Journal of Cell Science 112, 4581-4588 (1999) 4581 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0794 Cell cycle regulation of PML modification and ND10 composition Roger D. Everett1,*, Patrick Lomonte1, Thomas Sternsdorf2, Roel van Driel3 and Anne Orr1 1MRC Virology Unit, Church Street, Glasgow G11 5JR, Scotland, UK 2Heinrich-Pette-Institut, Martinistrasse 52, 20251 Hamburg, Germany 3E.C. Slater Institute, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands *Author for correspondence (e-mail: [email protected]) Accepted 2 October; published on WWW 30 November 1999 SUMMARY The nuclear sub-structures known as ND10, PODs or PML differential modification of PML during the cell cycle. PML nuclear bodies can be rapidly modified by diverse stimuli, and Sp100 normally tightly co-localise in ND10 in and the resultant structural changes correlate with events interphase cells, but they become separated during mitosis. such as cellular transformation and successful virus Interphase cells treated with phosphatase inhibitors or infection. We show that the ND10 components PML and subjected to heat shock also show structural changes in Sp100 undergo profound biochemical changes during the ND10, accompanied by alterations to the normal pattern of cell cycle. Both proteins are conjugated to the ubiquitin- PML modification. Taken with previous findings on the like protein SUMO-1 during interphase, but they become effects of infection by herpes simplex virus and adenovirus de-conjugated during mitosis and an isoform of PML of on ND10 structure and PML modification, these results distinct electrophoretic mobility appears. This mitosis- suggest that the many factors which have been shown to specific form of PML is highly labile in vitro, but is partially modify ND10 structure may do so by interaction with the stabilised by phosphatase inhibitors. Treatment of biochemical mechanisms that act on ND10 components interphase cells with phosphatase inhibitors induces the during the cell cycle. production of a PML isoform of similar gel mobility to the mitosis-specific species, and taken together these results suggest that phosphorylation is an important factor in the Key words: ND10, PML, Cell cycle INTRODUCTION 1999). Furthermore, immunofluorescence experiments showed that PML and Sp100 become separated during mitosis and the ND10, otherwise known as nuclear dots (NDs), PML nuclear remaining PML foci are not co-stained with an anti-SUMO-1 bodies or promyelocytic oncogenic domains (PODs) are reagent (Sternsdorf et al., 1997a,b). The implication was that punctate structures with a diameter of about 0.5 µm. There are the SUMO-1 modification status of PML and Sp100 varies usually between 5 and 20 ND10 per nucleus, each containing during the cell cycle, but this was not verified directly. several proteins of which the most studied are PML and Sp100 In this paper we report a systematic investigation of the (reviewed by Sternsdorf et al., 1997a). Interest in PML arose intracellular distribution and biochemical characteristics of because most cases of promyelocytic leukaemia are caused by endogenous PML and Sp100 through the cell cycle. We found a chromosomal translocation which results in the expression of that interphase SUMO-1-conjugated isoforms of both PML a fusion protein linking the normal PML protein to the retinoic and Sp100 were completely eliminated during mitosis and that acid receptor α (for references, see Sternsdorf et al., 1997a). a novel modified PML species appears. The mitosis-specific Both PML and Sp100 have been associated with the repression PML isoform was highly labile in vitro, but it was stabilised of gene expression (Mu et al., 1994; Alcalay et al., 1998; by inhibition of phosphatases. Treatment of interphase cells Vallian et al., 1998; Lehming et al., 1998; Seeler et al., 1998), with heat shock and the phosphatase inhibitor calyculin A also while PML has recently been implicated in apoptotic pathways caused loss of SUMO-1 modified PML isoforms and the (Quignon et al., 1998). The status of ND10 can be modulated appearance of PML species with a distinct gel mobility. We by a number of factors, including stress, virus infection, suggest that the disruption of ND10 that occurs at the interferon treatment and cell cycle progression (reviewed by beginning of mitosis is a consequence of deconjugation of Sternsdorf et al., 1997a). Both PML and Sp100 are covalently SUMO-1 from both PML and Sp100, and increased modified by the small ubiquitin-like protein SUMO-1, and it phosphorylation of PML. Reversal of this process leads to re- has been suggested that this modification is an important factor establishment of ND10 in the early stages of G1. These for the localisation of PML at ND10 (Sternsdorf et al., 1997b; biochemical changes provide a model by which ND10 Kamitani et al., 1998a,b; Muller et al., 1998; Duprez et al., structure and composition could be changed rapidly by diverse 4582 R. D. Everett and others stimuli through modulation of the PML and Sp100 staining. Instead, the Sp100 foci were often separated from modification pathways which operate during cell division. PML or they were surrounded by diffuse clouds of PML, giving the impression of release of PML from ND10 into the surrounding nucleoplasm (best seen in Fig. 1C,D and inset). MATERIALS AND METHODS After breakdown of the nuclear envelope, mitotic cells contained irregular accumulations of PML which did not Cell culture methods contain Sp100, while Sp100 gave a diffuse staining pattern Human epithelial Hep2 cells were grown and synchronised using with relative exclusion from the condensed chromatin region. thymidine and aphidicolin as described previously (Everett et al., Fig. 1E shows mitotic cells in prometaphase (lower left) and 1999). The degree of synchronisation was monitored on identically anaphase, while Fig. 1C includes a metaphase cell. These treated cultures by visualisation, FACS analysis and western blotting distributions of PML and Sp100 were maintained throughout for cyclin B (data not shown). Mitotic cells were also produced by mitosis until the earliest stages of G1. Immediately after or treatment with 25 ng/ml nocadazole, and by ‘mitotic shake off’, first during cytokinesis Sp100 remained diffuse and PML often discarding cells dislodged after overnight growth in a freshly seeded flask, then harvesting cells dislodged 1 hour later. Cell lines formed irregular foci in the vicinity of the cleavage furrow (not constitutively expressing a myc-tagged version of SUMO-1 were shown). On the basis of the proximity of the daughter cells and isolated after transfection of plasmid pCIPIC1 (Everett et al., 1998) the extent of reformation of the nuclear envelope, the G1 cell into HeLa cells, then screening colonies isolated after selection for pairs in Fig. 1E (upper right), Fig. 1C (upper right) and Fig. the G418 resistance gene carried in the plasmid. These lines were 1F (uppermost) probably represent the sequential events during unstable even in the presence of continuous G418 selection which the nuclear envelope re-forms and foci of PML and (400 µg/ml), but up to 20% of the cells expressed readily detectable Sp100 appear in the developing nucleus, eventually forming amounts of tagged SUMO-1. ND10 of normal appearance. However, a proportion of the Immunofluorescence, microscopy and antibodies PML protein, probably derived from the extrachromosomal foci during mitosis, remained outside the nuclear envelope in Cells seeded onto glass coverslips were prepared for immunofluorescence and examined by confocal microscopy as cytoplasmic punctate bodies which never contained Sp100. described previously (Everett et al., 1999). The following antibodies Thus cells in early G1 could be identified not only by being were used at the indicated dilutions: anti-PML rabbit serum r8 neighbouring cell pairs with small nuclei, but also by the (Boddy et al., 1996) (1/1000); anti-PML monoclonal antibody 5E10 presence of cytoplasmic PML. (Stuurman et al., 1992) (1/10); anti-Sp100 rabbit serum spGH (Sternsdorf et al., 1995) (1/1000); anti-Sp100 rat serum sp26 The presence of SUMO-1 in ND10 is cell cycle (Grötzinger et al., 1996) (1/1000); anti-myc tag monoclonal antibody regulated 9E10 (used at 1/1000) was purchased from Santa Cruz Laboratories. Given that the relative locations of PML and Sp100 change The secondary antibodies and the dilutions used were: FITC- through the cell cycle, and previous evidence linking their conjugated sheep anti-mouse IgG and goat anti-rabbit IgG (Sigma), localisation to the presence of conjugated SUMO-1, we both at 1/100; Cy3-conjugated goat anti-rabbit (1/2000), goat anti-rat (1/500) and goat anti-mouse (1/500); Cy5-conjugated goat anti-mouse investigated the localisation of SUMO-1 during the cell cycle. and goat anti-rabbit (1/500). All Cye-dye reagents were obtained from To improve detection, we isolated cells lines which express Amersham. myc-tagged SUMO-1 and found characteristic staining of SUMO-1 at the nuclear envelope (on account of its conjugation Western blotting to RanGAP1 (Saitoh et al., 1997)) and also at a number of SDS-polyacrylamide gel electrophoresis and detection of PML by intranuclear foci. In interphase cells there was a high degree of western blotting was as described previously (Everett et al., 1998). co-localisation of SUMO-1 with PML in ND10 (Fig. 1G,H), Sp100 was detected using rabbit serum SpGH. although ND10 without apparent SUMO-1 were also observed (inset, Fig. 1H). The foci of PML in mitotic cells did not co- stain for SUMO-1 (Fig. 1H,I), and the cytoplasmic foci of PML RESULTS in cells in early G1 were also mostly not associated with SUMO-1 (inset, Fig. 1I). PML and Sp100 separate during early prophase Examination of PML and Sp100 in synchronised cells showed Modification of PML and sp100 is cell cycle that, in agreement previous studies, ND10 throughout S phase dependent contained tightly co-localised PML and Sp100 (Fig.