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First published online March 21, 2007
doi: 10.1242/10.1242/jcs.000026

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Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells

¹ Nuclear Organization Group, Swammerdam Institute for Life Sciences, University of Amsterdam, BioCentrum Amsterdam, Kruislaan 318, 1098SM Amsterdam, The Netherlands
² Laboratoire de Biologie Cellulaire, IJPB, INRA, Route de St Cyr, 78026 Versailles Cedex, France

View larger version (56K): [in this window] [in a new window]   Fig. 1. Micrographs of cultured protoplasts, dividing cells and callus formation. (A) Freshly isolated protoplasts. (B) Protoplasts after 120 hours in culture showing active divisions (arrows). The absence of chloroplasts denotes a dedifferentiated state. (C) Microcallus after 15 days. Bar, 20 µm.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Protoplast nuclei contain chromocenters that are reduced in number and size. (A) DAPI-stained nuclei show up to three chromocenters in protoplasts (middle), and up to ten chromocenters in leaf (left) and cultured cells (right). Bar, 5 µm. (B) Relative heterochromatin fraction (RHF) in leaves (black bars), protoplasts (gray bars) and cultured cells (white bars). (C) Confocal images of living cells expressing H2B::YFP from leaf (left), protoplasts (middle) and after 72 hours in culture (right). Bar, 5 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 3. FISH localization of repetitive sequences in leaf and protoplasts. DAPI staining (left column); FISH signal (middle column); merge (right column). (A,B) The repeats of the pericentric BAC F28D6 localize at chromocenters in leaves (A) but are dispersed in protoplast nuclei (B). (C,D) The Athila transposon localizes at chromocenters in leaves (C) but is dispersed in protoplasts (D). (E) Both 5S (red) and 45S (green) rDNA repeats localize at chromocenters in leaves. (F) In protoplasts, 5S rDNA shows decondensed signals, whereas 45S rDNA signals remain condensed at the chromocenters. (G,H) The 180 bp centromere repeat localizes in chromocenters of leaf nuclei (G), but is dispersed in protoplast nuclei (H). Bar, 5 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Localization of repetitive sequences in cells cultured for 120 hours. DAPI staining (left column); FISH signal (middle column); merge (right column). (A) 45S rDNA (green signal) is condensed and located at large chromocenters. (B-D) FISH signals of the 180 bp repeat (red) and 5S rDNA (green) show partial (B,C) to full (D) condensation at chromocenters. Note that partially condensed 180 bp repeats colocalize with faint chromocenters (arrowheads in B). The large chromocenters (arrows) contain 45S repeats. (E,F) BAC F28D6 (E) and the Athila transposon (F) are only partially localized at chromocenters. Bar, 5 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. DNA methylation pattern in nuclei and in repeat regions. (A) DAPI staining (upper panels) and immunolocalization of 5-methylcytosine (lower panels) in leaf (left), protoplast (middle) and cultured cell (right). Bar, 5 µm. (B) Southern blot analysis of leaf, protoplast (pp) and cultured (cc) cells. DNA was digested with MspI (M) and HpaII (H). The blots were hybridized with DNA probes from the 180 bp repeat, 45S rDNA and Athila transposon.

View larger version (52K): [in this window] [in a new window]   Fig. 6. Distribution pattern and concentration of histone H3K9 and H3K4 dimethylation. (A) DAPI staining (upper panels) and immunolocalization of H3K9me2 (lower panels). (B) DAPI staining (upper panels) and immunolocalization of H3K4me2 (lower panels). Bar, 5 µm. (C) Western blot analysis of histone-enriched protein extracts from leaves, protoplasts and cultured cells (24 hours, 48 hours, 96 hours) using antibodies against H3K9me2 and H3K4me2.

View larger version (42K): [in this window] [in a new window]   Fig. 7. The heterochromatic transgene Locus A containing a multicopy HPT array is decondensed in protoplast nuclei. FISH signals (red; top and bottom panels) for the HPT sequence localize at mini-chromocenters (brightly fluorescent areas) in nuclei of Arabidopsis Line A. DAPI-stained (top and middle panels) minichromocenters are no longer visible in protoplasts and the FISH signal for the HPT sequence is decondensed. The bottom four panels are higher magnifications of the HPT regions. Bar, 5 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 8. Chromatin dynamics during protoplast culture. Schematic representation of an interphase chromosome before and after protoplast formation and in the ddm1 mutant, showing repeat regions (colors) and gene-rich regions (gray). The loop organization of gene-rich euchromatin is based on previous studies (Fransz et al., 2002). For simplicity only chromosome 4 is shown, which has all major tandem repeats (180 bp, 45S rDNA and 5S rDNA). Most repeat regions become decondensed in protoplasts. Only the 45S rDNA domain remains partly condensed. Cultured cells show various levels of chromatin condensation. The sequence of recondensation events is 45S rDNA, 180 bp, 5S rDNA and interspersed repeats, such as transposable elements. aThe total size of repeat sequences in chromocenters is based on the total genomic size of the repeats shown in Table S2 in supplementary material. bValues are normalized to the maximal value of dataset (=leaf). cThe total size of high copy transposons in Arabidopsis is unknown. In the ddm1 mutant all repeats, except the low copy repeats, are in chromocenters (Soppe et al., 2002). Hence, the fraction of repeats in chromocenters of ddm1 is higher compared with chromocenters of cultured cells and lower compared with chromocenters in leaf nuclei.