First published online 15 February 2005
doi: 10.1242/jcs.01685
Journal of Cell Science 118, 1021-1031 (2005)
Published by The Company of Biologists 2005
Large-scale chromatin decondensation induced in a developmentally activated transgene locus
Eva Wegel1,
Ruben H. Vallejos2,
Paul Christou3,
Eva Stöger4 and
Peter Shaw1,*
1 Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, Norwich, NR4 7UH, UK
2 Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), CONICET-UNR-F-LILLO, Suipacha 531, S202LRK Rosario, Argentina
3 Fraunhofer Institute for Molecular Biology and Applied Ecology, IME, Grafschaft, Auf dem Aberg 1, 57392 Schmallenberg, Germany
4 Institute of Molecular Biotechnology, BiologieVII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany
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Fig. 1. Probes used for locus and transcript detection in interphase nuclei.
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Fig. 2. FISH analysis of metaphase chromosomes. Metaphase spreads were hybridized with probes detecting the genomic fragments (green) and the vector sequences (red). (A) Loci encoding HMW-glutenin subunits in wild-type wheat (arrowheads). (B) Transgene loci (orange, hybridizing with both genomic and vector sequences) and gene loci (green, arrowheads) of loci encoding HMW-glutenin subunits in line E. Bar, 10 µm.
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Fig. 3. Transgene locus structure in non-expressing and expressing tissue. (top) Line E. (bottom) Line F. (Left) Chromosomes with transgene loci. Two loci very close together are visible in line E (arrowheads); visualized using a probe against genomic fragments of 1Ax1 and 1Dx5. Line F contains two chromosomes with transgene loci; visualized using a probe against vector sequence. Tissue sections of line E and F were hybridized with pHMW-1Ax1 and pHMW-1Dx5 to detect the transgene loci in the diploid root (as non-expressing tissue) and in the triploid endosperm at 6 dpa (before the onset of expression) and 13 dpa (after the onset of expression). The image shows projections of serial confocal sections through one layer of nuclei. A blue line is drawn around the edge of each nucleus. The insertion sites in E are visible as one or two foci in root tips and young endosperm, whereas each locus in line F is visible as one focus. In expressing endosperm tissue, the loci in both lines are decondensed into many foci. Bar, 10 µm; section spacing is 0.6 µm.
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Fig. 4. Locus decondensation in lines E and F. After hybridization with pHMW-1Ax1 and pHMW-1Dx5 to detect the transgene loci the fluorescent area per optical section was measured in 79 and 18 randomly selected nuclei of lines E and F, respectively, at 6 dpa and in 59 and 18 nuclei of lines E and F, respectively, at 14 dpa. The total number of voxels in the fluorescent region per nucleus was multiplied by the voxel volume and the resulting fluorescent volume is shown on the y-axis.
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Fig. 5. DNA decondensation in transcriptionally active loci. Endosperm nuclei of line E were hybridized with probes for pHMW-1Ax1 and pHMW-1Dx5, detecting the whole plasmids (green, A) or with probes detecting the flanking sequences and the vector (green, B) and counterstained with DAPI (blue). Overlay of several confocal sections showing only one of the three loci per nucleus. An overlay of all optical sections through the nucleus is shown for the DAPI staining in A and B to illustrate the similar sizes of the two nuclei. (A) Condensed locus at 6 dpa before the start of expression. (B) Transcriptionally active locus at 16 dpa in a rarely seen linear conformation. In the straightened chromatin fibres, the complex structure of the locus, which is broken up by genomic sequences, becomes visible. Bar, 10 µm; section spacing is 0.4-0.6 µm.
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Fig. 6. Time course of HMW-glutenin-subunit expression in endosperm. HMW-glutenin-encoding transcripts in seed sections of the wild type and line E were detected with an antisense probe for the coding region of 1Ax1. The sense probe was used as control for unspecific labelling. Left column, wild-type labelled with anti-sense probe; middle column, wild-type labelled with sense probe; right column, line E labelled with anti-sense probe. (A-C) 5 dpa. Before the onset of expression, no RNA signal is visible in the endosperm. The pericarp and some aleurone cells show unspecific labelling. (D-F) 8 dpa. Onset of expression in endosperm cells, unspecific labelling in the pericarp. (G-I) 10 dpa. Strong expression in wild-type endosperm, whereas the RNA signal in line E is much weaker and mainly concentrated around the nuclei instead of the whole of the cytoplasm. (J-L) 12 dpa. Very weak expression in line E, in contrast to the wild type. (M-O) 16 dpa. The wild type is still expressing strongly, whereas expression in line E has ceased. Bar, 0.5 mm.
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Fig. 7. Disappearance of cytoplasmic HMW-glutenin-encoding RNA at the onset of silencing. Sections from wax-embedded endosperm of line E were hybridized with an antisense probe for the coding region of 1Ax1 and detected with Cy3 (red). The nuclei were counterstained with DAPI. The images comprise only the nucleus and the cytoplasm immediately around it. (A) 10 dpa. HMW-glutenin-encoding RNA can be seen inside the nucleus and in the cytoplasm around it. (B) 14 dpa. RNA is still visible inside the nucleus, whereas it has disappeared from the cytoplasm. Bar, 10 µm.
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Fig. 8. Detection of siRNAs corresponding to the 1Ax1 coding region. Total seed RNA of the wild-type (WT) and line E extracted before (8 dpa) and after (14 dpa) the onset of silencing was hybridized with an antisense probe for the coding region of 1Ax1. Two RNA bands of  22 bp and 25 bp (which are indicative of PTGS) are visible in line E at 14 dpa (lane 4).
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Fig. 9. Localization of transgene loci and their transcripts during endosperm development. Endosperm nuclei counterstained with DAPI (blue) were hybridized with probes for the gene flanking regions and vector sequences of pHMW-1Ax1 and pHMW-1Dx5 to detect the locus (green) and with an antisense probe for the 1Ax1 coding region to detect the transcript (red). The insets are 2x enlargements of the loci marked by an arrowhead in the overlay. The sizes of the nuclei vary considerably owing to the cell-cycle phase they are in and because of endoreduplication at later stages. A and B are probably G1 nuclei, and C-E and G are probably G2 nuclei. F shows an endoreplicated nucleus. Each image is a projection of serial confocal sections. (A) Non-expressing nucleus in tissue starting to express. One locus is seen as one focus in the centre of the nucleus, whereas the two loci above and below are seen as two foci each. (B) Nucleus starting to transcribe the transgene in the same tissue as in A. All three loci are seen as two foci. Only the left-hand part of the locus denoted by the arrowhead is transcribing. (C) Nucleus in which all three loci are transcribing and condensed. The inset shows that some areas of the transgene locus are not transcriptionally active. (D) Transcriptionally active nucleus in the centre of the endosperm starting to decondense. One region in the locus in the inset shows no transcript. (E,F) Nuclei with fully decondensed loci, which are largely but not completely transcriptionally active. (G) Dying nucleus with dispersed transgene DNA and very weak RNA labelling. (H) Nucleus with degrading chromatin and no hybridization signal for the transgenes and their transcripts. Bar, 10 µm; section spacing is 0.4-0.6 µm.
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© The Company of Biologists Ltd 2005
