Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.

BioCEV Imaging Methods Core Facility Průmyslová 595 Vestec 252 50 Czech Republic

Department of Cell and Molecular Biology Uppsala University Uppsala 751 24 Sweden

Department of Immunology Genetics and Pathology Uppsala University Uppsala 751 08 Sweden

Institute of Computer Science Polish Academy of Sciences Warsaw 012 48 Poland

Laboratory of Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Kotlářská 2 Brno 61137 Czech Republic

Mendel Centre for Plant Genomics and Proteomics CEITEC Masaryk University Kamenice 5 Brno 62500 Czech Republic

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Ahmad, K. and Henikoff, S. (2002) The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell, 9, 1191-1200.

Bernstein, B.E., Mikkelsen, T.S., Xie, X.H. et al. (2006) A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell, 125, 315-326.

Berr, A. and Schubert, I. (2007) Interphase chromosome arrangement in Arabidopsis thaliana is similar in differentiated and meristematic tissues and shows a transient mirror symmetry after nuclear division. Genetics, 176, 853-863.

Borysko, E. and Bang, F.B. (1951) Structure of the nucleolus as revealed by the electron microscope; a preliminary report. Bull. Johns Hopkins Hosp. 89, 468-473.

Campos, E.I., Smits, A.H., Kang, Y.H. et al. (2015) Analysis of the histone H3.1 interactome: a suitable chaperone for the right event. Mol. Cell, 60, 697-709.

de Carcer, G. and Medina, F.J. (1999) Simultaneous localization of transcription and early processing markers allows dissection of functional domains in the plant cell nucleolus. J. Struct. Biol. 128, 139-151.

Chandrasekhara, C., Mohannath, G., Blevins, T., Pontvianne, F. and Pikaard, C.S. (2016) Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis. Genes Dev. 30, 177-190.

Chen, Z.J., Comai, L. and Pikaard, C.S. (1998) Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids. Proc. Natl Acad. Sci. USA, 95, 14891-14896.

Chica, C., Szarzynska, B., Chen-Min-Tao, R., Duvernois-Berthet, E., Kassam, M., Colot, V. and Roudier, F. (2013) Profiling spatial enrichment of chromatin marks suggests an additional epigenomic dimension in gene regulation. Front. Life Sci. 7, 80-87.

Cohen, S.M., Chastain, P.D. 2nd, Cordeiro-Stone, M. and Kaufman, D.G. (2009) DNA replication and the GINS complex: localization on extended chromatin fibers. Epigenetics Chromatin, 2, 6.

Copenhaver, G.P., Doelling, J.H., Gens, S. and Pikaard, C.S. (1995) Use of RFLPs larger than 100 kbp to map the position and internal organization of the nucleolus organizer region on chromosome 2 in Arabidopsis thaliana. Plant J. 7, 273-286.

Derenzini, M., Romagnoli, T., Ceccarelli, C. and Eusebi, V. (1988a) Fixatives and silver stainability of NOR proteins at the light microscopic level. J. Histochem. Cytochem. 36, 1453-1454.

Derenzini, M., Romagnoli, T., Mingazzini, P. and Marinozzi, V. (1988b) Interphasic nucleolar organizer region distribution as a diagnostic parameter to differentiate benign from malignant epithelial tumors of human intestine. Virchows Arch B Cell Pathol. Incl. Mol. Pathol. 54, 334-340.

Duc, C., Benoit, M., Detourne, G. et al. (2017) Arabidopsis ATRX modulates H3.3 occupancy and fine-tunes gene expression. Plant Cell, 29, 1773-1793.

Duc, C., Benoit, M., Le Goff, S., Simon, L., Poulet, A., Cotterell, S., Tatout, C. and Probst, A.V. (2015) The histone chaperone complex HIR maintains nucleosome occupancy and counterbalances impaired histone deposition in CAF-1 complex mutants. Plant J. 81, 707-722.

Dvorackova, M., Raposo, B., Matula, P., Fuchs, J., Schubert, V., Peska, V., Desvoyes, B., Gutierrez, C. and Fajkus, J. (2018) Replication of ribosomal DNA in Arabidopsis occurs both inside and outside the nucleolus during S phase progression. J. Cell Sci. 131, jcs202416.

Earley, K.W., Pontvianne, F., Wierzbicki, A.T., Blevins, T., Tucker, S., Costa-Nunes, P., Pontes, O. and Pikaard, C.S. (2010) Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation. Genes Dev. 24, 1119-1132.

Exner, V., Taranto, P., Schönrock, N., Gruissem, W. and Hennig, L. (2006) Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development. Development, 133, 4163-4172. https://doi.org/10.1242/dev.02599

Franek, M., Kovarikova, A., Bartova, E. and Kozubek, S. (2016) Nucleolar reorganization upon site-specific double-strand break induction. J. Histochem. Cytochem. 64, 669-686.

Fransz, P., De Jong, J.H., Lysak, M., Castiglione, M.R. and Schubert, I. (2002) Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate. Proc. Natl Acad. Sci. USA, 99, 14584-14589.

Fransz, P., ten Hoopen, R. and Tessadori, F. (2006) Composition and formation of heterochromatin in Arabidopsis thaliana. Chromosome Res. 14, 71-82.

Gordon, G.W., Berry, G., Liang, X.H., Levine, B. and Herman, B. (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702-2713.

Harding, S.M., Boiarsky, J.A. and Greenberg, R.A. (2015) ATM dependent silencing links nucleolar chromatin reorganization to DNA damage recognition. Cell Rep. 13, 251-259.

Highett, M.I., Rawlins, D.J. and Shaw, P.J. (1993) Different patterns of rdna distribution in pisum-sativum nucleoli correlate with different levels of nucleolar activity. J. Cell Sci. 104, 843-852.

Huang, J., Ma, L., Yang, F., Fei, S.Z. and Li, L. (2008) 45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp. PLoS One, 3, e2167.

Jackson, S.A., Wang, M.L., Goodman, H.M. and Jiang, J. (1998) Application of fiber-FISH in physical mapping of Arabidopsis thaliana. Genome, 41, 566-572.

Jacob, Y. and Michaels, S.D. (2009) H3K27me1 is E(z) in animals, but not in plants. Epigenetics, 4, 366-369.

Lafontaine, J.G. (1958) Structure and mode of formation of the nucleolus in meristematic cells of Vicia faba and Allium cepa. J. Biophys. Biochem. Cytol. 4, 777-784.

Lawrence, R.J., Earley, K., Pontes, O., Silva, M., Chen, Z.J., Neves, N., Viegas, W. and Pikaard, C.S. (2004) A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol. Cell, 13, 599-609.

Leitch, A.R., Mosgoller, W., Shi, M. and Heslop-Harrison, J.S. (1992) Different patterns of rDNA organization at interphase in nuclei of wheat and rye. J. Cell Sci. 101(Pt 4), 751-757.

Li, J., Langst, G. and Grummt, I. (2006) NoRC-dependent nucleosome positioning silences rRNA genes. EMBO J. 25, 5735-5741.

Liang, Y.M., Wang, X., Ramalingam, R., So, K.Y., Lam, Y.W. and Li, Z.F. (2012) Novel nucleolar isolation method reveals rapid response of human nucleolar proteomes to serum stimulation. J. Proteomics, 77, 521-530.

Liu, N., Fromm, M. and Avramova, Z. (2014) H3K27me3 and H3K4me3 chromatin environment at super-induced dehydration stress memory genes of Arabidopsis thaliana. Mol. Plant, 7, 502-513.

Mandakova, T. and Lysak, M.A. (2008) Chromosomal phylogeny and karyotype evolution in x=7 crucifer species (Brassicaceae). Plant Cell, 20, 2559-2570.

Mathieu, O., Jasencakova, Z., Vaillant, I., Gendrel, A.V., Colot, V., Schubert, I. and Tourmente, S. (2003) Changes in 5S rDNA chromatin organization and transcription during heterochromatin establishment in Arabidopsis. Plant Cell, 15, 2929-2939.

Mathieu, O., Probst, A.V. and Paszkowski, J. (2005) Distinct regulation of histone H3 methylation at lysines 27 and 9 by CpG methylation in Arabidopsis. EMBO J. 24, 2783-2791.

McKeown, P.C. and Shaw, P.J. (2009) Chromatin: linking structure and function in the nucleolus. Chromosoma, 118, 11-23.

Medina, F.J., Cerdido, A. and de Carcer, G. (2000) The functional organization of the nucleolus in proliferating plant cells. Eur. J. Histochem. 44, 117-131.

Mohannath, G., Pontvianne, F. and Pikaard, C.S. (2016) Selective nucleolus organizer inactivation in Arabidopsis is a chromosome position-effect phenomenon. Proc. Natl Acad. Sci. USA, 113, 13426-13431.

Moissiard, G., Cokus, S.J., Cary, J. et al. (2012) MORC family ATPases required for heterochromatin condensation and gene silencing. Science, 336, 1448-1451.

Montacie, C., Durut, N., Opsomer, A. et al. (2017) Nucleolar proteome analysis and proteasomal activity assays reveal a link between nucleolus and 26S proteasome in A. thaliana. Front. Plant Sci. 8, 1815.

Mozgova, I., Mokros, P. and Fajkus, J. (2010) Dysfunction of chromatin assembly factor 1 induces shortening of telomeres and loss of 45S rDNA in Arabidopsis thaliana. Plant Cell, 22, 2768-2780.

Neves, N., Viegas, W. and Pikaard, C.S. (2005) Nucleolar dominance and rRNA gene dosage control: a pardigm for transcriptional regulation via an epigentic on/off switch. Ann. Plant Rev. 19, 201-222.

Nie, X., Wang, H., Li, J., Holec, S. and Berger, F. (2014) The HIRA complex that deposits the histone H3.3 is conserved in Arabidopsis and facilitates transcriptional dynamics. Biol. Open, 3, 794-802.

Otero, S., Desvoyes, B., Peiro, R. and Gutierrez, C. (2016) Histone H3 dynamics reveal domains with distinct proliferation potential in the Arabidopsis root. Plant Cell, 28, 1361-1371.

Pavlistova, V., Dvorackova, M., Jez, M., Mozgova, I., Mokros, P. and Fajkus, J. (2016) Phenotypic reversion in fas mutants of Arabidopsis thaliana by reintroduction of FAS genes: variable recovery of telomeres with major spatial rearrangements and transcriptional reprogramming of 45S rDNA genes. Plant J. 88, 411-424.

Pendle, A.F., Clark, G.P., Boon, R., Lewandowska, D., Lam, Y.W., Andersen, J., Mann, M., Lamond, A.I., Brown, J.W. and Shaw, P.J. (2005) Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol. Biol. Cell, 16, 260-269.

Perry, R.P. (1966) Nucleolus: structure and function. Science, 153, 214-219.

Pfab, A., Breindl, M. and Grasser, K.D. (2018) The Arabidopsis histone chaperone FACT is required for stress-induced expression of anthocyanin biosynthetic genes. Plant Mol. Biol. 96, 367-374.

Pontvianne, F., Abou-Ellail, M., Douet, J. et al. (2010) Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana. PLoS Genet. 6, e1001225.

Pontvianne, F., Blevins, T., Chandrasekhara, C. et al. (2013) Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes Dev. 27, 1545-1550.

Pontvianne, F., Blevins, T., Chandrasekhara, C., Feng, W., Stroud, H., Jacobsen, S.E., Michaels, S.D. and Pikaard, C.S. (2012) Histone methyltransferases regulating rRNA gene dose and dosage control in Arabidopsis. Genes Dev. 26, 945-957.

Probst, A.V., Fagard, M., Proux, F. et al. (2004) Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats. Plant Cell, 16, 1021-1034.

Pruitt, R.E. and Meyerowitz, E.M. (1986) Characterization of the genome of Arabidopsis thaliana. J. Mol. Biol. 187, 169-183.

Risueno, M.C., Medina, F.J., Diaz, M. and de la Espina, S. (1982) Nucleolar fibrillar centres in plant meristematic cells: ultrastructure, cytochemistry and autoradiography. J. Cell Sci. 58, 313-329.

Roudier, F., Ahmed, I., Berard, C. et al. (2011) Integrative epigenomic mapping defines four main chromatin states in Arabidopsis. EMBO J. 30, 1928-1938.

Schofer, C. and Weipoltshammer, K. (2018) Nucleolus and chromatin. Histochem. Cell Biol. 150, 209-225.

Schwartz, Y.B. and Pirrotta, V. (2007) Polycomb silencing mechanisms and the management of genomic programmes. Nat. Rev. Genet. 8, 9-22.

Sequeira-Mendes, J., Araguez, I., Peiro, R., Mendez-Giraldez, R., Zhang, X., Jacobsen, S.E., Bastolla, U. and Gutierrez, C. (2014) The Functional topography of the Arabidopsis genome is organized in a reduced number of linear motifs of chromatin states. Plant Cell, 26, 2351-2366.

Shaw, P.J. and Jordan, E.G. (1995) The nucleolus. Annu. Rev. Cell Dev. Biol. 11, 93-121.

Shi, L., Wang, J., Hong, F., Spector, D.L. and Fang, Y. (2011) Four amino acids guide the assembly or disassembly of Arabidopsis histone H3.3-containing nucleosomes. Proc. Natl Acad. Sci. USA, 108, 10574-10578.

van Sluis, M. and McStay, B. (2019) Nucleolar DNA double-strand break responses underpinning rDNA genomic stability. Trends Genet. 35, 743-753.

Smith, S. and Stillman, B. (1989) Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell, 58, 15-25.

Soppe, W.J., Jasencakova, Z., Houben, A., Kakutani, T., Meister, A., Huang, M.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.

Stepinski, D. (2014) Functional ultrastructure of the plant nucleolus. Protoplasma, 251, 1285-1306.

Stroud, H., Otero, S., Desvoyes, B., Ramirez-Parra, E., Jacobsen, S.E. and Gutierrez, C. (2012) Genome-wide analysis of histone H3.1 and H3.3 variants in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA, 109, 5370-5375.

Sullivan, L.L., Boivin, C.D., Mravinac, B., Song, I.Y. and Sullivan, B.A.(2011) Genomic size of CENP-A domain is proportional to total alpha satellite array size at human centromeres and expands in cancer cells. Chromosome Res. 19, 457-470.

Tagami, H., Ray-Gallet, D., Almouzni, G. and Nakatani, Y. (2004) Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell, 116, 51-61.

Tessadori, F., Chupeau, M.C., Chupeau, Y., Knip, M., Germann, S., van Driel, R., Fransz, P. and Gaudin, V. (2007) Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells. J. Cell Sci. 120, 1200-1208.

Thorstensen, T., Fischer, A., Sandvik, S.V., Johnsen, S.S., Grini, P.E., Reuter, G. and Aalen, R.B. (2006) The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9. Nucleic Acids Res. 34, 5461-5470.

Watanabe-Susaki, K., Takada, H., Enomoto, K. et al. (2014) Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells. Stem Cells, 32, 3099-3111.

Wollmann, H., Holec, S., Alden, K., Clarke, N.D., Jacques, P.É. and Berger, F. (2012) Dynamic deposition of histone variant H3.3 accompanies developmental remodeling of the Arabidopsis transcriptome. PLoS Genet. 8, e1002658. https://doi.org/10.1371/journal.pgen.1002658

Wollmann, H., Stroud, H., Yelagandula, R. et al. (2017) The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana. Genome Biol. 18, 94.

Yelagandula, R., Stroud, H., Holec, S. et al. (2014) The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis. Cell, 158, 98-109.

Zentner, G.E. and Henikoff, S. (2014) High-resolution digital profiling of the epigenome. Nat. Rev. Genet. 15, 814-827.

Zentner, G.E., Saiakhova, A., Manaenkov, P., Adams, M.D. and Scacheri, P.C. (2011) Integrative genomic analysis of human ribosomal DNA. Nucleic Acids Res. 39, 4949-4960.

Zhao, Z., Dammert, M.A., Grummt, I. and Bierhoff, H. (2016) lncRNA-induced nucleosome repositioning reinforces transcriptional repression of rRNA genes upon hypotonic stress. Cell Rep. 14, 1876-1882.

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