Figure 1.
Fluorescence in situ hybridization (FISH) on metaphase chromosomes of Q. robur using 18S and 5S rDNA probes.
FITC green signals represent the 18S rDNA probe, and Fluoro-Red signals correspond to the 5S rDNA probe (not relevant in this study). A. NOR-1 locus (arrows) is located terminally on a submetacentric chromosome pair. NOR-2 (arrowheads) resides near centromere of a small metacentric chromosome. Observe the signal size and strength dimorphism of NOR-2 sites. The rDNA knob (localized FISH signal) is situated proximally on one site (A and D, yellow arrow), and the rest of rDNA chromatin is decondensed (punctuate FISH signal). On another site, rDNA knob is present both at the proximal and the terminal (satellite) part of the site (white arrows, A and D) and the dispersed rDNA fraction resides between the two (visible as a punctuate FISH signal corresponding to SC). B and E. Satellites (distal part of the NOR-1 site) are indicated with arrows in DAPI stained metaphases. C and F. Schematic representations of chromatin topology at the major rDNA locus corresponding to figures A and D. G. and H. Graphical representations of measured NOR-1 and NOR-2 sizes (G, arbitrary OD units normalized to pixel area) and volume (H, arbitrary OD units normalized to voxel volume) demonstrate that the two loci can be unambiguously identified. Number of asterisks indicates p<0.0001; error bars represent the standard error of the mean. Scale bar is 5 µm.
Table 1.
Different patterns of rDNA chromatin organization at NOR-1 sites.
Figure 2.
Electron micrographs of in situ hybridization with biotin labeled 18S rDNA probe on sections through root tip meristematic tissue of Q. robur.
Hybridization area was detected with DAB. Labeled condensed chromatin lies outside or close to the nucleolus (A and B, black arrows), protruding into the nucleolus (B, black arrow) and inside the nucleolus near the nucleolar border (C, black arrows). Diffuse hybridization signal that corresponds to completely decondensed DNA is seen inside the nucleolus (A, B, C, white arrows). Unlabeled chromatin corresponding to the NOR-bearing chromosome can be seen near the nucleolus (B, black arrowhead) and protruding into it (A, black arrowhead). Letter «n» marks the nucleolus. Scale bar is 2.5 µm.
Figure 3.
Position of NOR-1 and NOR-2 in interphase nucleus of Q.robur.
A. Major 45S rDNA sites (NOR-1, arrowheads) are associated with the nucleolus while minor 45S rDNA sites (NOR-2, arrows) are positioned at the nuclear periphery. Statistical analysis of measured distances from the nucleolus (B, N = 220) or the nuclear periphery (C, N = 240) reveals differences between the two rDNA loci. Number of asterisks indicates p<0.0001; error bars represent the standard error of the mean. Letter «n» marks the nucleoli. Scale bar is 5 µm.
Figure 4.
Correlation between the position of a NOR locus relative to the nucleolus and its transcriptional activity.
A. Locations of wheat (yellow-green) and rye (red) NOR loci in nuclei of triticale revealed by FISH. Statistical analysis of measured distances from the nucleolus (B, N = 42) or the nuclear periphery (C, N = 42) revealed significant differences between wheat and rye rDNA loci, concordant with the established transcriptional activity of the wheat loci and silencing of the rye locus due to nucleolar dominance. Number of asterisks indicates p<0.01 (**) and p<0.0001 (***); error bars represent the standard error of the mean. D. The graphical illustration of the correlation between the position of a NOR locus in relation to the nucleolus and its transcriptional activity in triticale and the common oak. Letter “n” marks the nucleoli. Scale bar is 5 µm.
Figure 5.
Level of DNA methylation in NOR-1 and NOR-2 of Q.robur.
A. Immunofluorescence using an antibody against 5-mC coupled with FISH using the 18S rDNA probe revealed the presence of 5-mC in both 45S rDNA loci. B. Co-localization analysis of 5-mC and FISH signals showed stronger co-localization in minor rDNA sites (NOR-2) when compared to major rDNA sites (NOR-1); N = 26. Number of asterisks indicates p<0.01; error bars represent the standard error of the mean. Letter “n” marks the nucleoli. Scale bar is 5 µm.
Figure 6.
Nuclear repositioning of NOR-2 of Q.robur following inhibition of DNA methylation.
A. After the treatment with 5-aza-2′-dC, NOR-2 sites (arrowheads) repositioned much closer to nucleoli, as confirmed by statistical analysis (B). Statistical analysis shows equal distance of the both NOR loci from the nuclear periphery (C). Number of asterisks indicates p<0.0001; error bars represent the standard error of the mean. Letter “n” marks the nucleoli. Scale bar is 5 µm.
Figure 7.
Decrease in DNA methylation level in NOR-1 and NOR-2 of Q.robur following treatment with 5-aza-2′-dC.
A. Immunofluorescent detection of 5-mC coupled with FISH using 18S rDNA probe revealed the reduction of 5-mC levels in NOR-2 sites. B. Similar levels of 5-mC in both NOR loci were confirmed by co-localization analysis. Error bars represent the standard error of the mean. Letter “n” marks the nucleoli. Scale bar is 5 µm.
Figure 8.
Histone modifications associated with NOR-1 and NOR-2 loci in root tip cycling cells of Q.robur.
IFF detection of H3K9ac (A), H3K4me3 (B), H3K9me1 (C) and H3K27me2 (D) and graphs showing Pearson’s coefficient of co-localization of respective histone marks with NOR loci. Green signals correspond to immunofluorescence of histone antibodies and red signals represent 18S rDNA FISH signals. Number of asterisks indicates p<0.05; error bars represent the standard the error of the mean. Letter “n” marks the nucleoli. Scale bar is 5 µm.
Figure 9.
Histone modifications associated with NOR-1 and NOR-2 loci in root tip cycling cells of Q. robur after treatment 5-aza-2′-dC.
IFF detection of H3K9ac (A), H3K4me3 (B), H3K9me1 (C) and H3K27me2 (D) and graphs showing Pearson’s coefficient of co-localization of respective histone marks with the NOR loci. Green signals correspond to immunofluorescence of histone antibodies and red signals represent 18S rDNA FISH signals. Number of asterisks indicates p<0.01; error bars represent the standard error of the mean. Letter “n” marks the nucleoli. Scale bar is 5 µm.
Figure 10.
RT-PCR analysis of 18S rRNA transcription.
RT-PCR analysis detected an increased level of 18S rRNA transcripts after treatment of root tips with 5-aza-2′-dC. C – control, T – treatment with 5-aza-2′-dC. OD/AU is optical density (in arbitrary units) of RT-PCR product peaks after electrophoresis in agarose gel and staining with ethidium bromide. The 18S rRNA (1830 bp band) transcription increased more than twofold after treatment with 5-aza-2′-dC, while the transcription level of the Actin3 gene (655 bp, used as control) remained essentially at the same level.
Figure 11.
Silver staining of chromosomes and nucleolus in root tip cycling cells of Q. robur.
Silver stained nuclei before (A) and after treatment with 5-aza-2′-dC (B) show strong silver deposition within the whole area corresponding to a single large nucleolus. A. rDNA knobs of both NOR-1 sites seen at the nucleolar periphery (arrows) show mild silver deposition. B. After treatment with 5-aza-2′-dC rDNA chromatin of the knob was decondensed, extending from the NOR-bearing chromosome to the nucleolus which was completely stained with silver (arrowhead). Scale bar is 5 µm.