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
*Authors for correspondence (e-mail: p.freemont{at}ic.ac.uk; sheer{at}icrf.icnet.uk)
Accepted July 6, 2001
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SUMMARY |
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Key words: Immuno-FISH, Major histocompatibility complex, Nuclear organisation, PML nuclear body, Transcription
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INTRODUCTION |
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In addition to the defining Sp100 and PML proteins, PML bodies also contain a heterogeneous mix of functionally important proteins (Seeler and Dejean, 1999). The PML protein plays a pivotal role in accumulating components at PML bodies, since proteins such as Blooms (BLMs), CREB binding protein (CBP), DAXX and Sp100 do not localise to PML bodies in the absence of PML (Ishov et al., 1999; Zhong et al., 1999; Zhong et al., 2000b; Zhong et al., 2000a). Cells lacking BLM or Sp100 have otherwise normal PML body components (Ishov et al., 1999). There is also an essential role for the small ubiquitin-related protein SUMO-1 in PML body formation, since a PML mutant that cannot be SUMO-modified does not form PML bodies (Muller et al., 1998; Zhong et al., 2000b). The heterogeneity of proteins localising to PML bodies has made it difficult to propose a single function for this class of nuclear domain. The observation that proteins, such as the tumour suppressor BRCA1, only localise to PML bodies when overexpressed (Maul et al., 1998) suggests that PML bodies are a type of nuclear storage site, perhaps regulating the levels of active proteins within the nucleus (Maul, 1998). The pRB tumour suppressor protein for example, only localises to PML bodies in an inactive non-phosphorylated form (Alcalay et al., 1998). In support of this idea is the recent finding that misfolded proteins can accumulate in PML bodies prior to degradation by ubiquitin-mediated proteolysis via the proteosome (Anton et al., 1999).
PML is also implicated in regulating transcription. The recent demonstration that nascent RNA accumulates at the periphery of PML bodies suggests that PML bodies could act to concentrate regulatory factors for transcriptional events at the surface of PML bodies (Boisvert et al., 2000). The observation that highly acetylated chromatin can also be found associated with PML bodies is of particular interest (Boisvert et al., 2000), since PML bodies have been observed to contain the histone acetyltransferase CBP (LaMorte et al., 1998; Zhong et al., 2000b). Additional evidence for an association with transcriptionally active chromatin comes from the finding that PML bodies are often embedded in the SC35 enriched nuclear domains, where expressed genes are often localised (Ishov et al., 1997; Smith et al., 1999). The PML protein is also implicated in the regulation of certain genes, including the upregulation of genes involved in MHC class I processing (Zheng et al., 1998), and the inhibition of Sp1-mediated activation at the EGFR promoter (Vallian et al., 1998). Viral genomes also localise to PML bodies in order to transcribe their DNA, again indicating that PML bodies are transcription permissible domains (Ishov et al., 1997).
Given the accumulating evidence that PML regulates the expression of specific target genes, we set out to determine the 3D spatial relationship between PML bodies and gene dense/gene poor genomic regions in primary human fibroblast nuclei. We examined two gene-rich regions, namely, the major histocompatibility complex (MHC) on chromosome 6 and the epidermal differentiation complex (EDC) on chromosome 1; and the gene-poor 6p24 region on chromosome 6. We found that there is a significantly higher association between PML bodies and the MHC region in comparison to the other genomic regions examined. We have mapped the association locus to a region of at least 1.6 Mb that encompasses the centromeric end of the MHC. We also show that in a B-lymphoblastoid cell line, PML association occurs with a sub-region of the 1.6 Mb MHC locus when integrated into chromosome 18. These data show for the first time that PML bodies have specific genomic associations that are independent of transcription and support a model for PML bodies as functional domains.
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MATERIALS AND METHODS |
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Antibodies and DNA probes
The following antibodies were used in this study: Rabbit polyclonal anti-PML (Borden et al., 1995) to detect PML nuclear bodies, mouse anti-BrdUTP (Boehringer Mannheim) to detect BrdUTP incorporated into newly replicating DNA and mouse anti-p80 coilin to detect Cajal bodies (Almeida et al., 1998). Secondary antibodies used were anti-rabbit Alexa-488, anti-mouse Alexa-488, anti-mouse Alexa-546 (all from Molecular Probes) and anti-rabbit cy5 (Amersham Pharmacia). Genomic loci were detected using the following DNA probes: the MHC (LMP/TAP) region was detected using cosmids HA14, U15, U10 and M4 containing the LMP and TAP genes (Beck et al., 1996); the EDC region was detected using cosmids K0695, D01101, K1632, F0823, J1113 and F0969 containing the SPRR genes (South et al., 1999); a subsection of the 6p24 region was detected using cosmids A9.5, B5.10, B10.10, B12.10, E11.2, F1.6 (Davies et al., 1998; Olavesen et al., 1997); the DAXX region was detected using PACs 229D22, 36A2 and cosmids I0332, B2046, P0717 (Herberg et al., 1998a); the BAK region was detected using cosmid A094 (Herberg et al., 1998b); the TCP11 region was detected using cosmids 3N2, 3A2, 3B21 and 11H10 (Tripodis et al., 1998); the MHC class I region was detected using cosmids C0247, C0426, I1421 and A0622 adjacent to the classical class I gene HLA-A (Goldsworthy et al., 1996); the centromere of chromosome 6 was detected using a commercial biotinylated alpha satellite (Appligene Oncor); and band 18q11 was delineated using a partial chromosome paint specific for this chromosomal band (www.biologia.uniba.it/rmc).
Combined immunofluorescence and FISH (immuno-FISH)
For immuno-FISH of adherent cell lines, cells grown on chamber-slides were pre-extracted in CSK (0.1 M NaCl2, 0.3 M sucrose, 3 mM MgCl2, 10 mM Pipes, pH 6.8) (Carter et al., 1991) containing 0.5% Triton X-100 for 5 minutes on ice before fixation in 4% formaldehyde for 10 minutes. To facilitate detection of DNA sequences by FISH, we also performed additional nuclear permeabilisation steps consisting of repeated freeze-thaw in liquid nitrogen and treatment with 0.1 M HCl for 10 minutes (Kurz et al., 1996). Suspension cells were subjected to the same permeabilisation steps, but were not attached to slides by cytocentrifugation until after the fixation step in order to prevent distortion of the cells during centrifugation (Ferguson and Ward, 1992). These permeabilisation steps have previously been shown to be optimal for preservation of nuclear structure and detection of DNA by FISH (Carter et al., 1991; Kurz et al., 1996; Verschure et al., 1999). For visualisation of genomic loci by FISH, cells were denatured in 70% deionised formamide, 2x SSC at 72°C for 2 minutes and washed for 1 minute in cold 2x SSC before addition of denatured DNA probe. Hybridisation was at 37°C overnight, followed by washes at 42°C in 50% formamide, 2x SSC for 3x 5 minutes and in 2x SSC for 3x 5 minutes. Biotin-labelled DNA probes were detected using streptavidin Alexa-546 (Molecular Probes) and digoxigenin-labelled DNA probes were detected using anti digoxigenin FITC (Boehringer Mannheim). Detection of nuclear proteins by immunofluorescence was performed simultaneously with the FISH detection steps.
Microscopy and statistical analysis
Images were captured as optical sections using a Zeiss LSM 510 confocal laser scanning microscope equipped with a Plan Apo 63x/NA 1.4 objective. Sections were collected at 0.4 µm intervals through each nuclei and processed using the newly developed Image3D program. This produces 3D co-ordinates of centroid positions for PML bodies and each genomic locus as shown in Fig. 3. The Euclidean distance between each pair of fluorescent foci was calculated from their co-ordinates.
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RESULTS |
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MHC-PML association can occur throughout the cell cycle
From our studies it is clear that the MHC region does not associate with PML bodies in all nuclei examined (Fig. 2). This could suggest that the observed MHC-PML association is either a transient event, occurring during a certain stage of the cell cycle, or a dynamic event, occurring throughout the cell cycle. To investigate this further, fibroblasts at two different stages of the cell cycle were examined and the MHC-PML association at each stage compared. To differentiate between G1 and S-phase nuclei, bromodeoxyuridine (BrdU) was incorporated into unsynchronised mammalian fibroblasts, labelling only those cells in S-phase with actively replicating DNA. Immuno-FISH was used to visualise PML bodies, MHC regions and the newly replicating DNA simultaneously. Cells in G1 were identified by both the absence of BrdU incorporation and the presence of two MHC regions (Fig. 5E). Cells in S-phase were visualised by the presence of BrdU incorporation (Fig. 5F).
We find that the association of PML bodies with the MHC region is statistically significant at both stages of the cell cycle (t=4.97 in G1, t=6.04 in S-phase; Table 2). However, a comparison of MHC-PML associations between both cell populations (based on MHC) is complicated by the increased number of PML bodies in S-phase cells compared with G1 [also noted previously (Terris et al., 1995)], resulting in a significant decrease in
PML and
MHC distances in S-phase cells (Table 2). Instead, we compared the MHC-PML association in S and G1-phase cells based on the difference between
PML and
MHC distances within each cell population (
PML
MHC=0.46 µm in G1 compared with
PML
MHC=0.42 µm in S-phase; Table 2). We find that these distances are not significantly different (P=0.76) and conclude that the observed MHC-PML association, within the sample of fibroblasts studied, occurs throughout the cell cycle and that there is no significant difference in MHC-PML association between S and G1-phase cells.
Mapping the PML association along the short arm of chromosome 6
We next examined PML body association with other genomic regions along the short arm of chromosome 6 (Fig. 6). Adjacent regions were examined in combination with the LMP/TAP locus and their relative PML associations compared. The adjacent DAXX, BAK and TCP11 loci, (500 kb, 700 kb and 1600 kb centromeric of the LMP/TAP region respectively; see Fig. 1B), have remarkably similar mean PML distances compared to those for the LMP/TAP region (Table 1). Each of the genomic regions are as close to a PML body as is the LMP/TAP region, with the DAXX locus marginally closer (P=0.01; Table 1). In contrast, more distant genomic regions, such as the MHC class I region located
3 Mb telomeric of the LMP/TAP region, show an increase in their mean minimal genomic locus-PML distance (Fig. 6), and are not as closely associated with a PML body as is the LMP/TAP region (P=0.0002, Table 1). Similarly, genomic regions several tens of megabases distant of the MHC region such as the 6p24 region (as discussed previously) and the centromere region of chromosome 6, are not as closely associated with PML bodies when compared with the LMP/TAP region (Table 1; Fig. 6).
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DISCUSSION |
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For this study, we chose three genomic regions, the MHC, the EDC and the 6p24 region, on the basis that they differ in gene density and transcriptional activity in primary fibroblasts. We used a combination of immunofluorescence and FISH (immuno-FISH) to co-label PML bodies and specific chromosome regions, and devised a statistical method based on minimal distances to provide a quantitative basis for our analyses. The method we have developed treats fluorescent foci as single points in cartesian space and thereby overcomes any difficulties associated with differential or inefficient fluorescence of labelling fixed cells. By analysing these centroid co-ordinates, we were able to directly compare the distances between specific genomic regions and PML bodies and carry out a number of statistical tests.
From this statistical analysis, we find that the MHC gene cluster is more strongly associated with PML bodies than is the EDC region on chromosome 1 or the transcriptionally silent region 6p24. This finding is of particular interest, since it has previously been shown that PML can upregulate the transcription of the MHC class II TAP and LMP genes (Zheng et al., 1998). One possible explanation for our observations could be the position of gene-rich regions (compared with gene-poor regions) within chromosomal territories. Both individual genes and gene-rich regions have been shown to be preferentially localised at the periphery of chromosomal territories (Kurz et al., 1996; Volpi et al., 2000), similarly, PML bodies tend to be positioned either at the border of (or are excluded from) chromosomal territories (Bridger et al., 1998). Thus PML bodies localised to the inter-chromosomal space are by inference in close proximity to gene-coding DNA. However, this does not explain the clear preferential association of the MHC region for PML bodies both in normal primary fibroblasts and in B-cells where MHC class II sequences have been artificially integrated into chromosome 18. These latter data show that the observed MHC-PML associations appear sequence specific and can be mediated by particular genomic sequences, irrespective of their chromosomal location. Theoretical matrix attachment region (MAR) sequences (van Drunen et al., 1999) have recently been identified in the MHC class II region and have been shown to bind to the nuclear matrix (R. Horton and R. Donev, personal communication). Since PML bodies are also tightly associated with the nuclear matrix (Stuurman et al., 1992), it is possible that the MHC region may associate with PML bodies through its MAR sequences. The association of genes and transcription factors with the nuclear matrix has been suggested to lead to the formation of nuclear domains that facilitate transcriptional control (Stein et al., 1998). Our data now provide support to the intriguing possibility that PML bodies associate with certain gene-rich clusters as part of a functional compartment involved in gene regulation. We are currently investigating the minimum sequence requirements for PML body association.
Previous studies have suggested that PML can upregulate the transcription of the MHC class II TAP and LMP genes (Zheng et al., 1998). One could speculate that any MHC-PML body association is related to these previous observations, where PML protein is required to actively promote transcription of specific MHC genes (via unknown mechanisms). However, some doubt has arisen about the role of PML in regulating LMP and TAP transcription. APL cells that lack functional PML express normal levels of MHC class I molecules (Larghero et al., 1999), as do PML/ cells derived from knockout mice (Ruggero et al., 2000). Our studies also show that the observed MHC-PML association is not directly dependent upon the presence of LMP and TAP sequences, since the association is maintained even when a substantial portion of the class II region containing these genes is deleted. Interestingly, in the primary fibroblasts studied, the number of MHC genes that are transcriptionally active is considerably higher compared with the EDC region. So it is possible that the transcriptional activity of a particular genomic locus influences the frequency of PML body association. Our experiments using positive and negative regulators of transcription would argue against this, as changes in transcriptional status did not significantly alter the observed MHC-PML association. We note that both IFN and global transcriptional inhibitors have pleiotropic effects and do not specifically target MHC genes. Also, it is well established that PML is upregulated by IFN
(Lavau et al., 1995), but we see little change in the MHC-PML body association after IFN
treatment. This suggests that the observed PML body associations appear independent of the concentration of PML protein. The static nature of both chromatin (Shelby et al., 1996) and PML bodies (Plehn-Dujowich et al., 2000) would suggest that once associations have been formed, they are maintained irrespective of the status of the cell. This is further highlighted by our results during different stages of the cell cycle, where little change in MHC-PML association is observed.
What is the functional significance of a MHC-PML body association? If PML bodies play a role in the transcriptional regulation of the MHC, it is difficult to explain why other functionally unrelated proteins, with no obvious involvement in transcription, localise to PML bodies (Borden et al., 1998). Although there may be functional heterogeneity between different PML bodies, another possibility could be that PML bodies coordinate and regulate the concentrations of active proteins (Maul, 1998). Evidence for this includes the observation that misfolded viral proteins, ubiquitin and proteosomal components can localise to PML bodies, suggesting that PML bodies serve as sites for protein degradation (Anton et al., 1999). Of particular interest are recent observations that transcriptional activators can be targeted in vivo for rapid ubiquitin-mediated degradation at sites of active transcription (Molinari et al., 1999; Salghetti et al., 2000). Combining these observations with our own data leads to the following plausible hypothesis. PML bodies associate with certain gene-rich genomic regions and in particular the MHC, to form part of a functional compartment involved in regulating the transcription of that particular region. We note that PML bodies are often associated with other nuclear domains including Cajal bodies (Grande et al., 1996) supporting the notion of discrete functional compartments comprising several multi-protein complexes or bodies juxtaposed to specific chromosomal regions. Specific proteins including transcription factors, co-activators and co-repressors could be targeted for degradation at PML bodies via the proteasome-mediated pathway or stored for subsequent use via the SUMO-1 modification pathway. The observed specific MHC-PML association could thus be necessary for transcriptional regulation of certain genes within the MHC region. The demonstration that PML interacts with the transcription factor Sp1, inhibiting its transactivation properties (Vallian et al., 1998), and the fact that Sp1 is involved in the constitutive expression of a number of genes within the MHC region, including LMP and TAP (Wright et al., 1995), would support this theory.
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ACKNOWLEDGMENTS |
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