chromatin-association of the polycomb group protein bmi1 is cell … · 2001-05-03 ·...

INTRODUCTION

Despite the recognition that Polycomb-group (Pc-G) proteinsplay an important role in sustaining developmentallyestablished expression boundaries of homeotic genes inDrosophila and Hox gene clusters in mammals, relatively littleis known about the molecular principles of this maintenancefunction. In Drosophila, Pc-G dependent silencing is mediatedvia polycomb responsive elements (PREs) (Zink and Paro,1989; Rastelli et al., 1993; Simon et al., 1993; Chan et al.,1994; Chiang et al., 1995; Müller, 1995; Strutt and Paro, 1997).Several lines of evidence predict an extensive interactionbetween known Pc-G members in both flies and mammals.Their colocalization on polytene chromosomes and ininterphase nuclei of cultured insect cells as well as mammaliancells provides a visual indication of physical association(Franke et al., 1995; Alkema et al., 1997a; Gunster et al., 1997;Buchenau et al., 1998). Furthermore, copurification andcoimmunoprecipitation of several Pc-G protein classes yieldsdirect evidence for their biochemical interaction (Franke et al.,1992; Alkema et al., 1997a; Gunster et al., 1997; Kyba andBrock, 1998). Studies in Drosophila have shown that, whenmutated, the Pc gene product seems to dislodge Pc-Gcomplexes in embryonal cells; this correlates with a severely

disturbed larval development (Franke et al., 1995). Takentogether, these data have prompted the idea that multiplebinding sites with varying binding affinities for Pc-G membersmay assist in recruitment of multiple factors into largernucleation sites for Pc-G repression (Zink and Paro, 1995;Pirotta, 1997, 1998; Strutt and Paro, 1997; Van Lohuizen,1998). How Pc-G proteins recognize and interact with targetgenes is still largely unclear. Most Pc-G proteins fail torecognize specific DNA sequences, although recently theDrosophila Pc-G gene pleiohomeotic was found to encode aprotein with homology to a mammalian transcriptionalregulator with sequence-specific DNA binding properties(Brown et al., 1998). Moreover, a conserved sequence motifwas reported to occur in Polycomb-responsive elements(Mihaly et al., 1998) suggesting that at least some Pc-G groupmembers may be in direct DNA contact.

Polycomb function has been conserved in mammals. TheBmi1 proto-oncogene was the first functional mammalian Pc-G identified (Van Lohuizen et al., 1991b; Brunk et al., 1991).Subsequent gain- and loss-of-function mutational analysis inthe mouse showed that morphological transformations of thevertebra along the antero-posterior axis of the skeleton areassociated with Hox gene expression boundary shifts (Van derLugt et al., 1994, 1996; Alkema et al., 1995). Several Pc-G

4627Journal of Cell Science 112, 4627-4639 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS4692

The human proto-oncogene Bmi1 is a member of themammalian Polycomb Group (Pc-G) genes. Thesubnuclear distribution of the BMI1 protein was studiedin several primary human and tumor-derived cell linesusing immunohistochemical and biochemical methods. Inprimary and tumor cells, nuclear BMI1 shows a fine-graindistribution over chromatin, usually dense in interphasenuclei and significantly weaker along mitotic chromosomes.In addition, BMI1 preferentially associates with severaldistinct heterochromatic domains in tumor cell lines. Inboth primary and tumor cell lines a marked cell cycle-regulation of Pc-G-chromatin interaction is observed:nuclear BMI1-staining dissipates in late S phase and is re-

established early in G1-phase. Chromatin-association ofBMI1 inversely correlates with its phosphorylationstatus in a cell cycle-dependent fashion: at G1/S,hypophosphorylated BMI1 is specifically retained in thechromatin-associated nuclear protein fraction, whereasduring G2/M, phosphorylated BMI1 is not chromatin-bound. Our findings indicate a strict cell cycle-controlledregulation of Pc-G complex-chromatin association andprovide molecular tools for improving our understandingof Pc-G complex regulation and function in mammaliancells.

Key words: Polycomb, Chromatin, Phosphorylation

SUMMARY

Chromatin-association of the Polycomb group protein BMI1 is cell cycle-

regulated and correlates with its phosphorylation status

Jan Willem Voncken1, Dieter Schweizer2, Louise Aagaard3, Lydia Sattler2, Michael F. Jantsch2

and Maarten van Lohuizen1,* 1The Netherlands Cancer Institute, Division of Molecular Carcinogenesis, NL-1066 CX Amsterdam, The Netherlands2Department of Cytology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria3Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Dr Bohrgasse 7, A-1030 Vienna, Austria *Author for correspondence (e-mail: [email protected])

Accepted 13 October; published on WWW 30 November 1999

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gene null-mutant studies in mice have revealed comparable,yet not fully identical, morphological transformations,accompanied by expression boundary shifts of distinct Hoxgenes, again suggesting the existence of multiple complexeswith diverse target gene specificity (reviewed in van Lohuizen,1998). Pc-G function is likely to be more complex in mammalsthan in insects. This increased complexity is partly due to themultiplication of Hox gene clusters in mammals as well as theduplication of many Drosophila Pc-G homologues (reviewedby Gould, 1997; van Lohuizen, 1998). To date, no cis-actingpolycomb responsive elements have been identified inmammalian DNA.

Despite increasing knowledge on processes controlled byPc-G proteins, relatively little is known about regulation of Pc-G protein complexes themselves. In human cell lines, a non-uniform Pc-G protein distribution is visible in interphasenuclei, which disappears at mitosis (Alkema et al., 1997a;Gunster et al., 1997). In the present study, we examined thesubnuclear distribution of BMI1 in primary human and tumorcells in relation to cell cycle progression. We demonstrate thatchromatin-association of Pc-G proteins in primary and tumorcell lines is subject to a strict cell cycle-dependent regulation.In addition, chromatin association of BMI1 throughout thecell cycle inversely correlates with its phosphorylationstatus, indicating a pronounced cell cycle-dependent post-translational modification. By employing more refinedimmunohistological techniques, we show that detection ofthe endogenous BMI1 protein is possible, also on mitoticmetaphase chromosomes, albeit at significantly reduced levels.The implications of our findings with respect to recent reportson the biological significance of Pc-G chromatin associationby others and to mechanistic models of Pc-G function inmammalian cells are discussed.

MATERIALS AND METHODS

Plasmid DNAA (myc)3H6-epitope-tagged expression vector was generated frompKW2T (a derivative of pRK7; Genentech). The resulting CMV(cytomegalo virus)-(myc)3H6[NotI] vector contains a unique NotIcloning site immediately following the amino-terminal tag. A PCR-amplified DNA fragment was inserted in-frame at the NotI siteencoding the human BMI1 protein (Alkema et al., 1993). The CMVenhancer/promoter directs BMI1 overexpression in mammalian cells(Aagaard et al., 1999). An LZRS-Bmi1-IRES-EGFP viral vector(Jacobs et al., 1999) was used to obtain high polyoma-tagged (2PY)BMI1 expression in primary human cells.

Cell culture and transient transfectionHeLa, U2-OS and MCF-7 cells were grown at 37°C, 5% CO2 inDulbecco’s modified Eagle’s medium (DMEM) supplemented with10% fetal calf serum (FCS). Primary amniotic cell cultures andprimary peripheral white blood (PWB) cells were obtained at theUniversity Hospital of Vienna (Austria). Confluent low passagenumber diploid primary human TIG3 fibroblast cultures (Fukami etal., 1995) were split 1:4 (one passage equals two populationdoublings, PDL). PWB cells were cultured in PB-max medium(Gibco) for 72 hours. Cells were either grown on multiwell slides(Alkema et al., 1997a) or spun onto glass slides in a Cytospin(Shandon). For transient expression, HeLa cells (1×105) weretransfected with 3 µg of the CMV-driven (myc)3-Bmi1 plasmid usingLipofectase (Gibco); transfection efficiencies varied between 2-15%

(not shown). 48 hours after transfection, cells were processed forindirect immunofluorescence. Isolation of recombinant Bmi1 andcontrol virus and viral infection of primary human TIG3 cells werecarried out essentially as described (Jacobs et al., 1999). Infectionefficiency was estimated at approximately 100% by GFP-positivity ofcells. To attain synchronization, primary and tumor cell cultures werearrested in early G1 using lovastatin (40 nM, 48 hours; Keyomarsi etal., 1991), or accumulated in G1 either by serum starvation or contactinhibition. S-phase enrichment was achieved by double thymidineblock (2× 2 mM; 12-16 hours); G2/M specific cell suspensions wereobtained by treatment with either nocodazole (50 ng/ml; 16 hours) orcolcemid (0.02-0.05 µg/ml; 16 hours) followed by mitotic ‘shake-off’.

Orthophosphate labeling, immunoprecipitation andwestern analysisCell suspensions from a spontaneous BMI1-overexpressing tumor(derived from Bmi1-transgenic mouse) were starved for 1.5 hours, andsubsequently grown for 3.5 hours in minimal medium supplementedwith 10% dialyzed FCS, in the presence of 10 mCi 32P-orthophosphate. Cell extracts were made in ELB buffer (Alkema etal., 1997a). Total lysates were incubated with preformed protein-sepharose/antiserum complexes for 1 hour at 4°C. Other cell extractsfor western analysis were made in RIPA buffer (Alkema et al., 1997a).Separation of soluble and chromatin-bound nuclear fractions wasessentially carried out as previously described (Muchardt et al., 1996).

Indirect immunofluorescenceIndirect immunofluorescence detection of the endogenous BMI1protein in HeLa cells, and of (myc)3-tagged BMI1 protein intransiently transfected HeLa cells, respectively, essentially followed acytospin procedure that is outlined elsewhere (Earnshaw et al., 1984;Haaf et al., 1990) with some minor modifications. Cells wereincubated for 3 hours with colcemid (0.1 mg/ml; Gibco), harvestedby trypsinization or by ‘shake off’ and hypotonically swollen for 30minutes at 37°C in RBS buffer (10 mM Tris, pH 7.4, 10 mM NaCl,5 mM MgCl2). Cytospin-spread nuclei and chromosomes were fixedin 2% formaldehyde in PBS, washed, permeabilized with 0.5% NP-40, blocked in culture medium containing 10% FCS for 30 minutesat room temperature, after followed by incubation with the respectiveprimary and secondary antibodies. Alternatively, cells grown onmultiwell coverslips were formaldehyde-fixed as described before(Alkema et al., 1997a). Primary TIG3 cells harboring LZRS/IRES-EGFP viral integrations were grown on 0.1% gelatin coated multi-well slides and extracted either in 0.5% Triton-X in KCM buffer(Jeppesen et al., 1992) and fixed in formaldehyde or directly in 100%methanol at −20°C for 3-5 minutes. Antisera used are indicated in thefigure legends. Immunofluorescently labeled mitotic cells werecounterstained with the DNA dye 4′-6-diamidino-2-phenylindole(DAPI) alone or in combination with distamycin A (Schweizer et al.,1978; Schweizer and Ambros, 1994). Preparations were mounted inVectashield AntiFade (Vector Laboratories).

Immunofluorescent labeling of unfixed cellsTo enhance detection of endogenous BMI1 protein on M-phasechromatin, a reversed fixation/staining technique was employed (i.e.‘post-fixation’; Jeppesen, 1994; Wreggett et al., 1994). A metaphase-enriched cell fraction was harvested by mitotic ‘shake-off’ (modifiedafter Jeppesen et al., 1992). Cells were hypotonically swollen for 30minutes at room temperature in 75 mM KCl, spun onto microscopeslides and immediately immersed in KCM-buffer (120 mM KCl, 20mM MgCl2, 10 mM, Tris pH 8.0, 0.5 mM EDTA, 0.1% Triton X-100). For interphase and mitotic chromosome immunofluorescence,preparations were blocked for 30 minutes with 10% FCS in KCM,and sequentially incubated with the appropriate primary andsecondary antisera (see above). Chromosomes were then fixed in 4%formaldehyde in KCM for 10 minutes, counterstained with DAPI, orwith DAPI plus distamycin A, and mounted in Vectashield AntiFade.

J. W. Voncken and others

4629Cell cycle-dependent chromatin association of BMI1

Microscopic analysis and documentation was done as describedbelow.

Fluorescent in situ hybridization (FISH)Following immunofluorescence microscopic analysis, coverslipswere removed by floating-off in PBS at ambient temperature.Preparations were again fixed in buffered paraformaldehyde (4%)for 15 minutes (Schweizer and Ambros, 1994). Paracentromericconstitutive heterochromatin on human chromosome 1 (1qh) wasvisualized using a previously described FISH procedure (Strehl andAmbros, 1993). A human chromosome 1-specific subregional probepUC1.77 (Cooke and Hindley, 1979) was used to probe for region1q12. The DNA probe was labeled by nick-translation either withbiotin-11-dUTP (Sigma) or with digoxigenin-11-dUTP (Boehringer,Mannheim). For detection of biotin-labeled probes a mouse anti-biotin antibody in conjunction with a secondary rabbit anti-mousetetramethyl-rodamine isothiocyanate (TRITC)-conjugated antibody(1:20 and 1:30, respectively; DAKO, Glostrup, Denmark) were used.Detection of digoxigenin-labeled probes was achieved using acombination of sheep anti-digoxigenin (1:100; Dakopatts no. R270;or Sigma, St Louis, Missouri) and FITC-conjugated rabbit anti-sheep antibodies (1:100; DAKO, Glostrup, Denmark). FollowingFISH, the preparations were fixed with 4% paraformaldehyde,

counterstained with DAPI and mounted in Vectashield AntiFadesolution.

Cytological characterization of cell linesChromosome analyses and karyotyping of cultured cells were donefollowing conventional methods for mitotic stimulation, metaphaseaccumulation and for metaphase chromosome spreading uponmethanol/acetic acid (3:1) fixation. Fluorescent chromosomeR-banding and C-banding of chromosome spreads was achievedusing the chromomycin/distamycin-A/DAPI tristaining technique(Schweizer and Ambros, 1994). Chromosome 1-identification bybanding techniques was confirmed by sequential application of FISHusing the human chromosome 1-specific satellite probe pUC1.77(Cooke and Hindley, 1979).

Fluorescence microscopy and image acquisitionFluorescence microscopical examination was performed on a ZeissAxioplan or Axioskop. Pictures were taken both on black and whiteand on color films. Alternatively, pictures were digitalized by meansof a cooled CCD camera (Photometrics). For this purpose, imageswere acquired separately for each fluorochrome using IPLab software,pseudocolored (Gene Join) and, when required, processed and mergedusing Adobe Photoshop 4.0 software.

Fig. 1. Nuclear concentrationof BMI1 at heterochromatinof chromosome 1 in tumorcell lines. (A) EndogenousBMI1 distribution in HeLainterphase nuclei. In additionto a fine-grained staining,BMI1 (red signal; left) isconcentrated on average atfour larger domains in HeLanuclei. Counterstaining withDA-DAPI (right) to highlightall major human C-bands (seeMaterials and Methods)complementsimmunolocalization of BMI1.The merge (middle) showscolocalization of BMI1 withheterochromatin.(B) Chromosome 1 q12-association of BMI1.Consecutive immuno-FISHusing the human chromosome1-specific probe pUC1.77 forsubregion 1q12 (see Materialsand Methods). BMI1 protein(left), DA-DAPI (middle),1q12 FISH-signal (right) inHeLa cells and in U2-OScells. The mouse mAb (F6,1:100; Alkema et al., 1997a)was used to detect BMI1; thesecondary antibody was Cy3-conjugated.

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RESULTS

BMI1 is enriched at paracentric heterochromatin ofchromosome 1 in tumor cell lines The nuclear localization of the BMI1 protein was studied indetail in three tumor cell lines. The BMI1 protein is exclusivelyseen in nuclei; the protein is excluded from the nucleolus. InHeLa cervical carcinoma cells, in addition to a fine-grainedpattern, the majority of BMI1-positive nuclei exhibits three(54%) or four signals (36%; Fig. 1A). These subnucleardomains are associated with distamycin-DAPI-positiveheterochromatin, marking chromosomes 1, 9 and 16, andthe short arm of chromosome 15. Only a fraction of all DA-DAPI positive chromocenters are BMI1-positive (Fig. 1A).Conversely, however, all bright BMI1 fluorescent signalsare associated with DA-DAPI positive heterochromatin.Consecutive immuno-FISH using satellite probe pUC1.77reveals a FISH labeling pattern fully congruent with the BMI1staining pattern (Fig. 1B), proving BMI1 protein concentrationat heterochromatic chromosome region 1q12 (1qh). Similarresults were obtained with U2-OS osteosarcoma cells andMCF-7 breast epithelial carcinoma cells. FISH data in HeLaand U2-OS cells were confirmed by conventional cytologicalanalysis (not shown). Since the three aneuploid cell linesexhibit significant differences in dosage of chromosome region1q12, it is concluded that this variation is a reflection ofdifferences between cell lines in karyotype rather thanexpression levels (see Discussion). The above data are inagreement with a recently published report (Saurin et al.,1998).

The localization of BMI1 at paracentromericheterochromatin is cell cycle-regulatedClose to mitosis, the intensity of the nuclear BMI1 signaldecreases significantly (Alkema et al., 1997a). To establish indetail at what cell cycle phase Pc-G-chromatin dissociationoccurs, synchronized and asynchronously growing U2-OScells were simultaneously immunostained for BMI1 and eithercell-cycle marker PCNA or phosphorylated histone H3. ThePCNA staining pattern in nuclei going through S phasedisplays characteristic alterations (Celis and Celis, 1985; Celiset al., 1986). Likewise, phosphorylation of histone H3 can beused as an immunocytochemical marker for late S, G2/M phase(Hendzel et al., 1997). In G0/ G1, nuclear PCNA staining isvirtually absent in U2-OS nuclei; at these stages, Pc-G-paracentromeric heterochromatin association is maximallyvisible (Fig. 2A). As cells move into S phase, the PCNA signalis initially detectable as a granular pattern, but subsequentlyredistributes into a punctate pattern, revealing foci close to thenuclear membrane. At late S phase histone H3 phosphorylationis initiated at clearly visible foci (Hendzel et al., 1997).Interestingly, at this transition, detection of 1qh-Pc-Gassociation becomes increasingly difficult in U2-OS nuclei: in32.8% of nuclei phosphorylation of histone H3 has beeninitiated, while approximately the same number of nuclei(33.8%) displays a clearly diminished or absent Pc-G-heterochromatin association (Fig. 2A, asterisks, B). Whenhistone H3 phosphorylation has spread over the entire genomeand the PCNA signals have dissipated, the Pc-G signal isvirtually undetectable and remains low throughout G2/M, butis re-established during the ensuing G1 phase. The latter is

demonstrated by the re-occurrence of Pc-G domains in earlyG1-arrested cells (Fig. 2A, bottom, C). These findings suggesta remarkable cell cycle-dependent regulation of Pc-G-paracentromeric heterochromatin association.

Distribution of endogenous BMI1 on metaphasechromosomes of HeLa and U2-OS cellsAbsence of BMI1 staining at mitosis could, besidesdissociation from chromatin, be due to cell cycle-dependentinaccessibility of the protein for the antibody, or to damage orloss of epitopes during the preparation procedure. We thereforeapplied a more sensitive immunocytological technique aimedat detection of less abundant endogenous proteins, in whichchromatin immuno-staining precedes fixation (Jeppesen, 1994;Wreggett et al., 1994). In control experiments, antibodiesagainst phosphorylated histone H3 (see Fig. 2B) or a CRESTscleroderma patient serum (not shown) in conjunction with aBMI1 specific antiserum was used. Clearly detectable stainingwith these antisera proves accessibility of the respectiveantigens. Using the more sensitive ‘post-fixation’ technique afine-grain BMI1 protein staining was visible over metaphases,albeit significantly weaker compared to interphase nuclei (Fig.3A). Importantly, BMI1-concentration at 1q12 on metaphasechromosomes was only seen when ‘post-fixation’ wasemployed. As depicted in Fig. 2A, all FISH signals colocalizedwith BMI1 signals and, vice versa, all BMI1 signals mergedperfectly with a FISH signal indicating association withchromosome 1 paracentromeric heterochromatin. The mitoticchromatin staining and colocalization were confirmed in U2-OS (Fig. 3B) and MCF-7 cells (not shown).

Immunolocalization of ectopically overexpressedBMI1 on interphase and mitotic chromosomes ofHeLa cellsTo substantiate the concentration of endogenous BMI1 at 1qh,we ectopically expressed epitope-tagged BMI1 in tumor celllines. This approach serves as an immunocytological controland allows an assessment of the influence of BMI1 expressionlevels on staining patterns. Besides a fine-grain overallchromatin labeling, all antigen-positive interphase nuclei ofHeLa cells expressing myc-tagged BMI1 exhibit distinct brightsignals (on average, four). These results demonstrate that thestaining pattern in cells ectopically expressing tagged BMI1protein (1-2 orders of magnitude above the level of theendogenous protein; L. A., unpublished) is qualitatively similarto that of the endogenous protein. The findings are also inline with our previous observations that haemagglutinin(HA)-tagged BMI1 perfectly colocalizes with endogenousproteins in transfected U2-OS cells (Alkema et al., 1997a). Inaddition, analysis of antigen-positive ‘post-fixed’ metaphasechromosomes reveal an immunostaining pattern similar to thatof endogenous BMI1: a pronounced enrichment of myc-BMI1within the paracentromeric heterochromatic region in the longarm of human chromosome 1 next to a weaker distributionalong the chromosome arms (Fig. 4A,B). Consecutiveimmuno-FISH results on metaphase and interphase chromatinwere fully congruent (Fig. 4B and not shown). These dataconfirm the specific concentration of BMI1 at 1qh duringinterphase and, at a lower intensity, at mitosis. In addition, thisdemonstrates that ectopic expression of the BMI1 protein doesnot alter its overall subnuclear distribution pattern.



Fig. 2. Cell cycle-dependent chromatin-association of BMI1. (A) Cell cycle-specific parallel staining of U2-OS nuclei for PCNA and BMI1protein. At late S phase (asterisk) the BMI1 signal disappears and remains undetectable by conventional fixation methods throughout G2 and Mphases. Nuclear BMI1 staining is re-established at early G1 (eG1). Antisera used: PCNA (mAb PC10; Santa Cruz Biotechnology, Inc.) andBMI1 (pAb bmi38; 1:30). (B) Parallel staining of U2-OS nuclei for phosphorylated histone H3 and BMI1. While at G1, Pc-G domains areclearly visible, BMI1-chromatin association is undetectable at late S and G2/M phases. Antisera used: phosphorylated histone H3 (pAb;Hendzel et al., 1997) and BMI1 (mAb F6). (C) Pc-G chromatin association is re-established early during G1 phase. Early G1-arrest (eG1) ofU2-OS cells was established with lovastatin, early S-phase arrest by a double thymidine block.

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Subnuclear distribution of endogenous BMI1 proteinin primary cells Interphase and metaphase chromatin of human primary cellswas examined immunohistologically for BMI1 distribution.Interphase nuclei of primary peripheral white blood cells showa fine-grained pattern, qualitatively comparable to that of long-term cultured cell lines, although considerably weaker inintensity (Fig. 5A). Metaphase spreads of these cells did notreveal BMI1 positive staining, although counter-staining withantiserum against phosphorylated histone H3 was positive (notshown). Similar findings were made in primary amniotic cellsand in an EBV-immortalized normal B-cell derived cell line(Fig. 5A; not shown). Again a weak, fine-grained pattern wasvisible in these cells. Finally, diploid, primary human TIG3fibroblasts show the same fine-grained staining pattern: thelarge nuclear domains as seen in tumor cells are absent (Fig.5A). The low intensity of BMI1 staining most likely relates tolow levels of gene expression in vivo. Indeed, infection ofprimary TIG3 cells at low PDL with retroviral expressionvectors carrying the Bmi1 gene, gave rise to higher, althoughvarying, levels of BMI1 expression throughout the culture.However, in synchronized cultures only a fine-grained stainingpattern was observed; none of the low passage primary cellsshowed the nuclear Pc-G domains characteristic of tumor celllines (Fig. 5A). The nuclear staining pattern of BMI1 wasconfirmed with two independent antisera (anti-BMI1 and anti-2PY epitope tag; not shown). Lack of BMI1 domains in nucleiof primary cells suggests that preferential BMI1 staining athuman chromosome region 1q12 is tumor-specific oralternatively, might be associated with changes in localchromatin structure as a result of long term establishment inculture. To test the latter possibility, synchronized TIG3 cellsexpressing high levels of BMI1 (TIG3/bmi) were examined atlow (<30) and high PDL (>60). Remarkably, a low percentage

(5-10%) of high passage number TIG3/bmi cells show clearlyvisible nuclear domains, while none were found in low passagenumber TIG3/bmi; this finding was confirmed with unrelatedantisera (Fig. 5B). However, consecutive immuno-FISHanalysis indicated that these nuclear sites were notparacentromeric α-satellite repeats at position 1q12 (data notshown). The above data argue that the nuclear distribution ofBMI1 in primary human cells is fine-grained and thatformation of distinctive Pc-G domains is secondary toprolonged culturing.

Nuclear staining of BMI1 in primary cell nuclei issubject to cell cycle-regulation We next examined BMI1-chromatin interaction in primarycells in relation to cell cycle progression. As described above,primary TIG3 cultures were counterstained at different cellcycle stages for PCNA or phosphorylated histone H3. PrimaryTIG3 cells are subject to replicative senescence at higher PDL(Fukami et al., 1995). We have previously shown thatoverexpression of BMI1 yields a proliferative advantage toprimary mouse and human fibroblasts and delays the Hayflicklimit in TIG3 cells (Jacobs et al., 1999). As a result, at highpassage, more than 80% of cells in G0/ G1 display a strongnuclear staining for BMI1 versus 40-50% of TIG3/bmi cells atlow passage. This enabled us to trace the global nuclear BMI1staining intensity throughout the cell cycle in primary cells. Asshown in Fig. 6A, the BMI1 signal drops significantly at lateS phase, when the PCNA pattern has become located in distinctfoci close to the nuclear membrane and subsequently begins tobreak down. BMI1-chromatin association is virtuallyundetectable by standard methods throughout G2/M, at whichtime phosphorylation of histone H3 has become widespread(Fig. 6B and not shown). These data indicate that, like in tumorcells, in primary human cells the biochemical interaction


Fig. 3. Association ofendogenous BMI1 protein onHeLa mid- (A) and late- (B)metaphase chromosomes.Sensitive indirectimmunofluorescence (‘post-fixation’; see Materials andMethods) on metaphasechromatin for BMI1 (leftpanels) reveals, in addition to adiffuse fine-grained patternalong chromosome arms andon chromatids, a BMI1concentration at fourheterochromatic areas(arrows). Corresponding DA-DAPI staining (middle panel)highlights, in addition to 1qh(arrows), heterochromaticregions on chromosomes 9, 16and 15 (short arms). Merge ofDA-DAPI and 1qh-specificFISH signals (right panels).Antisera used as in legend toFig. 1.


between Pc-G proteins and chromatin is also regulated in a cellcycle-dependent fashion.

The chromatin-association of BMI1 correlates withits phosphorylation statusMany cellular proteins undergo cell cycle-related post-translational modifications, which in turn can affect molecularinteractions. Interestingly, cell cycle-specific chromatin bindingwas also reported for trx-G proteins (Muchardt et al., 1996). Weasked whether BMI1 is subject to cell cycle-regulated post-translational modification and whether this correlates withchromatin-association. Endogenous BMI1 protein is readilydetectable by western analysis in many tumor-derived cell linesand is represented in multiple bands (Fig. 7A; see also Alkemaet al., 1997a). Comparison of cellular extracts at distinct phasesof the cell cycle reveals a difference in migration speed ofBMI1: the majority of BMI1 appears as faster migrating bandsduring G1 and early S phases (Fig. 7A). At mitosis, whenparacentromeric heterochromatin association of BMI1 in tumorcells is microscopically undetectable, the slow migrating fromof BMI1 is most abundant. In U2-OS extracts, the polyclonal(pAb) bmi38 and the monoclonal (mAb) bmiF6 antisera displaya slight preference for the differentially migrating forms ofBMI1, which complicates assessment of protein levelsthroughout the cell cycle. We therefore also studied epitope-tagged BMI1 in primary cells at low PDL. When expressed inprimary TIG3 cells, PY-tagged BMI1 shows similar cell cycle-

dependent migration differences as those in U2-OS cells (Fig.7C). Total cellular BMI1, detected with an mAb, a pAb or thePY-tag-specific antibody, shows a similar abundance of slowermigrating BMI1 in M-phase arrested cells. Importantly, thisdemonstrates that ectopically expressed BMI1 protein is alsosubject to cell cycle-dependent regulation. Furthermore, thesedata suggest that BMI1 is not subject to massive degradationduring mitosis (Fig. 7C,E). Direct immunoprecipitationfollowing orthophosphate labeling identifies BMI1 as aphospho-protein in vivo (Fig. 7D). Treatment of the M-phasespecific form of BMI1 with calf intestinal phosphatase showsthat, by removal of phosphate groups, faster migration of BMI1is restored to that seen in G1/S phase extracts (Fig. 7E). Theenzymatic removal of phosphate groups is concentrationdependent and specifically prevented by phosphatase inhibitors(Fig. 7E). When chromatin-association of BMI1 is mostpronounced, from G1 onward into S phase, it would follow thatBMI1 in U2-OS cells is relatively hypophosphorylated. Anintermediate phosphorylation degree is obvious in extracts ofearly G1-arrested cells (Fig. 7B), confirming the observationthat Pc-G domains in U2-OS cells are being visibly restored atthis time (Fig. 2C). When subjected to differential nuclearextraction, most of the hypophosphorylated BMI1 (G1 phase)is retained in the chromatin-bound fraction (Fig. 7F).Conversely, at M phase BMI1 is readily extracted in the solublenuclear fraction. Thus, the microscopically visible dissociationof Pc-G complexes at mitosis is supported by biochemical data,

Fig. 4. Immunolocalization ofepitope-tagged BMI1 intransiently transfected HeLacells.(A) Detection of myc-tagged BMI1 on mitoticmetaphase chromosomes.Epitope-tagged BMI1 shows afine-grained association withmetaphase chromatin andadditional concentration at thecentric region in four largemetacentrics and a smallchromosome (left panel,arrowheads). ChromosomalDNA was counterstained withDA-DAPI to highlightcentromeric heterochromatin(right panel). The merge picture(middle panel) showscolocalization of BMI1domains with paracentromericheterochromatin. (B) Imagesshowing completecolocalization of epitope-taggedBMI1 (left) withheterochromatin ofchromosome 1 (subregion1q12) as identified by DA-DAPI positive staining (middlepanel) and FISH (merge DA-DAPI and 1qh FISH, rightpanel).

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indicating actual detachment of phosphorylated BMI1 fromchromatin. MPH-chromatin interaction follows similardynamics (Fig. 7F). In addition, at least some Pc-G protein-protein interactions appear to have been retained at M phase,since MPH is readily immunoprecipitated with antibodiesagainst BMI1 (Fig.7G) or M33 (data not shown). Taken

together, the above findings demonstrate that cellular BMI1 is phosphorylated in a cell cycle-dependent manner, andthat phosphorylated BMI1, likely in association with other Pc-G proteins, is physically dislodged as a complex from chromatin at G2/M phase in both primary and tumor celllines.


Fig. 5. Distribution of BMI1 inprimary human cells.(A) Chromatin of primaryamniotic cells (top), peripheralblood lymphocytes (middle) andprimary human TIG3 fibroblastinterphase nuclei (bottom panel)show a fine-grained distributionpattern of endogenous BMI1.Shown are also the correspondingdiploid FISH signals in amnioticand PBL cells. Overexpression ofBMI1 (ectopic; lower right) inTIG3 fibroblasts at low passagedoes not alter the nucleardistribution of BMI1.(B) Spontaneous concentration ofBMI1 in nuclear subdomains inhigh passage TIG3/bmi cells, asevidenced by immuno-stainingwith unrelated antisera (2PY-epitope tag specific mAbAK1310; 1:3); this chromatinassociation is, however, unrelatedto 1q12 binding (FISH notshown).

Fig. 6. Cell cycle-dependentBMI1-chromatin association inprimary human cells.(A) Dissociation of BMI1from chromatin at late S phasein high PDL TIG3/bmifibroblasts. The panels showtwo adjacent cells at differentstages of the cell cycle (upperright cell, G1 and lower leftcell, late S). Shown are thePCNA (upper) and BMI1signal (middle); the DAPIsignal is shown at the bottom.(B) Parallel staining ofTIG3/bmi nuclei for PCNAand BMI1 protein.Asynchronously growingTIG3/bmi cells were costainedfor the cell cycle markerPCNA and BMI1 (pAb bmi38;1:30). At late S phase theBMI1 signal dissipates andremains undetectablethroughout G2/M phase.Photographs were take withequal exposure times.


DISCUSSION

Subnuclear distribution of BMI1 in primary andtumor cells In an earlier study, we reported on accumulation of endogenousBMI1 in distinct subnuclear domains in human U2-OS cells(Alkema et al., 1997a). Several other tumor cell lines show acomparable BMI1 distribution pattern (e.g. K562, HeLa, MCF-7, SOAS-2 and SW480). Although a possible connection tospecific chromatin regions remained unclear, the exactlyoverlapping nuclear patterns of several other Pc-G proteins(Alkema et al., 1997a; Gunster et al., 1997; Satijn et al., 1997;Schoorlemmer et al., 1997; Saurin et al., 1998) suggested thatthese domains reflect sites at which these Pc-G proteins interactin large Pc-G complexes.

Here we show that the domains at which BMI1 isconcentrated in human tumor cell lines are at paracentricheterochromatin on chromosome 1 (1qh). These results are in

full agreement with recently published findings of anindependent study (Saurin et al., 1998). We find that primaryhuman cells do not display the 1qh concentration of nuclearPc-G protein typical of human tumor cell lines, but instead afine-grained nuclear Pc-G staining pattern. The 1qh-associatednuclear Pc-G domains therefore most likely represent anacquired feature of tumor cell lines. Immuno-FISH data andkaryotype analysis presented here for HeLa, U2-OS and MCF-7 cells, strongly suggest that the variation in size and numberof heterochromatin associated Pc-G domains results fromkaryotypic differences between tumor cell lines, and furtherunderwrite this notion. Also, we have observed that localsubnuclear Pc-G accumulation can in fact be evoked at lowfrequency in primary cells by prolonged culturing (see Results)or by exposure to demethylating agents (data not shown), butthis association is 1qh-unrelated. This effect on nuclear Pc-Gredistribution is most likely due to acquired changes inchromatin structure (see also below). Endogenous BMI1 levels

Fig. 7. Cell cycle-dependent phosphorylation ofBMI1. (A) Cell extracts of cell cycle-arrestedU2-OS cells; immunoblots were stained with anmAb or a pAb for BMI1. Corresponding histoneH3-phosphorylation (P-H3) at M phase is shownat the bottom. G1, contact inhibition; M (left),nocodazole; M (right), colcemid; S, doublethymidine block. (B) Cell extracts correspondingto cells shown in Fig. 2C; early G1-arrest(lovastatin), S-phase arrest (double thymidineblock). (C) Protein extracts of TIG3/bmi cellsarrested in G1 (contact inhibition), S (doublethymidine block) and M-phase (colcemid).Immunodetection of ectopically expressed BMI1was done with three unrelated antisera (mAb F6,pAb bmi38 and mAb 2PY-tag). Shown are also apositive (asynchronously growing TIG3/bmi cellextract) and a negative control (TIG3/gfp extract)for the epitope tag-specific antiserum.(D) Immunoprecipitation from orthophosphate-labeled cell extract shows that BMI1 is aphosphoprotein. Phosphorylated BMI1 is readilyimmunoprecipitated with a peptide antiserum(116) but not with the 116 serum pre-incubatedwith peptide (pept). Similar results were obtainedwith two independent BMI1 monoclonal antisera(B7, F6); normal rabbit serum (nrs) and normalmouse serum (nms) served as additional controls.(E) Dephosphorylation of BMI1 from M-phasearrested TIG3/bmi cells by alkaline phosphatase.For immunodetection of hypo- andhyperphosphorylated BMI1, the mAb and pAbwere used in combination. A G1-arrestedTIG3/bmi cell extract was used as a control.Equal protein loading was confirmed in allinstances by Ponceau-S staining.(F) Phosphorylated BMI1 is released fromchromatin in M-phase U2-OS cells. Non-phosphorylated BMI1 is extracted in thechromatin-bound fraction in G1. The upper panelshows that similar dynamics apply to the Pc-G protein MPH (MPH-SM 70092 antiserum; Alkema et al., 1997a). Detection of BMI1 wascarried out as described in Fig. 7E. (G) Protein-protein interactions within Pc-G complexes are not affected by phosphorylation. BMI1 wasimmunoprecipitated from U2-OS cells arrested in G1 or M phases. MPH was detected using standard western technology. Pre-clearing wascarried out using protein A sepharose beads prebound with both normal mouse and rabbit serum. Immunoprecipitaion of BMI1 was carried outwith both mAb (BMI-F6) and pAb (BMI-38) separately precoupled to protein A sepharose beads.

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tend to be higher in tumor-derived human cell lines comparedto primary cells and may contribute to Pc-G-accumulation at1qh. However, overexpression of BMI1 per se does not alterits staining pattern: the nuclear distribution of ectopicallyexpressed BMI1 in tumor cell lines and, more importantly, inprimary human cells at low passage, is identical to that of theendogenous protein in the respective culture types. Thecombined data suggest that a fine-grained distribution of BMI1in interphase nuclei of primary human cells is the defaultsituation, as has been reported for Drosophila embryonal cells(Buchenau et al., 1997), murine fibroblasts (Wang et al., 1997)and primary murine cells (our unpublished observations).

The nature and possible significance of Pc-Ginteraction with heterochromatic satellite repeatsSpecific satellite DNA binding proteins, such as the chromatin-binding factor GAGA (Platero et al., 1998) and the product ofproliferation disrupter (prod) (Török et al., 1997), have beenidentified in Drosophila. We suggest that the preferential 1qh-association of BMI1 is mediated by specific proteininteraction(s) with a repeated sequence motif occurring inchromosome 1-specific α-satellite DNA. Alternatively, it ispossible, because of satellite DNA instability and highdivergence rate of alphoid tandem repeats, that chromosomeregion 1q12 by chance has acquired and amplified potential Pc-G binding sites (Waye et al., 1987; Csink and Henikoff, 1998).Since we see no specific DNA binding with BMI1 (ourunpublished results), it is likely that the putative protein-satellite DNA interaction is conferred by human BMI1-interacting protein(s) or the complex as a whole rather thanBMI1 itself. We and others have recently identified proteinsthat bind the RING-finger motif of BMI1, thus confirming itsrole in protein-protein rather than in protein-DNA interaction(Hemenway et al., 1998; M. van Lohuizen et al., unpublishedresults). Interestingly, the RING-finger domain is required forproper subnuclear localization of BMI1 (Alkema et al., 1997a).Specific in vivo DNA-binding had not been reported for any ofthe thus far known Pc-G proteins, although recently, theDrosophila Pc-G gene pleiohomeotic was found to encode aprotein with homology to the mammalian sequence-specificDNA-binding transcriptional regulator YY1 (Brown et al.,1998). Furthermore, a common sequence motif occurs inpolycomb responsive elements (PREs; Mihaly et al., 1998) andin the YY1 binding consensus. As yet unknown Pc-G complexproteins may recognize analogous sequence elements in α-satellite repeats or mediate binding of PcG-complexes to DNA.An interesting candidate protein with the latter potential maybe the recently identified RYBP1 (Garcia et al., 1999).

The absence of Pc-G domains in primary cells (see above)argues that these subnuclear domains in fact represent anacquired feature of tumor cells in culture. Of interest in thisrespect is that in many types of human cancers pericentromericrearrangements of chromosome 1 occur at high frequency(Brito-Babapulle and Atkin, 1981). Pericentromericchromosomal anomalies involving rearrangements betweensatellite repeat sequences on chromosomes 1, 9 and 16 haverecently been linked to DNA hypomethylation (Weizhen et al.,1997 and references therein). However, global demethylationby itself appears not sufficient to induce Pc-G/1qh-associationin primary cells (our unpublished observations). Mostestablished cell lines studied to date for nuclear Pc-G

distribution are tumor derived (Alkema et al., 1997a; Gunsteret al., 1997; Satijn et al., 1997; Saurin et al., 1998). Paracentricsatellite repeat sequences in these cells are likely to behypomethylated and possibly rearranged and may haveacquired PcG binding capacity. Of interest, in this context, isthe correlation between BMI1 expression levels and itstumorigenicity (van Lohuizen et al., 1991a; Alkema et al.,1997b) and the apparent expressional regulation of Pc-G genesduring normal haematopoietic development (Lessard et al.,1998). In addition, hBmi1 gene amplification was reported inhuman malignancy recently (Beà et al., 1999). Recentmechanistic insights in Polycomb and trithorax-Group functionconnect both deregulation of Pc-G and trx-G genes with cellcycle control and tumorigenesis (reviewed in van Lohuizen,1999). Given these correlations, it will be of considerableinterest to explore α-satellite/Pc-G-association as a possiblediagnostic marker for tumorigenicity.

A model recently put forward (Brown et al., 1997; Marshallet al., 1997) suggests that centromeric heterochromatin formsa transcriptionally repressive subnuclear domain and thatrepressed genes are selectively recruited into such domains. Itis possible that BMI1 target gene(s) are silenced through Pc-G-association at heterochromatin. Active recruitment of genesin a trans-fashion into repressive genomic domains isprecedented in telomere proximity silencing in yeast (Gotta etal., 1997) and somatic pairing as a result of the PEV browndominant mutation in Drosophila (Dernburg et al., 1996;Martin-Morris et al., 1997; Henikoff, 1997). In addition,Drosophila polycomb responsive elements have been recentlyfound to exhibit trans-interactions (Sigrist and Pirrotta, 1998;Pirotta, 1998). The preferential association of BMI1 withchromosome region 1q12 in human cell lines provides anexperimental basis to further test fragments from 1qh-specificα-satellite DNAs in DNA-binding assays or for ectopic actionas PREs.

Cell cycle-dependent distribution of BMI1 correlateswith its phosphorylation statusWe here report a remarkable cell cycle-dependent associationof Pc-G proteins to chromatin in both primary and tumor cells,which correlates well with changes in its phosphorylationstatus. Parallel-staining studies on growing cells with cellcycle-specific markers (PCNA, phosphorylated histone H3)demonstrate that at late S phase the majority of Pc-G proteinsvisibly dissociate from chromatin. This dissociation occurs inboth tumor cell lines and in primary cells expressing BMI1ectopically, arguing the observation is not an artifact or theconsequence of establishment in culture or immortalization/transformation. At G2/M, BMI1 is phosphorylated.Hyperphosphorylated BMI1 does not bind chromatin, asestablished by differential nuclear fractionation. These dataprovide direct biochemical evidence that BMI1-chromatin-association is lost during mitosis and thus corroborate theimmunohistochemical findings. Importantly, our findings alsoindicate that cell cycle-dependent regulation of Pc-G-chromatin association takes mainly place at level of nuclearredistribution. At least some of the protein interactions withinPc-G complexes remain during mitosis, suggesting thatphosphorylation determines Pc-G complex-chromatininteraction: Pc-G proteins are released from chromatin ascomplexes rather than as single units. This may have direct



bearing on the rapid re-establishment of Pc-G-chromatinbinding following nuclear division.

Using more sensitive detection methods, we find that a smallfraction of the BMI1 protein is detectable as a fine dotted layerover metaphase chromosomes and at heterochromatic region1q12 of HeLa, U2-OS and MCF-7 cells. Chromosomes wouldbe stripped of most Pc-G-protein complexes at mitosis, leavinga small, but biologically significant amount of transcriptionalrepressors in place, thereby epigenetically marking relevantloci for transcriptional repression in ensuing cell generations.Our combined data support this view and are in full agreementwith an analogous mechanism proposed for Pc-G-mediatedtranscriptional memory function in Drosophila, where a smallpercentage of Pc-G proteins was shown to remain physicallyattached to chromatin (Buchenau et al., 1998). Taken together,these data confirm that the biochemical interaction of Pc-Gproteins with chromatin and its cell cycle-dependent characterhave been conserved throughout phylogenetically distinct taxa,and add to the importance of phosphorylation in the regulationof chromatin-association.

Implications of phosphorylation-associated Pc-Gprotein-chromatin interactionThe phosphorylation-linked removal of Pc-G complexes atmitosis may simply be required to facilitate chromosomalcondensation and subsequent segregation, a process in whichphosphorylation of histone H3 at Ser10 was recently shown toplay a crucial role (Wei et al., 1999). Significantly, however,members of the trithorax group (trx-G) proteins, a diverseprotein family that functions as counter-actors of Pc-Grepression, are also subject to cell cycle-dependent post-translational modification: hbrm and BRG-1 are specificallyphosphorylated at mitosis (Muchardt et al., 1996). Thismodification correlates well with the chromosomal exclusion ofthe human SNF/SWI complex at G2-M. It was speculated thatthis dissociation could be part of a mechanism leading totranscriptional arrest at mitosis. Based on the observation thatPc-G complexes undergo a similar cell cycle-dependentdisplacement, we suggest an alternative explanation. Sincechromatin dissociation coincides in time with (completion of) denovo DNA-synthesis, the dissociation of Pc-G and trx-Gcomplexes from chromatin during late S-/G2-M phase wouldpresent a dividing cell with an opportunity for specific changesin gene expression patterns. This notion is in line with recentreports that PCNA directs CAF-1-mediated nucleosomeassembly onto newly synthesized DNA (Shibahara and Stillman,1999 and references therein), during a window in the cell cycleat which changes in heritable gene expression can beimplemented through modulation of chromatin structure(reviewed by Wolffe, 1991).

Elegant studies using purified Pc-G complexes have yieldedimportant insights in how Pc-G and trx-G proteins maycounteract each other (Shao et al., 1999). When positioned onnucleosomes prior to exposure to the chromatin remodelingfactors of the SWI/SNF family, Pc-G complexes appear to actas a molecular lock. Conversely, once exposed to SWI/SNFfactors, repression by Pc-G complexes does not occur. Inaddition, both chromatin-remodeling activities appearindependent of histone tails in vitro (Shao et al., 1999). Pc-G-mediated reporter gene silencing in Xenopus oocytes(Strouboulis et al., 1999) and in mammalian cells (our

unpublished observations) is trichostatin A-insensitive, andvarious Pc-G protein-specific antisera fail to coprecipitateHDAC activity from U2-OS and E12.5 embryo extracts (ourunpublished observations). These combined findings suggestthat histone (de)acetylation probably plays a minor role, if at all,in these expressional-maintenance processes by Pc-G and trx-Gproteins. It is conceivable that phosphorylation-dependentchromatin interaction is crucial for proper regulation of Pc-G andtrx-G action and provides coupling to cell cycle progression.Histone H3 was recently identified as a downstream target of aMAPKAP-kinase, which acts in a signaling cascade independentof mitosis (Sassone-Corsi et al., 1999). This exciting findingindicates a potential link between extracellular signaling andchromatin remodeling. In the context of Pc-G and trx-Gfunction, this finding raises the intriguing possibility that as yetunidentified protein kinases and phosphatases play crucial rolesduring development to direct commitment and differentiation-associated gene expression. Importantly, the here describedphosphorylation-linked chromatin dissociation of Pc-Gcomplexes gives a first insight into how Pc-G proteins aredynamically regulated, possibly by extracellular cues.

In summary, we conclude that Pc-G proteins are distributedin a fine-grained pattern along interphase chromosomes ofhuman primary cells, as seen in primary cells of other species.In addition, tumor cells have acquired concentrated Pc-Gbinding at 1qh, a feature that may be a consequence of cellulartransformation. Our data support the notion that the biochemicalinteractions between Pc-G complexes and chromatin and its cellcycle-dependent regulation have been conserved throughoutevolution. The observation that BMI1 is phosphorylated in astrictly cell cycle-dependent manner should pave the way to abetter understanding of how Pc-G group complexes areregulated.

We thank P. F. Ambros, H. Willard, B. Earnshaw and T. Jenuweinfor expert help and advice. We thank T. Ide for TIG3 cells and G.Nolan for providing phoenix packaging cell lines and retroviralvectors. We are grateful to J. Fuchs, F. Klein, M. Lambrou and L.Oomen for help in image acquisition. G. Steiner kindly provided theCREST scleroderma serum; the anti-phosphorylated histone H3antiserum was a kind gift of D. Allis. J.W.V. was supported by a grantfrom the Dutch Organization for Scientific Research (NWO).

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