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• W2024678090 abstract "Promyelocytic leukemia (PML) protein is implicated in transcriptional regulation, apoptosis, DNA repair, and tumor suppression. It is not known, however, whether PML and other components of PML bodies function within the vicinity of the bodies or elsewhere in the nucleoplasm. In this study, we demonstrate that chromatin organization around PML bodies influences their morphology, dynamics, and structural integrity by a SUMO-1-independent mechanism. Following transcriptional inhibition and during early apoptosis, chromatin retracts from the periphery of PML bodies, coinciding with the formation of new PML-containing structures through fission of supramolecular PML-containing microbodies. Both fission and fusion of microbodies with parental PML bodies indicate a loss of structural integrity of the bodies, dependent on the state of the surrounding chromatin. This is supported by the observation that treatment of live cells with DNase I could reproduce the structural instability of PML bodies. In addition, PML bodies, which are normally surrounded by chromatin and are positionally stable, become more dynamic following these treatments, presumably due to the loss of chromatin contacts. Overexpression of SUMO-1, a modification required for PML body formation, did not prevent PML body fission, indicating that chromatin-based integrity of PML body structure occurs through a SUMO-1-independent mechanism. Promyelocytic leukemia (PML) protein is implicated in transcriptional regulation, apoptosis, DNA repair, and tumor suppression. It is not known, however, whether PML and other components of PML bodies function within the vicinity of the bodies or elsewhere in the nucleoplasm. In this study, we demonstrate that chromatin organization around PML bodies influences their morphology, dynamics, and structural integrity by a SUMO-1-independent mechanism. Following transcriptional inhibition and during early apoptosis, chromatin retracts from the periphery of PML bodies, coinciding with the formation of new PML-containing structures through fission of supramolecular PML-containing microbodies. Both fission and fusion of microbodies with parental PML bodies indicate a loss of structural integrity of the bodies, dependent on the state of the surrounding chromatin. This is supported by the observation that treatment of live cells with DNase I could reproduce the structural instability of PML bodies. In addition, PML bodies, which are normally surrounded by chromatin and are positionally stable, become more dynamic following these treatments, presumably due to the loss of chromatin contacts. Overexpression of SUMO-1, a modification required for PML body formation, did not prevent PML body fission, indicating that chromatin-based integrity of PML body structure occurs through a SUMO-1-independent mechanism. The promyelocytic leukemia (PML) 1The abbreviations used are: PML, promyelocytic leukemia; RARα, retinoic acid receptor α; ESI, electron spectroscopic imaging; DMEM, Dulbecco’s modified Eagle’s medium; ActD, actinomycin D; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; CREB, cAMP-response element-binding protein; TUNEL, terminal dUTP nick-end labeling.1The abbreviations used are: PML, promyelocytic leukemia; RARα, retinoic acid receptor α; ESI, electron spectroscopic imaging; DMEM, Dulbecco’s modified Eagle’s medium; ActD, actinomycin D; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; CREB, cAMP-response element-binding protein; TUNEL, terminal dUTP nick-end labeling. protein was first identified as the fusion partner of the retinoic acid receptor α (RARα) in patients suffering from acute promyelocytic leukemia (1Melnick A. Licht J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar). In cells expressing the PML-RARα fusion, PML nuclear bodies, which appear as 5-20 discrete foci in normal cells, are redistributed into many punctate particles throughout the nucleoplasm. Remission of acute promyelocytic leukemia is achieved by either retinoic acid or arsenic trioxide treatment, which restores both the integrity of PML bodies and regulated cell growth (1Melnick A. Licht J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar). Furthermore, PML knock-out mice are susceptible to tumor-promoting agents and exhibit chromosome instability, firmly implicating PML in tumor suppression (2Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (97) Google Scholar, 3Wang Z.G. Rivi R. Delva L. Konig A. Scheinberg D.A. Gambacorti-Passerini C. Gabrilove J.L. Warrell Jr., R.P. Pandolfi P.P. Blood. 1998; 92: 1497-1504Crossref PubMed Google Scholar). Heat shock, heavy metal exposure, or viral infection can also affect the integrity of PML bodies (4Maul G.G. Yu E. Ishov A.M. Epstein A.L. J. Cell. Biochem. 1995; 59: 498-513Crossref PubMed Scopus (131) Google Scholar, 5Ishov A.M. Sotnikov A.G. Negorev D. Vladimirova O.V. Neff N. Kamitani T. Yeh E.T. Strauss III, J.F. Maul G.G. J. Cell Biol. 1999; 147: 221-234Crossref PubMed Scopus (671) Google Scholar, 6Nefkens I. Negorev D.G. Ishov A.M. Michaelson J.S. Yeh E.T. Tanguay R.M. Muller W.E. Maul G.G. J. Cell Sci. 2003; 116: 513-524Crossref PubMed Scopus (65) Google Scholar, 7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar). Thus, the integrity of PML bodies, even from their first description, is linked to the physiological state of the cell. Several models have been proposed for the function of PML bodies. In one model, PML bodies are thought to consist of aggregates of excess nuclear protein, thus having no function other than storage of proteins awaiting degradation. In a second model, PML bodies act as storage sites, modulating concentrations of nuclear proteins by sequestering them at PML bodies until required (8Negorev D. Maul G.G. Oncogene. 2000; 20: 7234-7242Crossref Scopus (232) Google Scholar). In an extension of this model, PML bodies may be sites of post-translational modification of PML body components. None of these models predict that the position and relative size of the bodies are important. We have observed, however, that position and size of bodies observed before stress are conserved following recovery from the stress event (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar). The above models also do not provide a basis for the positional stability of PML bodies. We propose another model, which does not exclude the previous models, but which implicates PML bodies in the regulation of nuclear functions through direct contacts with chromatin. Indirect support for this model includes the observation that chromatin-modifying proteins such as acetyltransferases (e.g. CREB-binding protein) (9Doucas V. Tini M. Egan D.A. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2627-2632Crossref PubMed Scopus (154) Google Scholar, 10von Mikecz A. Zhang S. Montminy M. Tan E.M. Hemmerich P. J. Cell Biol. 2000; 150: 265-273Crossref PubMed Scopus (109) Google Scholar, 11Boisvert F.M. Kruhlak M.J. Box A.K. Hendzel M.J. Bazett-Jones D.P. J. Cell Biol. 2001; 152: 1099-1106Crossref PubMed Scopus (128) Google Scholar) and deacetylases (e.g. type I histone deacetylases) (12Wu W.S. Vallian S. Seto E. Yang W.M. Edmondson D. Roth S. Chang K.S. Mol. Cell. Biol. 2001; 7: 2259-2268Crossref Scopus (128) Google Scholar) accumulate at PML bodies, implying a role in chromatin structure regulation. Furthermore, PML bodies may control the local concentration of HP1α in the vicinity of centromeres through regulation of HP1α shuttling to heterochromatin via Daxx (5Ishov A.M. Sotnikov A.G. Negorev D. Vladimirova O.V. Neff N. Kamitani T. Yeh E.T. Strauss III, J.F. Maul G.G. J. Cell Biol. 1999; 147: 221-234Crossref PubMed Scopus (671) Google Scholar, 13Seller J.S. Marchio A. Sitterlin D. Transy C. Dejean A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7316-7321Crossref PubMed Scopus (227) Google Scholar, 14Seeler J.S. Dejean A. Oncogene. 2001; 20: 7243-7249Crossref PubMed Scopus (135) Google Scholar, 15Hayakawa T. Haraguchi T. Masumoto H. Hiraoka Y. J. Cell Sci. 2003; 116: 3327-3338Crossref PubMed Scopus (113) Google Scholar). PML bodies play a direct role in transcriptional regulation through the recruitment of p53, the acetyltransferase CREB-binding protein, HIPK2 (16Kim Y.H. Choi C.Y. Kim Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12350-12355Crossref PubMed Scopus (142) Google Scholar) and MDM2 (17Louria-Hayon I. Grossman T. Sionov R.V. Alsheich O. Pandolfi P.P. Haupt Y. J. Biol. Chem. 2003; 278: 33134-33141Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The activated p53 may then leave the bodies and diffuse throughout the nucleoplasm, where it interacts with p53-activated genes. Alternatively, p53 may act on genes that are specifically localized in the immediate environment of a PML body. Similarly, transcriptional repressors such as Sp100 and Daxx (13Seller J.S. Marchio A. Sitterlin D. Transy C. Dejean A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7316-7321Crossref PubMed Scopus (227) Google Scholar, 18Li H. Leo C. Zhu J. Wu X. O'Neil J. Park E.J. Chen J.D. Mol. Cell. Biol. 2000; 20: 1784-1796Crossref PubMed Scopus (305) Google Scholar, 19Lehming N. Le Saux A. Schuller J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7322-7326Crossref PubMed Scopus (149) Google Scholar) may also serve genes in the immediate vicinity of PML bodies. Transcriptional activity on the surface of PML bodies is supported by observations of the accumulation of nascent transcripts on the surface of PML bodies (20Boisvert F.M. Hendzel M.J. Bazett-Jones D.P. J. Cell Biol. 2000; 148: 283-292Crossref PubMed Scopus (220) Google Scholar) and the association of specific gene loci with PML bodies (e.g. major histocompatibility complex class I gene family (21Shiels C. Islam S.A. Vatcheva R. Sasieni P. Sternberg M.J. Freemont P.S. Sheer D. J. Cell Sci. 2001; 114: 3705-3716Crossref PubMed Google Scholar) and TP53 gene locus (22Sun Y. Durrin L.K. Krontiris T.G. Genomics. 2003; 82: 250-252Crossref PubMed Scopus (29) Google Scholar)). Transcription on or near the surface of PML bodies, the localization of chromatin modifying proteins and transcription factors to PML bodies, and the association of specific gene loci with these structures strongly support a critical role for the association of chromatin with PML nuclear bodies in the maintenance of normal nuclear function. PML body formation requires specific domains and post-translational modifications of PML protein. One of these domains is an N-terminal RING finger, which is required for protein-protein interactions. This RING finger is followed by two B boxes and, together with a central coiled-coil domain, constitutes the RBCC (RING/Bbox/coiled-coil) region found within all PML protein isoforms (23Jensen K. Shiels C. Freemont P. Oncogene. 2001; 20: 7223-7233Crossref PubMed Scopus (375) Google Scholar). The function of the RBCC domain has been postulated to be involved with PML homodimerization/oligomerization as well as protein-protein interactions with other PML body-associated proteins. SUMO-1 (small ubiqutin-like modifier-1) modification of the PML protein occurs at lysines 65, 160, and 490. However, Lys160 appears to be the only modification site required for the formation of PML bodies. Expression of mutant PML proteins that have an amino acid substitution for Lys160 are targeted to the nucleus but are unable to form PML bodies (24Boddy M.N. Howe K. Etkin L.D. Solomon E. Freemont P.S. Oncogene. 1995; 13: 971-982Google Scholar, 25Sternsdorf T. Jensen K. Will H. J. Cell Biol. 1997; 139: 1621-1634Crossref PubMed Scopus (289) Google Scholar). Further insights into the mechanisms of PML body assembly and disassembly have also been derived from the live cell analysis of these subnuclear domains during cellular stress. During heat shock for example, PML bodies dissociate into small microstructures, which lack normal PML body components, such as Sp100 and SUMO-1 (4Maul G.G. Yu E. Ishov A.M. Epstein A.L. J. Cell. Biochem. 1995; 59: 498-513Crossref PubMed Scopus (131) Google Scholar, 6Nefkens I. Negorev D.G. Ishov A.M. Michaelson J.S. Yeh E.T. Tanguay R.M. Muller W.E. Maul G.G. J. Cell Sci. 2003; 116: 513-524Crossref PubMed Scopus (65) Google Scholar, 7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar). These small structures bleb or bud from the surface of the PML bodies and become mobile (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar). The remaining PML structures, or PML body remnants, are positionally stable for several hours, as are PML bodies in unstressed interphase nuclei (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar, 26Wiesmeijer K. Molenaar C. Bekeer I.M. Tanke H.J. Dirks R.W. J. Struct. Biol. 2002; 140: 180-188Crossref PubMed Scopus (51) Google Scholar). Upon recovery from stress, microstructures begin to reacquire SUMO-1 and Sp100 and can be observed to fuse with large immobile PML body remnants (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar). The hypothesis that SUMO-1 is essential to the integrity of PML bodies was also supported by results demonstrating that PML bodies, in cells overexpressing SUMO-1, are resistant to the microstructure formation in heat-stressed nuclei (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar). In this study, we demonstrate that the structural integrity of PML bodies not only relies on the post-translation modification of PML by SUMO-1 but also relies on the integrity and the state of chromatin condensation. Using correlative fluorescence microscopy and electron spectroscopic imaging (ESI), we demonstrate that the integrity of PML bodies is related to the degree of association of chromatin with the protein-based cores of PML bodies. In untreated cells, PML bodies are surrounded by chromatin, which explains their positional stability. Direct physical contacts between the protein core and chromatin fibers are prevalent. Inhibition of transcription causes a reduction in these contacts through the condensation of chromatin, correlating with the fission of new PML-containing microbodies from the parental bodies. This also occurs at the early stages of apoptosis, coinciding with the cleavage of euchromatin and associated chromatin condensation (27Hendzel M.J. Nishioka W.K. Raymond Y. Allis C.D. Bazett-Jones D.P. Th'ng J.P. J. Biol. Chem. 1998; 273: 24470-24478Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). These newly formed microbodies dissociate from existing PML bodies and are free to diffuse through the nucleoplasm and fuse with each other or with the parental PML bodies. Finally, we confirm that PML bodies break down in response to changes in chromatin integrity by introducing exogenous nuclease (DNase I) into cells. Immunofluorescence microscopy indicates that microbodies and the PML body remnants that are generated coincidentally with these chromatin changes are biochemically indistinguishable from PML bodies in untreated cells. Further, the formation of these microbodies is not affected by SUMO-1 overexpression. We conclude that chromatin integrity and condensation state alone can influence PML body morphology and stability. Cell Lines—SK-N-SH cells were grown in DMEM plus 10% fetal bovine serum to the desired degree of confluence. U2OS cells stably expressing GFP-PML-IV (a gift from Dr. J. Taylor, previously described in Ref. 7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar) were cultured in DMEM plus 10% fetal bovine serum containing 1600 μg/ml G418 (Wisent). Transcriptional inhibition of SK-N-SH cells was accomplished by treatment with either 5 μg/ml actinomycin D (Sigma), 80 μg/ml 5,6-dichlorobenzimidizole riboside (Sigma) or 100 μg/ml α-amanitin (Sigma) at 37 °C for 2 h. Apoptosis was induced in U2OS cells expressing GFP-PML-IV or SK-N-SH cells in DMEM containing 2 μm staurosporine (Sigma) at 37 °C. Saponin/DNase I Treatments and DNase I Transfection—U2OS or SK-N-SH cells grown on coverslips were treated on ice with 0.1% saponin/phosphate-buffered saline containing 250 μg/ml of DNase I for 5 min. Coverslips were then washed 3 times for 1 min each in ice-cold phosphate-buffered saline containing identical concentrations of DNase I. Coverslips were then incubated at room temperature in RPMI 1640 containing DNase I. For protein transfection of DNase I, either two (12 μl) or three (18 μl) reactions of Chariot™ protein transfection agent (Active Motif) were mixed with 30 μl of DNase I (5 mg/ml). The Chariot™ agent was allowed to couple with the DNase I for 30 min at room temperature. The mixture was placed on coverslips with serum-free media and incubated at 37 °C. After 1 h, DMEM plus 10% fetal bovine serum was added. At the specified time points of individual experiments, coverslips were fixed in 1% paraformaldehyde for 5 min at room temperature and permeabilized with phosphate-buffered saline-Triton X-100 (0.5%) for 10 min. For live cell experiments, after the appropriate incubation periods in DMEM plus 10% fetal bovine serum, coverslips were placed on an environmental chamber containing media and imaged. All fluorescence microscopy was performed using a Leica DMR upright microscope fitted with a Hamamatsu ORCA camera. Openlab version 3.1.3 (Improvision) software was used to collect fluorescence images. Images were processed using either ImageJ version 1.28 (National Institutes of Health) or Photoshop 6.0/7.0 (Adobe). TUNEL Assay and Cell Labeling—After fixation and permeabilization, TUNEL assay labeling was preformed on coverslips. 1 μl (20 units) of terminal transferase (Roche Applied Science) was mixed with 10 μl of 5× reaction buffer, 1.5 μl of CoCl2 (1 mm), 0.3 μl of Cy3-dCTP (1 mm) (Amersham Biosciences), 0.33 μl of dCTP (0.1 mm), and 36.9 μl water for each coverslip to be analyzed. The TUNEL reaction mix was placed on the coverslips and incubated in a humidity chamber at 37 °C for 1 h. The reaction was terminated by washing the coverslips in 300 mm NaCl for 15 min at room temperature. PML protein was labeled by using either 5E10 antibody (1:10) (a gift from Dr. R. Van Driel) or rabbit α-PML antibody (1:1000) (Chemicon). SUMO-1 and Sp100 protein were labeled using mouse α-GMP-1 (1:500) (Zymed Laboratories Inc.) and rabbit α-Sp100 (1:1000) (Chemicon), respectively. HSP70 levels were monitored by labeling with mouse α-HSP70 (1:500) (Stressgen). ESI—After labeling, cells were fixed (2% gluteraldehyde for 5 min at room temperature) and then dehydrated in a series of graded ethanol washes (30, 50, 70, and 95%). Coverslips were then infiltrated with Quetol 651 resin (EM Science) for 2 h, and then 2× 2-h Quetol mix with 2% DMP-30 (hardener), as previously described (20Boisvert F.M. Hendzel M.J. Bazett-Jones D.P. J. Cell Biol. 2000; 148: 283-292Crossref PubMed Scopus (220) Google Scholar). Resin containing coverslips were cured overnight at 70 °C. Blocks of resin containing cells were sectioned (Leica ultramicrotome) to 70 nm, and the sections were collected on copper grids (EM Sciences). Images were collected using a Tecnai 20 TEM (FEI) equipped with an energy-filtering spectrometer (Gatan) (28Bazett-Jones D.P. Hendzel M.J. Kruhlak M.J. Methods Companion Methods Enzymol. 1999; 17: 188-200Crossref Scopus (52) Google Scholar). Nitrogen and phosphorus maps are used to distinguish protein-based from nucleic acid-based structures, without the need for exogenous stains or labels (28Bazett-Jones D.P. Hendzel M.J. Kruhlak M.J. Methods Companion Methods Enzymol. 1999; 17: 188-200Crossref Scopus (52) Google Scholar). The precise locations of PML bodies in images were determined by correlative fluorescence microscopy and ESI (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar, 20Boisvert F.M. Hendzel M.J. Bazett-Jones D.P. J. Cell Biol. 2000; 148: 283-292Crossref PubMed Scopus (220) Google Scholar, 29Ren Y. Kruhlak M.J. Bazett-Jones D.P. J. Histochem. Cytochem. 2003; 51: 605-612Crossref PubMed Scopus (26) Google Scholar). PML Bodies Make Chromatin Contacts in Both Unstressed and Stressed Nuclei—We wished to determine whether PML bodies were free to move through the nucleoplasm or whether their mobility is restricted by a nuclear component. We followed the dynamic behavior of PML bodies in live U2OS cells stably expressing GFP-PML-IV. We observed that PML bodies retain their relative positions within the nucleus over time periods of a few hours (Fig. 1A), with the exception of minor movements due to cellular shape changes. The relative sizes of the PML bodies in a given cell are conserved over several hours. In some cells, new PML bodies appear during the course of imaging, consistent with reports that PML bodies increase in size and number during interphase (29Ren Y. Kruhlak M.J. Bazett-Jones D.P. J. Histochem. Cytochem. 2003; 51: 605-612Crossref PubMed Scopus (26) Google Scholar). These new structures also appear to maintain their positional stability within the nucleus. The positional stability of PML bodies could be explained by extensive contacts with chromatin or by attachment to an underlying protein-based structure or nuclear matrix (30Chang K.S. Fan Y.H. Andreeff M. Liu J. Mu Z.M. Blood. 1995; 85: 3646-3653Crossref PubMed Google Scholar). To determine whether we could observe such a structural basis for PML body positional stability, we used correlative fluorescence microscopy and ESI (31Drouin A. Schmitt A. Masse J.M. Cieutat A.M. Fichelson S. Cramer E.M. Exp. Cell Res. 2001; 271: 277-285Crossref PubMed Scopus (4) Google Scholar). ESI offers the ability to distinguish between protein-, DNA-, or RNA-based structures in the nucleus (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar, 20Boisvert F.M. Hendzel M.J. Bazett-Jones D.P. J. Cell Biol. 2000; 148: 283-292Crossref PubMed Scopus (220) Google Scholar). We observed that the protein-based cores of PML bodies are completely surrounded by and make physical contacts with chromatin fibers, predominantly 30-nm fibers. Nitrogen maps (green) and phosphorus maps (red) permit us to identify chromatin, ribonucleoprotein structures (RNPs), and protein-based structures (Fig. 1B). In merged nitrogen and phosphorus maps, chromatin appears as bright yellow fibers. RNPs appear less yellow (circled RNP in Fig. 1B), due to a lower P/N ratio than chromatin. RNPs, exhibiting a distinct morphology from chromatin, consisting of a more punctuate distribution of granules between 5 and 10 nm in diameter. Protein-based structures appear as green in the merged nitrogen and phosphorus maps. The high density of chromatin (arrowheads) around the bodies provides a physical barrier that may account for their positional stability. The protein cores demonstrate radial symmetry, and images from serial sections indicate that the cores of the bodies are spherical in shape (not shown). In addition, protein-based “spokes” are frequently observed, which make physical contacts with both the cores of the bodies and the chromatin on the periphery. A final feature that is typical of PML bodies is the accumulation of RNPs near their periphery (Fig. 1B, circled). To test whether the association of PML bodies with chromatin and RNPs may have functional consequences, we examined nuclei at the ultrastructural level from cells that had been heat-stressed. Under such conditions, most transcription is repressed, with the exception of genes involved with the heat shock response (32Morrimoto R.I. Science. 1993; 259: 1409-1410Crossref PubMed Scopus (1205) Google Scholar). We observed several major differences between PML bodies in stressed cells compared with control cells. First, the protein-based cores of over 80% of PML bodies lose their typical radial symmetry (Fig. 1C, circled structures). Instead, the protein cores display extended length/width ratios, confirmed in serial sections (not shown). In addition, the protein-based cores are less dense, displaying a more open and fibrous appearance (Fig. 1, compare the cores in B and C). In heat-stressed cells, it is difficult to delineate the edges of the PML accumulations from other protein-based fibers and globular depositions that are present both in the vicinity of PML bodies and throughout the nucleoplasm (within versus outside the circled regions in Fig. 1C). A second difference we observed relates to the organization of chromatin in heat-stressed nuclei. Large blocks of condensed chromatin were observed throughout the nucleoplasm (data not shown), supporting a previous study in which heat stress led to an increase in chromatin condensation (33Plehn-Dujowich D. Bell P. Ishov A.M. Baumann C. Maul G.G. Chromosoma. 2000; 109: 266-279Crossref PubMed Scopus (26) Google Scholar). In the vicinity of PML bodies, however, the chromatin is less densely distributed (Fig. 1, C and D). Instead of dense packing of 30-nm fibers, 10-nm fibers are observed, separated by large spaces. These chromatin fibers frequently make physical connections to the fibrous protein cores of PML bodies (arrowheads in Fig. 1C). The 10-nm nature of these fibers is illustrated in Fig. 1, D and E. Nucleosome-based fibers are illustrated in the higher magnification insets (i and ii). The basis for defining these structures as nucleosomes is their morphology (11-nm diameter), some displaying a doughnut-shaped appearance in phosphorus-enhanced images (consistent with en face projections) and their nitrogen and phosphorus content (34Bazett-Jones D.P. Hendzel M.J. Kruhlak M.J. Micron. 1999; 30: 151-157Crossref PubMed Scopus (32) Google Scholar). Finally, in heat-stressed cells, we never observe clearly identifiable accumulations of RNPs on the periphery of PML bodies. This is consistent with the loss of the 5′-fluorouridine incorporation on the periphery of PML bodies (data not shown), which is observed in nonstressed cells (20Boisvert F.M. Hendzel M.J. Bazett-Jones D.P. J. Cell Biol. 2000; 148: 283-292Crossref PubMed Scopus (220) Google Scholar). Chromatin Condensation by Transcriptional Inhibition or Apoptosis Causes a Loss in PML Body Integrity—Since major structural and compositional features of PML bodies correlate with transcriptional repression arising from heat stress, we decided to test for changes in PML body structure and composition by inhibiting transcription directly. SK-N-SH cells were incubated with 5 μg/ml actinomycin D (ActD), fixed, and labeled for PML (Fig. 2A). Using DAPI as a marker for chromatin, we observed a global reorganization of chromatin in the nuclei of ActD-treated cells (+ActD), which was characterized by a less uniform distribution of DAPI signal compared with control cells (-ActD). This visual impression was supported by line scans of DAPI-stained cells (Fig. 2A, inset). We also observed the appearance of numerous, small PML-containing structures following transcriptional inhibition. In the example shown, the number of PML-containing structures in untreated cells is less than 10 per cell (cell i and cell ii), whereas the number in treated cells is over 30 (cell iii and cell iv). All of the PML-containing structures contain two other PML body-associated components, Sp100 and SUMO-1, at levels indistinguishable from PML bodies in control cells (Fig. 2B). We chose these two biochemical markers, since they are labile markers following heat stress (7Eskiw C.H. Dellaire G. Mymryk J. Bazett-Jones D.P. J. Cell Sci. 2003; 116: 4455-4466Crossref PubMed Scopus (111) Google Scholar) and are lost in early stages of mitosis (preliminary data). We refer to these newly formed PML containing structures as “microbodies,” since they contain these characteristic markers for PML bodies. Similar results were obtained with other transcriptional inhibitors (α-amanitin and 5,6-dichlorobenzimidizole riboside) (data not shown). Since both heat stress and transcription inhibition with drugs lead to major changes in chromatin organization in the immediate vicinity of PML bodies and the organization of PML-containing structures, we wished to address whether the loss of euchromatin in the early stages of apoptosis by endogenous nucleases (27Hendzel M.J. Nishioka W.K. Raymond Y. Allis C.D. Bazett-Jones D.P. Th'ng J.P. J. Biol. Chem. 1998; 273: 24470-24478Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 35Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2785) Google Scholar, 36Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1409) Google Scholar) would also lead to the disruption of PML bodies. Cells expressing GFP-PML-IV were treated with the apoptotic inducer, staurosporine, and observed by fluorescence microscopy over time (Fig. 3A). Within 30 min of staurosporine treatment, new PML-containing structures begin to appear. These small PML accumulations contain significant levels of Sp100 and SUMO-1 (Supplemental Fig. 1), similar to the structures that form during transcriptional inhibition (Fig. 2A). We observed that these new structures are highly mobile, moving several hundred nanometers during 1-s intervals (Supplemental Movies 1-5). Furthermore, the large parental PML bodies present prior" @default.
• W2024678090 created "2016-06-24" @default.
• W2024678090 creator A5022101184 @default.
• W2024678090 creator A5078364744 @default.
• W2024678090 creator A5089464980 @default.
• W2024678090 date "2004-03-01" @default.
• W2024678090 modified "2023-10-09" @default.
• W2024678090 title "Chromatin Contributes to Structural Integrity of Promyelocytic Leukemia Bodies through a SUMO-1-independent Mechanism" @default.
• W2024678090 cites W1492302205 @default.
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