PML bodies associate specifically with the MHC gene cluster in interphase nuclei
Carol Shiels1,5,
Suhail A. Islam2,
Radost Vatcheva1,
Peter Sasieni3,
Michael J. E. Sternberg2,
Paul S. Freemont4,5,* and
Denise Sheer1,*
1 Human Cytogenetics Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK
2 Biomolecular Modelling Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK
3 Mathematics and Statistics Group, Imperial Cancer Research Fund, London, WC2A 3PX, UK
4 Molecular Structure Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK
5 Centre for Structural Biology, Imperial College of Science, Technology and Medicine, South Kensington, London, SW7 2AZ, UK
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Fig. 1. Schematic maps indicating the organisation of the MHC, EDC and 6p24 genomic regions and the location of the genomic probes used for FISH analysis. (A) Ideograms show the positions of the EDC on chromosome 1 and the MHC and 6p24 regions on chromosome 6. The EDC contains 27 genes and spans a genomic region of 2.5 Mb, it is mainly composed of genes involved in keratinocyte differentiation and can be visualised using genomic probes to the small proline rich region (SPRR). The 6p24 region is relatively gene-poor, with the 500 kb subsection studied flanked by only two known genes. The MHC is divided into three classes and contains many genes involved in the immune response. It is one of the most gene-rich regions of the genome, with 224 gene loci identified over a genomic region of 3.9 Mb. A more detailed map of the MHC class II and adjacent centromeric region is shown in (B), with the relative positions of the LMP/TAP, DAXX, BAK and TCP11 regions indicated. A number of genes mapping to this region are shown, for a more comprehensive list refer to the Sanger Centre website (www.sanger.ac.uk). The region deleted in the 721.174 cell line is shown as a solid bar and covers a large proportion of the MHC class II region from the DRA gene up to the LMP/TAP region.
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Fig. 2. Spatial distribution of PML bodies relative to the MHC and EDC genomic regions. Immuno-FISH was performed on three-dimensionally preserved fibroblast nuclei, detecting PML bodies by immunofluorescence (blue) and simultaneously visualising the MHC using genomic probes recognising the LMP/TAP region (green) and the EDC using genomic probes recognising the SPRR region (red). Projections of serial optical sections through each nucleus are shown. PML bodies can be observed either closely associated or overlapping with both the MHC and the EDC in a percentage of the nuclei investigated. A-D demonstrates the variability in PML body association, with PML bodies found in association with both MHC regions (A), both EDC regions (B), associating with one MHC region and one EDC region (C) and associating with neither genomic region (D).
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Fig. 3. Determining minimal intranuclear genomic locus-PML body distances. Images of three-dimensionally preserved fibroblast nuclei collected by confocal microscopy (A) were processed using the image3D program to determine the x,y,z centroid co-ordinates of each PML body, MHC and EDC region in the nucleus (B). Using these coordinates it was possible to calculate the distance between each genomic locus and its closest PML body: MHC1-PML=1.3 µm; MHC2-PML=3 µm; EDC1-PML=2.3 µm; EDC2-PML=1.7 µm. Note that these images are 2D representations of 3D images. Bar, 5 µm.
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Fig. 4. Comparison and distribution of minimal genomic locus-PML body distances. The distribution of minimum distances between each genomic locus and its nearest PML body is shown as a box and whiskers plot, where the box extends from the lower quartile (where 25% of observed genomic locus-PML body distances are smaller), to the upper quartile (where 25% of observed genomic locus-PML body distances are larger) and the horizontal line in the middle of the box is the median. The small circles show outlying points and the ‘whiskers’ extending from the box show the range of minimum genomic locus-PML body distances. Note that the distribution of minimal MHC-PML distances is clearly shorter in comparison to the minimal EDC-PML and 6p24-PML distances.
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Fig. 6. Graphical representation of minimal distances to nearest PML body for each genomic locus along the short arm of chromosome 6. A small circle marks the mean distance and the horizontal line extends 2 standard errors either side of the mean. The standard errors are calculated based on a single mean minimum distance from each nucleus. Note that the error bars for PML-PML and LMP/TAP-PML distances are shorter than the others, because these distances were measured in all the experiments. The dashed vertical lines are at the mean LMP/TAP-PML and mean PML-PML distances.
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Fig. 7. The spatial distribution of PML bodies and coiled bodies with respect to the MHC region. The relative associations of PML bodies (blue), coiled bodies (CB) (red) and MHC regions (green) were examined in three dimensionally preserved HeLa cell nuclei. Insets on the right (from top to bottom) show examples of associations between PML bodies and the MHC region, PML bodies and coiled bodies, and coiled bodies and the MHC region. A projection of optical sections through the nucleus is shown.
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Fig. 8. Specificity of the MHC-PML association. (A-B) MHC-PML association was investigated in the 721.174 B lymphoblastoid cell line (A) and the control B-lymphoblastoid cell line AHB (B) by immuno-FISH on three-dimensionally preserved nuclei. 721.174 cells have only one copy of the short arm of chromosome 6 containing a large deletion of the MHC class II region (from DRA to LMP/TAP). The MHC region was visualised using genomic probes recognising the adjacent DAXX region (red) and PML bodies were detected simultaneously by immunofluorescence (green). (C-D) MHC-PML association was investigated in a derivative of the 721.174 cell line (4D1D10.2), resulting from stable integration of an MHC YAC into chromosome 18. (C) The MHC class II YAC DNA on chromosome 18 was detected using genomic probes recognising the LMP/TAP region (green) and the residual MHC region on chromosome 6 using genomic probes recognising the DAXX region (red). (D) The relative association of PML bodies with each copy of chromosome 18 was determined using a partial chromosome paint (red) to delineate the YAC integration site on band 18q11 and the MHC class II YAC DNA was detected using genomic probes recognising the LMP/TAP region (green). The approximate chromosomal position of these regions is illustrated on the ideograms.
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© The Company of Biologists Ltd 2001
(B), 
-amanitin (C) and DRB (D). PML bodies were detected by immunofluorescence (green) and the MHC region was visualised simultaneously using genomic probes recognising the LMP/TAP region (red). Note in C and D the clustering of PML bodies observed in a percentage of cells after transcriptional inhibition. (E-F) The MHC-PML association was also examined in fibroblast nuclei during G1 phase (E) and S phase (F) of the cell cycle, detecting PML bodies (blue) and BrdU (green) by immunofluorescence, and visualising the MHC region by FISH using genomic probes recognising the LMP/TAP region (red). Projections of optical sections through each nucleus are shown.