Plant Journal 19, 463-472
  • Zhong X.B., P.F. Fransz, J. Wennekes-van Eden, M. S. Ramanna, A. van Kammen, Pim Zabel and J. H. de Jong (1998a) FISH studies reveal the molecular and chromosomal organisation of individual telomere domains in tomato. The Plant Journal. 13, 507-517
  • Wisman E., G.H. Cardon, P. Fransz and H. Saedler (1998) Insertional mutagenesis with the autonomous transposable element En/Spm in Arabidopsis thaliana. Plant Molec. Biol. 37, 989-999
  • Montijn B., ten Hoopen R., Fransz P.F., Oud J.L. and Nanninga N. (1998) Characterisation of the nucleolar organising regions during the cell cycle in two varieties of Petunia hybrida as visualised by fluorescence in situ hybridisation and silver staining. Chromosoma 107, 80-86
  • Armstrong S.J., Fransz P., Marshall D. and Jones G.H. (1998) Physical mapping of DNA repetitive sequences to mitotic and meiotic chromosomes of Brassica oleracea var. alboglabra by fluorescence in situ hybridization. Heredity 81, 666-673