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Pairing of lacO tandem repeats in Arabidopsis thaliana nuclei requires the presence of hypermethylated, large arrays at two chromosomal positions, but does not depend on H3-lysine-9-dimethylation

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Abstract

Fluorescent chromatin tagging by the lacO operator/lac repressor system in Arabidopsis thaliana is useful to trace distinct chromatin domains in living cells. Nevertheless, the tandem repeats of the tagging system may alter the spatial organisation of chromatin within nuclei by increasing homologous pairing as well as association with heterochromatin. Efficient homologous pairing occurs if lacO repeat arrays of ∼10 kb are present at two loci, either on the same chromosome or on different chromosomes. DNA hypomethylation of lacO repeats results in reduced homologous pairing. Because, in plants, DNA methylation can serve as a signal for H3-lysine9-dimethylation (H3K9me2), and subsequently for non-CG-context DNA methylation, SET-domain histone methyltransferase and chromodomain dna methyltransferase 3 (cmt3) mutations were introgressed. In suvh4 suvh5 suvh6 and cmt3 mutants, H3K9me2 associated with lacO repeats is diminished, but homologous pairing persists. Thus, neither H3K9me2 nor CMT3-mediated non-CG methylation are required at wild-type level for homologous pairing of lacO repeat loci.

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Acknowledgements

We thank Antonius J.M. Matzke, Gregor Mendel Institute, Vienna, and Eric Lam, Rutgers University, for contributing transgenic lines with lacO array inserts and Judith Bender, Brown University, and Gunter Reuter, MLU Halle-Wittenberg, for providing mutant lines. Technical support by Achim Bruder, Christa Fricke, Inge Glaser, Beate Kamm and Martina Kühne is gratefully acknowledged. This work was supported by the Land Sachsen-Anhalt in the frame of the “Exzellenznetzwerk Biowissenschaften” (G.J.; B.E.B.) and the Deutsche Forschungsgemeinschaft in the frame of the “Sonderforschungsbereich 648” (M.K.).

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  1. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstreet 3, 06466, Gatersleben, Germany

    Gabriele Jovtchev, Branimira Emilova Borisova, Markus Kuhlmann, Jörg Fuchs, Koichi Watanabe, Ingo Schubert & Michael Florian Mette

  2. Institute of Biodiversity and Ecosystem Research—BAS, Gagarinstreet 2, 1113, Sofia, Bulgaria

    Gabriele Jovtchev

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  1. Gabriele Jovtchev
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Correspondence to Michael Florian Mette.

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Suppl. Figure 1

a Composition of transfer DNA constructs used to generate transgenic lines CCP4.193, CCT431, 112, 125, 26 and EL702C. Transgene CCT431 originated by transposition from a CCP4-type transfer DNA (T-DNA) insertion. The maps are drawn approximately to scale; the size bar indicates 1 kb. The black bar labelled “ChIP-pPCR” at the map of EL702C indicates the area that was amplified to test for the association of lacO tandem repeats with H3K9me2 and H3K4me3. b Chromosomal positions of T-DNAs with lacO repeat inserts (green) in transgenic lines CCP4.193, CCT431, 125 + 26, 26, 112 and EL702C. (PPT 159 kb)

Suppl. Figure 2

Exemplary non-pairing and pairing events of large lacO repeat arrays in interphase nuclei from A. thaliana. Evaluation of homologous pairing for transgenic lacO repeat inserts (lacO probe, green) and their flanking regions (BAC probes, red or blue) by fluorescence in situ hybridisation. a Unpaired lacO inserts, b single ectopic pairing, c double allelic pairing, d double ectopic pairing, e single allelic and single ectopic pairing and f double allelic and double ectopic pairing; white arrows mark pairing events. The white scale bar indicates 3 μm. (PPT 281 kb)

Suppl. Figure 3

Individual ChIP qPCR determinations of H3K9me2 and H3K4me3 associated with lacO tandem repeat arrays and control sequences. Targeted sequences were a Ta3, b 180bp repeats, c lacO tandem repeats and d PFK. Labels identify transgenic and mutant states of analysed plant material. Lower case “l” after the label identifies leaves, lower case “s” seedlings as starting material for chromatin immunoprecipitation. Signal intensities in qPCR for immunoprecipitated chromatin were related to the signal intensities for the corresponding initial chromatin prepatations (input). Grey columns represent the results for ChIP preparations with specific antibodies (recognising H3K9me2 and H3K4me3, respectively), white columns correspond results from control preparations without addition of antibody. n.d. indicates “not determined”. (PPT 249 kb)

Suppl. Figure 4

Distribution of histone modifications H3K9me1,2 and H3K27me2 in interphase nuclei of suvh2, suvh4 and suvh2 suvh4 mutants. Bold black labels Identify the transgenic, italic letters the mutant state of A. thaliana lines (suvh24 indicates suvh2 suvh4 double mutant). Indirect immunolabeling (red signal) of histone H3K9me2 (left), H3K9me1 (middle) and H3K27me2 (right) was performed on flow-sorted 4C nuclei isolated from leaves. Bright spots detected in nuclei by DAPI staining (blue signal) represent heterochromatic chromocenters. The scale bar indicates 2.5 μm. (PPT 448 kb)

Suppl. Figure 5

Homologous pairing and heterochromatin association of transgenic lacO tandem inserts and/or their flanking regions in suvh2, suvh4 and suvh2 suvh4 mutants. The colour of columns indicates the type of probes. Bold black labels identify the transgenic, italic labels the mutant state of A. thaliana lines (suvh24 indicates suv2 suvh4 double mutant). Positions of lacO tandem repeat inserts are marked by horizontal green bars, and flanking BACs by red or blue vertical bars on the right side of the chromosomes. Top Frequencies of somatic homologous pairing. Plain green columns show the average allelic pairing, hatched green columns the average ectopic pairing frequency per lacO insert locus. Pairing frequencies detected with BAC probes are shown in grey for the absence (pooled) and in blue and red for the presence of transgenic lacO repeat inserts. The horizontal red dashed line indicates the average pairing frequency over the genome. Bottom Frequencies of association of lacO tandem repeat inserts with heterochromatic chromocenters (CC). The association frequencies are shown in grey for the absence (pooled) and in blue and red for the presence of transgenic lacO repeat inserts. Bars on columns indicate the standard deviation. (PPT 188 kb)

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Suppl.Table 1

Pairing frequencies of lacO inserts and corresponding flanking BACs for different transgenes, suvh4 suvh5 suvh6 and cmt3 mutants. (DOC 98 kb)

Suppl. Table 2

Association frequencies of lacO inserts and corresponding flanking BACs with heterochromatic chromocenters (CCs) for different transgenes, suvh4 suvh5 suvh6 and cmt3 mutants. (DOC 103 kb)

Suppl. Table 3

Pairing frequencies of lacO inserts and corresponding flanking BACs for suvh2, suvh4 and suvh2 suvh4 mutants. (DOC 59 kb)

Suppl. Table 4

Association frequencies of lacO inserts and corresponding flanking BACs with heterochromatic chromocenters (CCs) for suvh2, suvh4 and suvh2 suvh4 mutants. (DOC 60 kb)

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Jovtchev, G., Borisova, B.E., Kuhlmann, M. et al. Pairing of lacO tandem repeats in Arabidopsis thaliana nuclei requires the presence of hypermethylated, large arrays at two chromosomal positions, but does not depend on H3-lysine-9-dimethylation. Chromosoma 120, 609–619 (2011). https://doi.org/10.1007/s00412-011-0335-8

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