Interference with the Expression of a Novel Human Polycomb ......Interference with the Expression of...

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/13906168

Interference with the expression of a novel human polycomb protein, hPc2,

results in cellular transformation and apoptosis

Article in Molecular and Cellular Biology · November 1997

DOI: 10.1128/MCB.17.10.6076 · Source: PubMed

CITATIONS

110READS

68

10 authors, including:

Some of the authors of this publication are also working on these related projects:

Establishing of human liver organoid culture View project

SLE and anti-dsDNA antibodies View project

Johan van der Vlag

Radboud University Medical Center (Radboudumc)

257 PUBLICATIONS 9,406 CITATIONS

SEE PROFILE

Karien Hamer

Koninklijke Nederlandse Akademie van Wetenschappen

27 PUBLICATIONS 4,096 CITATIONS

SEE PROFILE

Hugo Masselink

Hogeschool Arnhem and Nijmegen

5 PUBLICATIONS 158 CITATIONS

SEE PROFILE

All content following this page was uploaded by Johan van der Vlag on 29 May 2014.

The user has requested enhancement of the downloaded file.

https://www.researchgate.net/publication/13906168_Interference_with_the_expression_of_a_novel_human_polycomb_protein_hPc2_results_in_cellular_transformation_and_apoptosis?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_2&_esc=publicationCoverPdfhttps://www.researchgate.net/publication/13906168_Interference_with_the_expression_of_a_novel_human_polycomb_protein_hPc2_results_in_cellular_transformation_and_apoptosis?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_3&_esc=publicationCoverPdfhttps://www.researchgate.net/project/Establishing-of-human-liver-organoid-culture?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_9&_esc=publicationCoverPdfhttps://www.researchgate.net/project/SLE-and-anti-dsDNA-antibodies?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_9&_esc=publicationCoverPdfhttps://www.researchgate.net/?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_1&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Johan-Vlag?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Johan-Vlag?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Johan-Vlag?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Karien-Hamer?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Karien-Hamer?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Koninklijke-Nederlandse-Akademie-van-Wetenschappen?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Karien-Hamer?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Hugo-Masselink?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_4&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Hugo-Masselink?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_5&_esc=publicationCoverPdfhttps://www.researchgate.net/institution/Hogeschool_Arnhem_and_Nijmegen?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_6&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Hugo-Masselink?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_7&_esc=publicationCoverPdfhttps://www.researchgate.net/profile/Johan-Vlag?enrichId=rgreq-5e2918bd38919b8aa5c52a9bb0a09c78-XXX&enrichSource=Y292ZXJQYWdlOzEzOTA2MTY4O0FTOjEwMjIxMTM0MjMwNzMzMUAxNDAxMzgwNDkxMjg0&el=1_x_10&_esc=publicationCoverPdf

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/97/$04.0010

Oct. 1997, p. 6076–6086 Vol. 17, No. 10

Copyright © 1997, American Society for Microbiology

Interference with the Expression of a Novel Human PolycombProtein, hPc2, Results in Cellular Transformation

and ApoptosisDAVID P. E. SATIJN,1 DANIEL J. OLSON,2 JOHAN VAN DER VLAG,1 KARIEN M. HAMER,1

CARO LAMBRECHTS,3 HANS MASSELINK,3 MARCO J. GUNSTER,1 RICHARD G. A. B. SEWALT,1

ROEL VAN DRIEL,1 AND ARIE P. OTTE1*

E. C. Slater Instituut, University of Amsterdam, 1018 TV Amsterdam,1 and Divisions of Experimental Therapy andMolecular Carcinogenesis of the Netherlands Cancer Institute, 1066 CX Amsterdam,3 The Netherlands, and

Department of Oral Molecular Biology, School of Dentistry, Cancer Center, Hormonal and Reproductive CancerDivision, Oregon Health Sciences University, Portland, Oregon 972012

Received 19 March 1997/Returned for modification 3 May 1997/Accepted 1 July 1997

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophiladevelopment. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closelyrelated to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1).However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcomacells, suggesting that the proteins are part of a common protein complex. To study the functions of the novelhuman Pc homolog, we generated a mutant protein, DhPc2, which lacks an evolutionarily conserved C-terminaldomain. This C-terminal domain is important for hPc2 function, since the DhPc2 mutant protein which lacksthe C-terminal domain is unable to repress gene activity. Expression of the DhPc2 protein, but not of thewild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypicchanges, altered marker gene expression, and anchorage-independent growth. Specifically in DhPc2-trans-formed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation resultsin apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression.Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 functioncan lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.

The Drosophila Polycomb (Pc) gene is a member of thePolycomb group (PcG) gene family. These genes are part of acellular memory system that is responsible for the inheritanceof gene activity by progeny cells (7, 19, 21, 28, 38). Stable andheritable transmission of gene activity is crucial for the main-tenance of the differentiated identity of cells over many cellgenerations. It has been proposed that PcG proteins represshomeotic gene expression via the formation of multimericcomplexes. This model is based on the observation that differ-ent PcG proteins, including Pc, bind in overlapping patterns onpolytene chromosomes in Drosophila salivary gland cells (32,48). In Drosophila, the PcG protein Polyhomeotic (Ph), but noother known protein, coimmunoprecipitates or cofractionateswith Pc (12). Recently, we found that two human counterpartsof Ph, HPH1 and HPH2, coimmunoprecipitate, cofractionate,and colocalize in nuclear domains with both the vertebratePcG protein BMI1 and a human Pc homolog, indicating thatthese proteins associate in vivo (16). A similar in vivo interac-tion between Bmi1 and a Ph homolog has been found in mice(1). These findings indicate the existence of a vertebrate PcGmultimeric protein complex.

The Pc protein binds to about 100 loci on polytene chromo-somes in Drosophila salivary gland cells (32, 48). These lociinclude the homeotic gene loci and other PcG gene loci (48),indicating that homeotic genes and PcG genes are target genesof Pc. Direct evidence for this idea stems from the observation

that Pc is associated with chromatin of the silent part of thehomeotic bithorax (Ubx) complex (27). Physical association ofthe Pc protein with chromatin of other target loci has not beenreported so far.

Several vertebrate homologs of Pc have been identified (15,30, 33), suggesting that repression of gene activity, mediated byPc, is evolutionarily conserved. This idea is supported by thefinding that a mouse Pc homolog, M33, is able to rescue theDrosophila Pc phenotype (25). Recently, it has become evidentthat M33 displays functions in mice similar to those of Pc inDrosophila. M33-deficient mice show homeotic transforma-tions of the axial skeleton, along with sternal and limb malfor-mations (6).

Many aspects of the molecular mechanism underlying therole of PcG proteins in the stable transmission of gene activityare enigmatic. An important clue about the molecular mech-anism underlying Pc action is the observation that the Pc pro-tein shares a homologous domain with the Drosophila hetero-chromatin-binding protein HP1 (29, 39). The shared motifbetween Pc and HP1 has been termed the chromodomain(chromatin organization modifier) (29). This discovery pro-vides an important, direct link between the regulation of geneactivity and chromatin structure. It suggests that Pc and HP1operate through common mechanisms, which may involve theformation of heterochromatin-like structures. The chromodo-main is essential for binding of the Pc protein to chromatin.When the chromodomain is either mutated or deleted, it nolonger binds to chromatin (24). Also, a conserved domainlocated in the C terminus of the Pc protein (30) is crucial forPc function. In Drosophila, several naturally occurring Pc mu-tants either are mutated in the C-terminal domain or lack this

* Corresponding author. Mailing address: E. C. Slater Instituut,University of Amsterdam, Plantage Muidergracht 12, 1018 TV Am-sterdam, The Netherlands. Phone: 31-20-5255115. Fax: 31-20-5255124.E-mail: [email protected].

6076

domain entirely (13). A mutant Pc gene lacking the C-terminaldomain is unable to repress gene activity (4, 24).

In the present study, we identified a novel human Pc ho-molog, hPc2. We showed that the hPc2 protein and a previ-ously characterized human Pc protein, CBX2 (15), colocalizein nuclear domains of human U-2 OS osteosarcoma cells. Tostudy the functions of hPc2, we designed an hPc2 mutantprotein that lacks the C-terminal domain, which is crucial forthe ability of hPc2 to repress gene activity. Expression of thishPc2 mutant in mammalian cell lines results in (i) cellulartransformation, (ii) enhanced expression of the c-myc proto-oncogene, and (iii) apoptosis upon serum deprivation. Ourdata suggest that hPc2 is a repressor of proto-oncogene activityand that interference with hPc2 function can lead to derepres-sion of proto-oncogene transcription and subsequently to cel-lular transformation.

MATERIALS AND METHODS

Isolation of the hPc2 gene. For screening a lgt10 human fetal brain cDNAlibrary (Clontech, Palo Alto, Calif.), a probe encompassing the entire codingregion of XPc (33) except for the chromodomain (ranging from amino acids [aa]60 to 558) was generated by PCR. The filters were hybridized overnight at 50°Cin 0.53 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–103 Den-hardt’s solution–10% dextran sulfate–0.1% sodium dodecyl sulfate (SDS)–100mg of denatured herring sperm DNA per ml–[a-32P]ATP-labelled probe (5 3 105

cpm/ml). After being washed twice for 60 min at 55°C in 0.53 SSC–0.1% SDS,the filters were exposed to X-ray films with intensifying screens for 2 days at270°C. Positive plaques were isolated and purified. The complete nucleotidesequence of the longest cDNA was determined by sequence analysis.

Analysis of hPc2 transcripts. Multitissue Northern blots containing approxi-mately 2 mg of poly(A)1 RNA per lane from different human tissues or humancell lines were obtained commercially (Clontech). The U-2 OS cell line was notpresent on the commercial Northern blot. Poly(A)1 RNA of U-2 OS was isolatedand blotted, and the expression pattern of hPc2 was analyzed. To allow a com-parison with the commercial Northern blot, we blotted poly(A)1 RNA of SW480cells, which are represented on the commercial blot and in which all three genesare strongly expressed. The blots were hybridized with [a-32P]dATP-labelledDNA probes and autoradiographed with X-ray films and intensifying screens at270°C. Northern blot analyses were performed with an hPc2 probe that excludesthe conserved chromodomain and C-terminal domain (coding for aa 60 to 530).

Production of the M33 and hPc2 polyclonal antibodies. A cDNA fragmentencoding aa 157 to 432 of M33 was cloned into the expression vector pET-23b(Novagen, Madison, Wis.). This fragment does not encompass the conservedchromodomain or the conserved C-terminal domain. A cDNA fragment encod-ing aa 60 to 558 of hPc2 was cloned in pET-23c (Novagen). This fragment doesnot encompass the conserved chromodomain. Fusion proteins were produced inEscherichia coli BL21 (DE), and the purified fusion proteins were used toimmunize rabbits and chicken. Sera were affinity purified over an antigen-cou-pled cyanogen bromide-Sepharose column (Pharmacia, Uppsala, Sweden). Fu-sion proteins or extracts of U-2 OS cells were separated by SDS-polyacrylamidegel electrophoresis (PAGE) and transferred to nitrocellulose. The blots wereprobed with a 1:10,000 dilution of T7 antibody (Novagen) or a 1:1,000 to 1:5,000dilution of affinity-purified anti-hPc2 or anti-M33 antibodies. The secondaryalkaline phosphatase–goat anti-rabbit or anti-chicken IgG (H1L) antibodies(Jackson Immunoresearch Laboratories) were diluted 1:10,000, and nitrobluetetrazolium–5-bromo-4-chloro-3-indolylphosphate toluidinium (NBT/BCIP)(Boehringer) was used as the substrate for detection.

Immunofluorescence staining of tissue culture cells. U-2 OS cells were cul-tured and labeled as described recently (16). Labeling has been analyzed byconfocal laser scanning microscopy, and single optical sections are shown (seeFig. 5). The first two pictures represent the two different detection channels ofthe dual image, and the third picture represents the false-color overlay. Forlabelling, donkey anti-rabbit immunoglobulin G coupled to Cy3 (Jackson Immu-noresearch Laboratories) and donkey anti-chicken IgG coupled to fluoresceinisothiocyanate (FITC) (Jackson) were used.

LexA fusion reporter gene targeted repression assay. The LexA fusion re-porter gene targeted repression assay was performed as described previously (4,35). The chloramphenicol acetyltransferase (CAT) reporter gene has been re-placed by the luciferase gene (LUC). U-2 OS cells were cultured in a 25-cm2 flaskand cotransfected with the heat shock factor (HSF)-inducible LUC reporterplasmid, 4 mg of the LexA fusion constructs, and 2 mg of the pSV/b-Gal construct(Promega) by the calcium phosphate precipitation method. The HSF-inducibleLUC reporter plasmid was activated by exposing the cells at 43°C for 1 h,followed by a 6-h recovery at 37°C, as described previously (4, 35). LUC activitywas normalized to b-galactosidase activity. The absolute values of LUC activityvaried between independent experiments. The LUC activity in cells transfectedwith the LUC reporter plasmid only was therefore set at 100%, and LUC

activities in cells which were cotransfected with indicated plasmids were ex-pressed as a percentage of this control value. The degree of repression is ex-pressed as the mean 6 the standard error of the mean (SEM).

Establishment and characterization of stable C57MG and U-2 OS cell lines.Indicated cDNAs were cloned into the pcDNA3 vector (Invitrogen, San Diego,Calif.), which carries the neomycin resistance gene and in which the indicatedcDNAs are under control of the enhancer from the immediate-early gene ofhuman cytomegalovirus (CMV). C57MG, U-2 OS, or Rat1a cells were trans-fected with Lipofectin (Gibco) as specified by the manufacturer, and stablytransfected lines were selected by culturing the cells for 2 weeks in mediumcontaining 800 mg of Geneticin (G418) (Gibco) per ml. The surviving cells wereclonally expanded for 2 to 4 weeks in medium containing 250 mg of G418 per ml.Individual C57MG and U-2 OS cell clones were selected and cultured in indi-vidual dishes. After five passages, the cell lines were characterized for [3H]thy-midine incorporation and gene expression. [3H]thymidine incorporation wasmeasured as described previously (26). The numbers were normalized to theprotein content. From C57MG cells, expressing low levels of c-myc, poly(A)1

RNA was isolated. From U-2 OS cells, expressing high levels of c-myc, total RNAwas isolated. Isolation of RNA and Northern analysis were performed by stan-dard procedures. The blots were hybridized with [a-32P]dATP-labelled DNAprobes, and the blots were autoradiographed with intensifying screens at 270°Cwith preflashed X-ray films.

Western blot analysis of hPc2 and c-myc proteins. The expression levels of thehPc2 and c-myc proteins were analyzed by using cell lysates of the stably trans-fected C57MG and U-2 OS clones (35). For hPc2 detection, the blots wereincubated with a 1:5,000 dilution of affinity-purified rabbit anti-hPc2 antibodies.The monoclonal 9E10 antibody was used for the detection of c-myc (8). Equalamounts of proteins were loaded, as measured by the bicinchoninic acid method(41) and visualized by Coomassie blue staining of a gel.

Soft agar growth assays. Cell lines were analyzed for anchorage-independentgrowth as described previously (34, 40, 44). Rat1a cells were transfected by thecalcium phosphate transfection procedure with full-length hPc2 (hPc2WT), theC-terminal deletion mutant (DhPc2), and a mutant lacking the chromodomain(Dchromo), which were all cloned in the pcDNA3 vector. As a positive control,the c-myc cDNA which was cloned in the pRc-CMV vector was transfected. Thecells were subjected to selection with 500 mg of G418 per ml. The cells were thencultured for 14 days, at which time the G418-resistant clones were counted. Theclones were trypsinized, and cells were counted. A total of 5 3 104 cells in 5 mlof 10% Dulbecco’s modified Eagle’s medium containing 0.4% (wt/vol) agarosewere seeded in 5-cm petri dishes containing 1% (wt/vol) agarose. The plates wereinspected 14 to 21 days after seeding of the cells, and the colonies were counted.The entire procedure, including the transfection of the cDNAs, was performedin triplicate.

Apoptosis assays. Exponentially growing cells were washed with phosphate-buffered saline, and culture medium containing 10% serum was exchanged forculture medium containing 0.1% serum. After 3 or 6 h, the cells were washedwith cold phosphate-buffered saline and incubated with FITC-conjugated an-nexin V (Nexins Research, Hoeven, The Netherlands) for 15 min at 4°C. Thebinding conditions were as recommended by the manufacturer. After beingwashed, the cells were fixed in 2% paraformaldehyde for 15 min. The cells werestill viable, since they excluded propidium iodine (36). The assay was repeatedfive times with similar results. Beside annexin V staining, C57MG/DPc cellsshowed hallmarks of apoptosis, such as membrane blebbing, cell shrinkage, andchromatin condensation after 24 to 48 h. As an independent assay to detectapoptosis, we monitored apoptosis-induced DNA strand breaks by the TUNEL(TdT-mediated dUTP nick end labeling) assay (Boehringer), in which the ter-minal deoxynucleotidyltransferase (TdT) enzyme activity is measured. Also usingthis assay, we observed apoptosis, specifically in serum-deprived DhPc2-trans-fected cells (36).

Nucleotide sequence accession number. The complete hPc2 sequence has beendeposited with GenBank under accession no. AF013956.

RESULTS

Isolation and characterization of a novel human Pc ho-molog, hPc2. To isolate human Pc homologs, we screened ahuman fetal brain cDNA library with a probe that encom-passes the coding region of a Xenopus Pc homolog, XPc, exceptfor the chromodomain (33). We isolated an 1,867-bp cDNAclone (Fig. 1). This clone contains a 1,674-bp open readingframe (Fig. 2A). The predicted 558-aa protein possesses aconserved region, the chromodomain, which is 96% identicalto the XPc chromodomain, 77% identical to the chromodo-main of the mouse Pc homolog, M33, and 55% identical to theDrosophila Pc chromodomain (Fig. 2A) (29, 30, 33). A con-served C-terminal domain is 100% identical to XPc at theprotein level, 73% identical to M33, and 67% identical to Pc(Fig. 2). Overall, the human protein is 48% identical to XPc.

VOL. 17, 1997 HUMAN Polycomb PROTEIN AND CELLULAR TRANSFORMATION 6077

When conservative changes are taken into account, the humanprotein is 80% similar to the XPc protein. Also, at the nucle-otide level the human cDNA has a striking 52% identity to theXPc cDNA in the entire coding region. We conclude, there-fore, that we have isolated the human homolog of the XPcprotein.

In contrast, the overall homology between the human Pcprotein and the mouse Pc homolog M33 is a mere 24% iden-tity. At the nucleotide level, the novel human Pc homolog has29% identity to the M33 cDNA. This degree of homology isconsiderably lower than that between the human Pc homologand the XPc cDNA. However, when conservative changes aretaken into account, the novel human Pc homolog is 68% sim-ilar to M33 at the protein level. This probably implies that theoverall three-dimensional shapes of the two proteins are verysimilar. This may in turn indicate that the two proteins arefunctionally equivalent.

A partially characterized human homolog of M33, CBX2(15), has 86% identity and 100% similarity to M33 at theprotein level (Fig. 2B). Significant homology between CBX2and our novel human Pc protein is limited to the conservedC-terminal domain (Fig. 2). The CBX2 protein has a mere25% overall identity but a significantly higher 80% similarity tothe novel human Pc homolog (Fig. 2B).

We conclude that there are at least two human Pc homologs.They encode proteins that are homologous only in the con-served C-terminal domain and presumably also in the chromo-domain. The hPc gene that we have isolated is highly homol-ogous to the XPc homolog, whereas the other human Pchomolog, CBX2, is more homologous to the murine Pc ho-molog, M33. For convenience, we named the CBX2 gene hPc1and the gene that we isolated hPc2.

Distribution of hPc2 transcripts in human tissues and can-cer cell lines. The hPc2 cDNA clone that we have identified is

1,867 bp in length, of which 1,674 bp (encoding aa 1 to 558)encompasses the open reading frame. To study the distributionof hPc2 mRNA, we selected a probe which has little homologyto the other vertebrate Pc genes, that is, between the conservedchromodomain and C-terminal domain ranging from aa 60 to530. On a Northern blot, a single transcript of approximately2.8 kb was detected in all the tissues and cell lines tested (Fig.3). In normal human tissues, the highest level of hPc2 expres-sion is found in the thymus (Fig. 3A, lane 2) and in peripheralblood leukocytes (lane 8). Expression levels are still pro-nounced in the spleen (lane 1), prostate (lane 3), testis (lane 4),ovary (lane 5) and small intestine (lane 6) but are very low inthe colon (lane 7). This expression pattern of hPc2 is verysimilar to the expression pattern of the vertebrate PcG genesBMI1 and HPH2. In human tissues, all three PcG genes areexpressed at a high level, except in the colon (16). The expres-sion levels of hPc2 in several human cancer cell lines vary morethan in normal human tissues (Fig. 3B). The hPc2 transcriptsare hardly detectable in the Burkitt’s lymphoma Raji (Fig. 3B,lane 5) and lung carcinoma (lane 7) human cell lines. In con-trast, the expression level of hPc2 is high in all other humancell lines.

In summary, the hPc2 gene is expressed in normal humantissues with almost equal abundance. The hPc2 mRNA levelsare more variable in the different human cell lines.

Characterization of antibodies against hPc2 and hPc1/M33.To further strengthen our conclusion that we have isolated anovel human Pc homolog that is different from the previouslyisolated human M33 homolog, CBX2 (hPc1) (15), we raisedpolyclonal antibodies against hPc2 and M33. We raised anti-bodies against M33 since the full-length cDNA of CBX2 hasnot been reported. The published CBX2 sequence is virtuallyidentical to M33 at the protein level, and we reasoned thatpolyclonal antibodies raised against M33 will recognize the

FIG. 1. Nucleotide sequence of hPc2 and its predicted amino acid sequence. The chromodomain in the N terminus of the protein and the conserved C-terminaldomain of the protein are shaded. A putative nuclear localization signal is underlined. The stop codon of the hPc2 gene is indicated by an asterisk.

6078 SATIJN ET AL. MOL. CELL. BIOL.

closely related human CBX2 protein as well. The predictedmolecular masses of the 558-aa hPc2 and the 519-aa M33 are61 and 55 kDa, respectively. The fusion proteins include a3-kDa T7 tag. A mouse monoclonal antibody against T7 rec-ognized an 85-kDa T7-hPc2 fusion protein and a 79-kDa T7-M33 fusion protein in extracts of the E. coli strain in which thefusion proteins were produced (Fig. 4, lanes 1 and 2, respec-tively). Both proteins have an aberrant mobility on SDS-PAGE. The predicted size difference of 6 kDa between hPc2and M33, however, remains visible on SDS-PAGE.

We raised polyclonal chicken antibodies against T7-taggedhPc2 and polyclonal rabbit antibodies against T7-tagged M33.The chicken anti-hPc2 antibody recognizes the T7-hPc2 fusionprotein but not the T7-M33 fusion protein (Fig. 4, lanes 3 and4, respectively). The rabbit anti-M33 antibody recognizes theT7-M33 fusion protein but not the T7-hPc2 fusion protein(lane 6 and 5, respectively). In extracts of U-2 OS humanosteosarcoma cells, the hPc2 antibody detected an 82-kDaprotein (lane 7) and the M33 antibody detected a 76-kDaprotein (lane 8). The detected molecular masses in extracts ofhuman cells are consistent with the molecular masses of thehPc2 and M33 fusion proteins. We will refer to the humanprotein that is detected by the anti-M33 antibody as hPc1/M33.Although M33 is a murine protein, the human CBX2 (hPc1)protein is virtually identical to M33 (Fig. 2B), and it is there-fore likely that our anti-M33 antibody detects the hPc1 proteinin extracts of human U-2 OS cells. In support of this notion, we

find that the hPc2 and M33 antibodies recognize proteins withidentical molecular masses of, respectively, 82 and 76 kDa inextracts of the murine P19 embryo carcinoma cell line (36) andthe murine mammary epithelial cell line C57MG (see also Fig.7). We conclude that the antibodies specifically recognize hPc2and hPc1/M33 in extracts of human and murine cells.

hPc2 and hPc1/M33 colocalize in nuclei of U-2 OS cells. Wenext analyzed the subcellular localization of the hPc2 proteinin relation to the hPc1/M33 protein by performing immuno-fluorescence labelling experiments. The use of chicken anti-hPc2 and rabbit anti-M33 allowed double-labelling experi-ments. We used human U-2 OS osteosarcoma cells in whichseveral human PcG genes are expressed at a high level (Fig.3B, lane 10) (1, 16). Both the hPc2 and hPc1/M33 proteinswere detected in the nucleus of U-2 OS cells, throughout thenucleoplasm. They are detected in a fine granular pattern aswell as in large, brightly labelled domains (Fig. 5). hPc2 andhPc1/M33 colocalize in these large domains. Using these hPc2-and M33-directed antibodies, we have shown previously thathPc2 coimmunoprecipitates and colocalizes with the vertebratePcG proteins BMI1 and HPH1 (35). Also, M33 protein coim-munoprecipitates and colocalizes with BMI1 (1). We thereforeconclude that hPc2 and hPc1/M33 colocalize in interphasenuclei of U-2 OS human osteosarcoma cells.

An hPc2 mutant lacking the conserved C-terminal region isnot able to repress gene activity. The PcG complex proteinsare known to be involved in repressing homeotic gene activity

FIG. 2. Comparison of the hPc2 protein with other Pc homologs. (A) The sequence is aligned to Xenopus Pc, Pc, M33, and the CBX2 (hPc1) protein sequences.Identical amino acids are shown, and nonidentical amino acids are indicated by dashes. The conserved chromodomain and C-terminal domain (COOH box) are boxed.(B) Alignment of the identified amino acids of CBX2 (hPc1) with corresponding regions of the hPc2 and M33 proteins. Double dots indicate identical amino acids,and single dots indicate conservative changes.


in Drosophila (21, 28). So far, no PcG protein has been foundto bind directly to DNA (28). To investigate the ability of PcGproteins to repress gene activity, they have been targeted toreporter genes as LexA or Gal4 fusion proteins (4, 35). Previ-ously, we have analyzed the ability of LexA-XPc and LexA-hPc2 fusion proteins to repress gene activity in different celllines by using CAT reporter constructs (35). We observed thatLexA-XPc and LexA-hPc2 repress HSF-induced CAT expres-sion to approximately 20% (35). In Drosophila, several natu-rally occurring Pc mutants either are mutated in the C-terminaldomain or lack this domain entirely (13). We tested the abilityof an hPc2 deletion mutant protein, DhPc2, that lacks the last30 aa of the C terminus (Fig. 2) to repress gene activity. U-2OS cells were transfected with a construct containing a tandemof four LexA operators, binding sites for the HSF transcrip-tional activator, and the hsp70 TATA promoter immediatelyupstream of the LUC reporter gene. The endogenous HSF wasused as a transcriptional activator. In the absence of HSF, noLUC activity was observed (36). Maximum LUC activity in thepresence of HSF was set at 100% (Fig. 6, Control). Cotrans-fection of LexA alone had no significant influence on HSF-

induced (Fig. 6, 97% 6 6% [mean 6 SEM], n 5 4) LUCactivity. We found that LexA-hPc2 repressed LUC activity toapproximately 20% (14% 6 4% [mean 6 SEM], n 5 4). WhenLexA-DhPc2 is targeted to the reporter gene, LUC activity isnot significantly repressed (90% 6 9% [mean 6 SEM], n 5 4).The results show that the C-terminal deletion mutant, unlikehPc2, is not able to repress HSF-induced LUC activity. This isin agreement with earlier data which also showed that a Dro-sophila Pc protein which lacks the C-terminal domain is unableto repress gene activity (4). This indicates that the conservedC-terminal region of hPc2 is involved in mediating gene re-pression.

Expression of DhPc2 results in cellular transformation. Tostudy potential functions of hPc2, we expressed the protein inseveral different mammalian cell lines. In Drosophila, severalnaturally occurring Pc mutants either are mutated in the con-served C-terminal domain or lack this domain entirely (13). Amutant hPc2 protein that lacks the C-terminal domain has lostits ability to repress gene activity (Fig. 6). On the other hand,the chromodomain is essential for binding of the Pc protein tochromatin (23). Based on the different functions of these twoconserved domains, we reasoned that a mutant hPc2 proteinthat possesses a functional intact chromodomain but does notcontain the C-terminal domain might still be able to bind to thechromatin but be unable to repress gene activity. Competitionof the mutant hPc2 protein with the endogenous wild-typehPc2 protein might interfere with wild-type hPc2 function.

To test this idea, we expressed wild-type hPc2 (hPc2WT)and the C-terminal deletion mutant (DhPc2) in the murinemammary epithelial C57MG (2, 3, 26) and NIH 3T3 cell linesand in the human U-2 OS osteosarcoma cell line. To testwhether the proteins are indeed properly overexpressed, weanalyzed the hPc2 protein levels of the different, individualclones by Western blotting (Fig. 7). We found higher levels ofthe hPc2 protein in hPc2WT- (Fig. 7, lane 2) and in DhPc2(lane 3)-transfected C57MG cells than in the untransfectedC57MG cells (lane 1). The 82-kDa protein which is detected inthe untransfected C57MG cells is presumably the murine ho-molog of hPc2. Similarly, we found higher levels of hPc2 pro-tein in hPc2WT- (lane 5) and DhPc2 (lane 6)-transfected U-2OS cells than in the untransfected U-2 OS cells (lane 4). These

FIG. 3. Expression patterns of hPc2 in human tissues and human cancer celllines. (A) Expression levels in the spleen, thymus, prostate, testis, ovary, smallintestine, colon, and peripheral blood leukocytes. As a probe, we used the entirehPc2 clone, except for the conserved chromodomain and the C-terminal domain.The filter was rehybridized with a probe for glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) to verify the loading of RNA in each lane. (B) Expressionlevels in promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenousleukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt’s lymphoma Raji,colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361cells. Lane 1 to 8 represent a commercially obtained Northern blot. We alsoisolated and blotted poly(A)1 RNA from U-2 OS. To allow comparison with thecommercial multiple-tissue Northern blot, we isolated and blotted poly(A)1

RNA from SW480 cells (lane 9).

FIG. 4. Antibodies directed against the hPc2 and hPc1/M33 proteins. Full-length cDNA clones of hPc2 and M33 were cloned into pET23 vectors to produceT7-tagged fusion proteins. The T7-hPc2 (lane 1) and T7-M33 (lane 2) fusionproteins were detected with a mouse monoclonal antibody against the T7 tag(Novagen). A chicken antibody raised against the T7-hPc2 fusion protein de-tected the T7-hPc2 protein (lane 3) but not the T7-M33 protein (lane 4). A rabbitantibody raised against the T7-M33 fusion protein recognized the T7-M33 pro-tein (lane 6) but not the T7-hPc2 protein (lane 5). In extracts of U-2 OSosteosarcoma cells, the chicken anti-hPc2 and rabbit anti-M33 detected endog-enous hPc2 (lane 7) and hPc1/M33 (lane 8) proteins, respectively. Analysis wasperformed with a 7% polyacrylamide gel. Molecular masses of the proteins areindicated (in kilodaltons).


results show that the transfected constructs are expressed inthe selected cell clones.

When cultured to confluence, DhPc2-transfected C57MGcells showed striking changes in morphology compared to theparental cell line. Whereas untransfected C57MG cells exhibita “cuboidal” morphology (Fig. 8), C57MG/DhPc2 cells exhibita “streaming” phenotype, characterized by highly elongatedcells (Fig. 8). This latter phenotype is commonly observed intransformed cells and is very similar to the phenotype obtainedwhen the wnt1 oncogene is transfected into C57MG cells (Fig.8) (2, 3, 26). No phenotypic changes were observed in cellstransfected with hPc2WT (Fig. 8). To test whether the pheno-typic changes induced by the DhPc2 mutant were specific forthe deletion of the C-terminal domain, we also transfected aconstruct containing a mutant lacking the chromodomain(Dchromo) but retaining the C-terminal domain. This did notresult in phenotypic changes (36). We also transfected theDhPc2-transformed cells with additional hPc2WT. This re-sulted in reversal of the transformed phenotype (36), suggest-ing that the cellular transformation caused by the DhPc2 mu-tant is due to a dominant-negative effect.

Similar phenotypic changes, characterized by elongated cells,were observed in NIH 3T3 and U-2 OS cells after transfectionwith DhPc2 but not after transfection with hPc2WT. However,transformed C57MG cells are well characterized in terms ofmorphology and changes in the expression of marker genes,and we therefore further characterized the C57MG cells.Transformed C57MG cells that are transfected with either theneuT, wnt1, or wnt2 gene have decreased expression of thewnt4 gene (2, 3, 14, 26). This indicates that wnt4 expression canbe used as a marker for cellular transformation of C57MG cellsresulting from the overexpression of proto-oncogenes. Consid-ering the striking similarities between DhPc2-induced andwnt1-induced phenotypic changes, we tested whether DPc-in-duced phenotypic changes are accompanied by changes in wnt4expression level. We found that wnt4 is strongly down-regu-lated in both C57MG/DhPc2 and C57MG/wnt1 cells whereasno changes in wnt4 levels were observed in C57MG/hPc2WTcells (Fig. 9A). In contrast, overexpression of wild-type hPc2did not have such an effect.

It has further been shown that after reaching confluence,C57MG/wnt1 cells continue to divide, as measured by [3H]thy-midine incorporation (2, 3, 26). Similarly, we found that

C57MG/DhPc2 but not C57MG/hPc2WT cells continued toproliferate at the same rate as C57MG/wnt1 cells (Fig. 9B).

We conclude that expression of the DhPc2 mutant proteinresults in a partial transformation of C57MG, NIH 3T3 andU-2 OS cell lines. A likely mechanism is that this transforma-tion is due to interference of the DhPc2 mutant protein withthe PcG protein complex.

Enhanced, deregulated expression of c-myc in DhPc2-trans-formed cells. The PcG protein complex is involved in repres-sion of gene activity, and our results suggest that interferencewith hPc2 protein function results in derepression of at leastone oncogene. Deregulated expression of this oncogene willsubsequently lead to cellular transformation. To test this idea,we probed a Northern blot containing poly(A)1 mRNA ofC57MG, C57MG/wnt1, C57MG/hPc2WT, and C57MG/DhPc2

FIG. 5. Colocalization of the hPc2 and hPc1/M33 proteins in nuclei of U-2 OS cells. Rabbit anti-M33 (A) and chicken anti-hPc2 (B) labelling is shown in singleconfocal optical sections. (A and B) Two different detection channels of the dual image; (C) false-color overlay. hPc2 and hPc1/M33 are homogeneously distributedin the nucleus but are also concentrated in large, brightly labelled domains. hPc2 and hPc1/M33 colocalize in these large domains (C) (yellow).

FIG. 6. DhPc2, lacking the C-terminal domain, is not able to repress HSF-induced LUC gene activity. LUC reporter expression is maximally induced byHSF in the absence of any LexA fusion protein (Control). Cotransfection ofLexA alone has hardly any influence on HSF-induced LUC expression, which isstill about 97% of the total (LexA). Cotransfection of LexA and hPc2 repressesLUC activity fivefold. Cotransfection of LexA and DhPc2 has no significantrepression effect on LUC activity, which is still 90% of the total. The barsrepresent the average degree of repression by LexA-hPc2 or LexA-DhPc2 in fourindependent experiments (mean 6 SEM). The actual values are indicated in thetext.


cells with cDNA fragments of several oncogenes. We foundthat in C57MG/DhPc2 cells the expression of the c-myc proto-oncogene is strikingly enhanced, concomitant with a decreasein the wnt4 expression level (Fig. 10). In C57MG/wnt1 cells,expression of c-myc was also increased, but this increase wassixfold lower than that in C57MG/DhPc2 cells. No significant

changes in the expression levels of the bmi1, bcl-2, c-fos, andc-jun oncogenes were observed (36). This underlines the spec-ificity of the effect of DhPc2 expression on c-myc expression inC57MG/DhPc2 cells.

The U-2 OS osteosarcoma cell line shows phenotypic char-acteristics of cellular transformation similar to those seen inC57MG after the transfection with DhPc2. We therefore alsoexamined the RNA levels of c-myc in the different U-2 OSclones. We found that in U-2 OS/DhPc2 cells the expression ofc-myc is enhanced (Fig. 11A, lane 3) compared to the controlU-2 OS cells (lane 1). Surprisingly, we detected reduced c-mycRNA levels in U-2 OS/hPc2WT cells (lane 2). This effect wasspecific for c-myc expression, since no reduced c-fos and c-junexpression in U-2 OS/hPc2WT cells was observed (36). Wealso analyzed the expression of the 67-kDa c-myc protein,using the 9E10 monoclonal antibody, which specifically detectsthe human c-myc protein (8). We found that also the c-mycprotein levels are elevated in U-2 OS/DhPc2 (Fig. 11B, lane 3)cells in comparison with control U-2 OS cells (lane 1). As withc-myc RNA levels, a lower level of c-myc protein was found inU-2 OS/hPc2WT cells (lane 2).

In many cell lines, as is the case for C57MG, the expressionlevels of c-myc are very low. Therefore, we needed to isolatepoly(A)1 RNA to be able to detect c-myc RNA in C57MGcells. On the other hand, c-myc levels in U-2 OS cells areknown to be relatively high. In many osteosarcomas, the c-mycgene is amplified (17, 37). Therefore, we were able to detect

FIG. 7. Western analysis of transfected hPc2 proteins from cell extracts ofC57MG and U-2 OS cells. Equal amounts of C57MG and U-2 OS cell extractswere Western blotted. The blots were incubated with a rabbit anti-hPc2 antibody.Endogenous hPc2 levels were detected in the untransfected U-2 OS cells (lane4). Endogenous levels of a murine hPc2 homolog were detected in untransfectedC57MG cells (lane 1). Elevated levels of the hPc2 protein were detected in thehPc2WT- (lanes 2 and 5) and in the DhPc2 (lanes 3 and 6)-transfected cell lines.Analysis was performed with a 10% polyacrylamide gel. Molecular masses of theproteins are indicated (in kilodaltons). The C-terminal 30-aa deletion was toosmall to be detected on these Western blots.

FIG. 8. Overexpression of a DhPc2 deletion mutant induces partial transformation of C57MG cells. The C57MG cells were stably transformed with the wnt1oncogene (C57MG/wnt1), the full-length hPc2 gene (C57MG/hPc2WT), and the DhPc2 deletion mutant, which lacks the conserved C-terminal domain (C57MG/DhPc2). The cells were cultured to confluence before the photographs were taken.


c-myc RNA expression in total RNA in U-2 OS cells. Thedifference in expression levels of c-myc in C57MG and U-2 OScells is also a likely explanation for our detection of a reductionin c-myc expression levels in U-2 OS/hPc2WT cells but not inC57MG/hPc2WT cells. In the last cell line, the c-myc levels arealready low.

In summary, we find that c-myc expression is elevated afterexpression of the DhPc2 protein in two different cell lines. Weconclude that interference with the expression of hPc2 resultsin deregulated, enhanced c-myc transcription. At the sametime, a reduction of c-myc RNA and c-myc protein levels isdetected in U-2 OS cells by the overexpression of hPc2WT,providing extra evidence for a role of hPc2 in the regulation ofc-myc expression.

DhPc2 induces anchorage-independent growth. Rat 1a cells,a fibroblast cell line, have been found to transform and displayanchorage-independent growth in soft agarose by overexpres-sion of c-myc alone (40, 44). Previous studies have shown thatBmi-1, a vertebrate PcG protein and oncoprotein, is able toinduce cellular transformation and anchorage-independentgrowth in Rat 1a cells (5). Since overexpression of DhPc2enhances c-myc transcription, it is possible that DhPc2, like

c-myc, is able to induce anchorage-independent growth of Rat1a cells. We therefore used Rat 1a cells to test the transform-ing effect of DhPc2 as an independent assay for cellular trans-formation.

Rat 1a cells were transfected with c-myc (Rat 1a-myc),hPc2WT (Rat 1a-hPc2WT), DhPc2 (Rat 1a-DhPc2), and anhPc2 mutant lacking the chromodomain (Rat 1a-Dchromo).The results demonstrate that DhPc2 (Rat 1a-DhPc2) as well asc-myc (Rat1a-myc) overexpression alone is sufficient to trans-form cells (Table 1). The number of colonies induced by DhPc2overexpression is comparable to the effect of c-myc overexpres-sion alone. In contrast, overexpression of hPc2WT andDchromo did not induce colonies of Rat1a cells (Table 1).These results demonstrate that DhPc2 is able to induce an-chorage-independent growth when expressed in Rat 1a cells.

Induction of apoptosis, specifically in DhPc2-transformedcells. We addressed the functional significance of the enhancedc-myc expression in C57MG/DhPc2 and U-2 OS/DhPc2 cells. Ithas been shown that overexpression of c-myc, in combinationwith exposure to culture medium containing a low serum con-centration, leads to apoptosis (9–11, 47). To test whether theenhanced expression of c-myc in the two different cell lines alsoresults in apoptosis, we cultured the cells in 0.1% serum. Weobserved extensive cell death of C57MG/DhPc2 cells 24 to 48 hafter serum deprivation (Fig. 12C). In contrast, the numbers ofC57MG cells and C57MG/hPc2WT cells had increased three-to fourfold 48 h after serum deprivation (Fig. 12A and B,respectively). One of the earliest hallmarks of apoptosis is theredistribution of phosphatidylserine from the inner face of theplasma membrane to the cell surface (20, 22). This redistribu-tion can be detected with an FITC conjugate of annexin V, aprotein that has a high affinity for phosphatidylserine (20, 22).After 4 to 6 h of serum deprivation, we observed a substantialnumber of C57MG/DhPc2 cells and cells that could be labelledwith annexin V-FITC at the outside of the cell, 9% 6 3% infive independent experiments (Fig. 12F). In contrast, no an-nexin V-FITC-positive cells were observed in C57MG andC57MG/PcWT cells 6 h after serum deprivation (Fig. 12D andE). Similar results were detected with U-2 OS cells (36). Weconclude that serum deprivation of C57MG/DhPc2 and U-2OS/DhPc2 cells results in extensive cell death, which showshallmarks of apoptosis.

FIG. 9. Changes in gene expression and growth characteristics in C57MG/DhPc2 cells. (A) Poly(A)1 mRNA of C57MG control cells (lane 1), C57MG cellstransfected with only the pcDNA3 plasmid carrying the neomycin resistancegene (lane 2), C57MG/wnt1 (lane 3), C57MG/hPc2WT (lane 4), and C57MG/DhPc2 (lane 5) cells was Northern blotted and probed with a fragment of thewnt4 gene. To verify equal RNA loading, the filter was hybridized with aGAPDH probe. (B) Untransfected C57MG cells and C57MG/NEO, C57MG/wnt1, C57MG/hPc2WT, and C57MG/DhPc2 cells were cultured to confluence,and [3H]thymidine was added to the culture medium. The cells were cultured foran additional 2 days before [3H]thymidine incorporation was determined. Thecounts were normalized to the protein content. The [3H]thymidine incorporationby control, untransfected C57MG cells is set at 100%. Representative results offour independent experiments are shown.

FIG. 10. c-myc expression is enhanced in C57MG/DhPc2 cells. Poly(A)1

mRNA of C57MG control cells (lane 1) and C57MG/wnt1 (lane 2), C57MG/hPc2WT (lane 3), and C57MG/DhPc2 cells (lane 4) was Northern blotted andprobed with fragments of the c-myc and wnt4 genes. To verify equal RNAloading, the filter was hybridized with a GAPDH probe.


DISCUSSION

Two human Pc homologs exist. The Pc protein is involved inthe stable and heritable repression of gene activity duringDrosophila development. To study the functions of vertebratePc homologs, we have isolated and characterized a novel hu-man Pc homolog. To isolate this gene, we used the Xenopushomolog of Pc, XPc, as a probe. Overall, the novel human Pchomolog has 48% identity and 80% similarity to the XPcprotein. Based on sequence homologies with another, previ-ously characterized human Pc homolog, CBX2 or hPc1, andthe murine Pc homolog, M33, we conclude that we have iso-lated a novel human Pc homolog, hPc2, which is distinct fromhPc1/M33. Whereas the hPc2 protein is most closely related toXPc, the hPc1 protein is most closely related to M33.

It appears to be a common feature among vertebrate PcGproteins that each of them exists as a pair of closely relatedproteins. For instance, the vertebrate PcG proteins Bmi1 (33)and mel-18 (45) have large identical regions (alignment shownin reference 33). Furthermore, we recently identified two hu-man proteins, HPH1 and HPH2, that both have extensivesequence homology to the Drosophila PcG protein Polyho-meotic (Ph) in two conserved homology domains (16). Strik-ingly, homology between the HPH1 and HPH2 proteins them-selves is restricted to these conserved homology domains;outside these domains, homologies are very limited. This isvery similar to what we report here for hPc1/M33 and hPc2.Overall homologies between hPc2 and hPc1/M33 do not ex-ceed the homologies between these vertebrate Pc homologsand Drosophila Pc. It is important, however, to note that whennot only identical amino acids but also conservative changesare taken into consideration, the similarity between hPc2 and

hPc1/M33 is 68%. This is significantly higher than the 29%identity. As already pointed out, this could indicate that theoverall three-dimensional structures of hPc2 and hPc1/M33 arevery similar, which may imply that the two proteins are func-tionally equivalent. It is also possible, however, that the strongconservation in the chromodomain and C-terminal domain isenough to provide functional equivalence to the proteins. Al-though homology between M33 and Drosophila Pc is restrictedto the chromodomain and the C-terminal domain, M33 is ableto partly rescue the Drosophila Pc phenotype when overex-pressed in the Pc mutant (25). This indicates that hPc1/M33can be considered a functional homolog of Pc, and it signifiesthe importance of the conserved regions. The potential func-tional relationship between the two human Pc homologs isfurther underlined by our finding that hPc1/M33 and hPc2colocalize in nuclei of human U-2 OS cells, suggesting thathPc1 and hPc2 are part of a human PcG protein complex. Inthis context, it is significant that the human PcG protein BMI1and the human Polyhomeotic-related HPH1 and HPH2 pro-teins also colocalize with hPc2 and hPc1/M33 in the samenuclear domains of several human cell lines (1, 16).

We conclude that there are at least two human Pc homologs.It is not clear why two closely related human Pc proteins exist.Since other vertebrate PcG-related proteins have been foundto exist as functional pairs as well, this may have functionalsignificance. It is possible that small differences induce subtlechanges in, for instance, their specificities for binding to targetgenes.

c-myc is a potential target gene of hPc2. In Drosophila, PcGproteins have been identified as repressors of gene expression.The only identified target genes of PcG proteins are homeoticgenes and gap genes (28, 31, 38, 48). These genes are allinvolved in developmental decisions. In this study we foundthat expression of a mutant hPc2 cDNA in two different mam-malian cell lines, U-2 OS and C57MG, results in deregulated,enhanced expression of a gene that controls a different process.This gene, c-myc, is involved in cell cycle and differentiationevents. The mutant hPc2 protein lacks a conserved C-terminaldomain that is crucial for the ability of the hPc2 protein torepress gene activity. Further, overexpression of the wild-typehPc2 cDNA results in decreased expression of c-myc. It istherefore likely that it is due to interference with hPc2 functionthat c-myc expression is deregulated and enhanced. Fromthese data, however, it cannot be concluded whether the effecton c-myc expression is a direct or indirect effect. It is temptingto speculate that hPc2 interacts directly with the c-myc locus.Unfortunately, the fact that Pc proteins binds to chromatin andnot to naked DNA excludes the use of standard methods, suchas DNA footprinting, to assess whether the hPc2 protein isphysically associated with the c-myc locus. Association of Dro-

FIG. 11. c-myc expression is enhanced in U-2 OS/DhPc2 cells. (A) TotalRNA of U-2 OS control (lane 1), U-2 OS/hPc2WT (lane 2), and U-2 OS/DhPc2(lane 3) cells was Northern blotted and probed with a fragment of the c-mycgene. To verify equal RNA loading, the filter was hybridized with a GAPDHprobe. (B) Cell extracts of U-2 OS control (lane 1), U-2 OS/hPc2WT (lane 2),and U-2 OS/DhPc2 (lane 3) cells were analyzed on a Western blot. The blotswere incubated with the mouse monoclonal antibody 9E10, which specificallyrecognizes the human c-myc protein. Molecular masses of the proteins areindicated (in kilodaltons).

TABLE 1. Colony formation by DhPc2-transfected Rat 1Acells in soft agarose

Construct No. of colonies/5 3 104

transfected cellsa

pRcCMV c-myc..................................................................451 6 52pcDNA3 .............................................................................. 0pcDNA3-hPc2WT.............................................................. 0pcDNA3-DhPc2..................................................................337 6 27pcDNA3-Dchromo............................................................. 0

a A total of 5 3 104 of each pool of transfected and geneticin-selected cellswere seeded into 0.4% top agarose, and colonies with diameters of .0.1 mmwere counted 14 to 21 days after seeding (34, 40, 44). The entire procedure,including the transfection of the cDNAs, was performed in triplicate, and themean 6 SEM is shown.


sophila Pc with chromatin of target genes has so far only beenproven for chromatin of the homeotic bithorax (Ubx) genelocus (27). In future studies, we will address the question of apotential, direct association of hPc2 with the c-myc locus byemploying the in vivo cross-linking method (27). This currentlack of knowledge does not, however, detract from our findingthat reveals a novel, hitherto unexpected level of regulation ofc-myc. This involves regulation of gene activity at the level ofchanges in chromatin structure, in this case involving one ofthe PcG proteins.

We observed extensive cell death, with the hallmarks ofapoptosis, specifically of C57MG/DhPc2 transformed cells af-ter serum deprivation. Apoptosis has been shown to occurupon overexpression of c-myc, in combination with serum de-privation (11–13). Apoptosis therefore appears to be a directconsequence of the deregulated, enhanced c-myc expression inC57MG/DhPc2 cells. Apoptosis has not been described forDrosophila Pc mutants. However, it is noteworthy that muta-tions in another PcG gene, polyhomeotic (Ph), lead to extensivecell death between 9.5 and 12 h after egg laying, specifically inthe ventral epidermis (42, 43). This precisely defined timewindow and the cell type specificity of cell death point toapoptosis as the underlying cause. It would be of interest toexamine this previously described phenomenon in the light ofour present results.

Involvement of hPc in cellular transformation and apopto-sis. We show that interference with hPc2 function by ectopicexpression of the hPc2 deletion mutant that lacks the con-served C-terminal domain results in cellular transformation ofmammalian cell lines. Concomitantly, the expression of thec-myc proto-oncogene is enhanced in these transformed cells.It is therefore tempting to speculate that one function of themammalian Pc proteins is to repress the transcription of cer-

tain proto-oncogenes. Interference with hPc2 function willthen result in derepression of transcriptionally repressedproto-oncogenes and subsequently in cellular transformation.Importantly, our results do not constitute the only link betweenPcG proteins and cellular transformation. Two other mamma-lian PcG proteins, bmi1 and mel-18, have been shown to beinvolved in tumorigenesis (5, 18, 46). However, molecularmechanisms that underlie oncogenesis due to changes in chro-matin structure have hardly been explored so far. It will bechallenging to search for tumor cell lines in which the functionof hPc2 is disturbed, either by mutations or by changed expres-sion levels.

ACKNOWLEDGMENTS

D.P.E.S. and D.J.O. contributed equally to this work.We thank René Bernards for suggesting the apoptosis experiments

and for providing probes and the Rat1a cells, Robert Kingston forplasmids, Andy McMahon for the wnt4 probe, Jan Kooter for criticallyreading the manuscript, and Thijs Hendrix for raising the antibodies.

A.P.O. is sponsored by a grant from the Royal Netherlands Acad-emy of Sciences (KNAW).

REFERENCES1. Alkema, M. J., M. Bronk, E. Verhoeven, A. P. Otte, L. J. van’t Veer, A. Berns,

and M. van Lohuizen. 1997. Identification of Bmi1-interacting proteins asconstituents of a multimeric mammalian Polycomb complex. Genes Dev.11:226–240.

2. Bradley, R. S., and A. M. C. Brown. 1995. A soluble form of Wnt-1 proteinwith mitogenic activity on mammary epithelial cells. Mol. Cell. Biol. 15:4616–4622.

3. Brown, A. M. C., R. S. Wildin, T. J. Prendergast, and H. E. Varmus. 1986. Aretrovirus vector expressing the putative mammary oncogene int-1 causespartial transformation of a mammary epithelial cell line. Cell 46:1001–1009.

4. Bunker, C. A., and R. E. Kingston. 1994. Transcription repression by Dro-sophila and mammalian Polycomb group proteins in transfected mammaliancells. Mol. Cell. Biol. 14:1721–1732.

FIG. 12. Apoptosis in serum-deprived C57MG/DhPc2 cells. (A to C) The 10% fetal calf serum in the culture medium of exponentially growing C57MG control cells(A), C57MG/hPc2WT (B) and C57MG/DhPc2 cells (C) was replaced with 0.1% fetal calf serum. The cells were counted after 24 and 48 h. Open symbols representcells continuing to grow in medium containing 10% serum, and solid symbols represent cells growing in 0.1% serum-containing medium. (D to F) C57MG control cells(D), C57MG/hPc2WT cells (E), and C57MG/DhPc2 cells (F) were grown for 6 h in 0.1% serum before they were vitally labelled with FITC-conjugated annexin V. Thetop panels show annexin V labelling, and the bottom panels show the phase-contrast images of the corresponding top panels.


5. Cohen, K. J., J. S. Hanna, J. E. Prescott, and C. V. Dang. 1996. Transfor-mation by the Bmi-1 oncoprotein correlates with its subnuclear localizationbut not its transcriptional suppression activity. Mol. Cell. Biol. 16:5527–5535.

6. Coré, N., S. Bel., S. J. Gaunt, M. Aurrand-Lions, J. Pearce, A. Fischer, andM. Djabali. 1997. Altered cellular proliferation and mesoderm patterning inpolycomb-M33-deficient mice. Development 124:721–729.

7. Duncan, I., and E. B. Lewis. 1982. Genetic control of body segment differ-entiation in Drosophila, p. 533–554. In G. Subtelny (ed.), Developmentalorder: its origin and regulation. Alan R. Liss, Inc., New York, N.Y.

8. Evan, G. I., G. K. Lewis, G. Ramsay, and M. Bishop. 1985. Isolation ofmonoclonal antibodies specific for human c-myc proto-oncogene product.Mol. Cell. Biol. 5:3610–3616.

9. Evan, G., E. Harrington, A. Fanadi, H. Land, B. Amati, and M. Bennett.1994. Integrated control of cell proliferation and cell death by the c-myconcogene. Philos. Trans. R. Soc. London Ser. B 345:269–275.

10. Evan, G., A. Wyllie, C. Gilbert, T. Littlewood, H. Land, M. Brooks, C.Waters, L. Penn, and D. Hancick. 1992. Induction of apoptosis in fibroblastsby c-myc protein. Cell 69:119–125.

11. Fanidi, A., E. A. Harrington, and G. Evan. 1992. Cooperative interactionbetween c-myc and bcl-2 proto-oncogenes. Nature (London) 359:554–556.

12. Franke, A., M. DeCamillis, D. Zink, N. Cheng, H. W. Brock, and R. Paro.1992. Polycomb and polyhomeotic are constituents of a multimeric proteincomplex in chromatin of Drosophila melanogaster. EMBO J. 11:2941–2950.

13. Franke, A., S., Messmer, and R. Paro. 1995. Mapping functional domains ofthe Polycomb protein of Drosophila melanogaster. Chromosome Res. 3:351–360.

14. Gavin, B. J., and A. P. McMahon. 1992. Differential regulation of the Wntgene family during pregnancy and lactation suggests a role in postnataldevelopment of the mammary gland. Mol. Cell. Biol. 12:2418–2423.

15. Gecz, J., S. J. Gaunt, E. Passage, R. D. Burton, C. Cudrey, J. J. Pearce, andM. Fontes. 1995. Assignment of a Polycomb-like chromobox gene (CBX2) tohuman chromosome 17q25. Genomics 26:130–133.

16. Gunster, M. J., D. P. E. Satijn, K. M. Hamer, J. L. den Blaauwen, D. deBruijn, M. J. Alkema, M. van Lohuizen, R. van Driel, and A. P. Otte. 1997.Identification and characterization of interactions between the vertebratePolycomb-group protein BMI1 and human homologs of Polyhomeotic. Mol.Cell. Biol. 17:2326–2335.

17. Isfort, R. J., D. B. Cody, and C. J. Doersen. 1995. Analysis of oncogenes,tumor suppressor genes, autocrine growth-factor production, and differen-tiation state of human osteosarcoma cell lines. Mol. Carcinog. 14:170–178.

18. Kanno, M., M. Hasegawa, A. Ishida, K. Isono, and M. Taniguchi. 1995. mel-18,a Polycomb group-related mammalian gene, encodes a transcriptional negativeregulator with tumor suppressive activity. EMBO J. 14:5672–5678.

19. Kingston, R. E., C. A. Bunker, and A. N. Imbalzano. 1996. Repression andactivation by multiprotein complexes that alter chromatin structure. GenesDev. 10:905–920.

20. Koopman, G., C. P. M. Reutelingsperger, G. A. M. Kuijten, R. M. J. Keeh-nen, S. T. Pals, and M. H. J. van Oers. 1994. Annexin V for flow cytometricdetection of phosphatidylserine expression on B cells undergoing apoptosis.Blood 84:1415–1420.

21. Lewis, E. B. 1978. A gene complex controlling segmentation in Drosophila.Nature (London) 276:565–570.

22. Martin, S. J., C. P. M. Reutelingsperger, A. J. McGahon, J. A. Rader, R. C.van Schie, D. M. LaFace, and D. R. Green. 1995. Early redistribution ofplasma membrane phosphatidylserine is a general feature of apoptosis re-gardless of the initiating stimulus: inhibition by overexpression of Bcl-2 andAbl. J. Exp. Med. 182:1545–1556.

23. Messmer, S., A. Franke, and R. Paro. 1992. Analysis of the functional role ofthe Polycomb chromo domain in Drosophila melanogaster. Genes Dev. 6:1241–1254.

24. Müller, J. 1995. Transcriptional silencing by the Polycomb protein in Dro-sophila embryos. EMBO J. 14:1209–1220.

25. Müller, J., S. J. Gaunt, and P. A. Lawrence. 1995. Function of the Polycombprotein is conserved in mice and flies. Development 121:2847–2852.

26. Olson, D. J., and J. Papkoff. 1994. Regulated expression of Wnt family

members during proliferation of C57mg mammary cells. Cell Growth Differ.5:197–206.

27. Orlando, V., and R. Paro. 1993. Mapping Polycomb-repressed domains in theBithorax complex using in vivo formaldehyde cross-linked chromatin. Cell75:1187–1198.

28. Paro, R. 1990. Imprinting a determined state into the chromatin of Drosoph-ila. Trends Genet. 6:416–421.

29. Paro, R., and D. S. Hogness. 1991. The Polycomb protein shares a homolo-gous domain with a heterochromatin-associated protein of Drosophila. Proc.Natl. Acad. Sci. USA 88:263–267.

30. Pearce, J. J. H., P. B. Singh, and S. J. Gaunt. 1992. The mouse has aPolycomb-like chromobox gene. Development 114:921–929.

31. Pelegri, F., and R. Lehmann. 1994. A role of Polycomb group genes in theregulation of Gap gene expression in Drosophila. Genetics 136:1341–1353.

32. Rastelli, L., C. S. Chan, and V. Pirotta. 1993. Related chromosome bindingsites for zeste, suppressors of zeste and Polycomb group proteins in Drosophilaand their dependence on Enhancer of zeste function. EMBO J. 12:1513–1522.

33. Reijnen, M. J., K. M. Hamer, J. L. den Blaauwen, C. Lambrechts, I. Schon-eveld, R. van Driel, and A. P. Otte. 1995. Polycomb and bmi-1 homologs areexpressed in overlapping patterns in Xenopus embryos and are able to in-teract with each other. Mech. Dev. 53:35–46.

34. Samuels, M. L., M. J. Weber, J. M. Bishop, and M. McMahon. 1993.Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human Raf-1protein kinase. Mol. Cell. Biol. 13:6241–6252.

35. Satijn, D. P. E., M. J. Gunster, J. van der Vlag, K. M. Hamer, W. Schul, M. J.Alkema, A. J. Saurin, P. S. Freemont, R. van Driel, and A. P. Otte. 1997.RING1 is associated with the Polycomb-group protein complex and acts asa transcriptional repressor. Mol. Cell. Biol. 17:4105–4113.

36. Satijn, D. P. E., and A. P. Otte. Unpublished data.37. Schon, A., L. Michiels, M. Janowski, J. Merregaert, and V. Erfle. 1986.

Expression of proto-oncogenes in murine osteosarcomas. Int. J. Cancer38:67–74.

38. Simon, J. 1995. Locking in stable states of gene expression: transcriptionalcontrol during Drosophila development. Curr. Opin. Cell Biol. 7:376–385.

39. Singh, P. B., J. R. Miller, J. Pearce, R. Kothary, R. D. Burton, R. Paro, T. C.James, and S. Gaunt. 1991. A sequence motif found in a Drosophila hetero-chromatin protein is conserved in animals and plants. Nucleic Acids Res.19:789–794.

40. Small, M. B., N. Hay, M. Schwab, and J. M. Bishop. 1987. Neoplastictransformation by the human gene N-myc. Mol. Cell. Biol. 7:1638–1645.

41. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner,M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C.Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Bio-chem. 150:76–85.

42. Smouse, D., C. Goodman, A. Mahowald, and N. Perrimon. 1988. polyho-meotic: a gene required for the embryonic development of axon pathways inthe central nervous system of Drosophila. Genes Dev. 2:830–842.

43. Smouse, D., and N. Perrimon. 1990. Genetic dissection of a complex neu-rological mutant, polyhomeotic, in Drosophila. Dev. Biol. 139:169–185.

44. Stone, J., T. de Lange, G. Ramsay, E. Jakobovits, J. M. Bishop, H. Varmus,and W. Lee. 1987. Definition of regions in human c-myc that are involved intransformation and nuclear localization. Mol. Cell. Biol. 7:1697–1709.

45. Tagawa, M., T. Sakamoto, K. Shigemoto, H. Matsubara, Y. Tamura, T. Ito,I. Nakamura, A. Okitsu, K. Imai, and M. Taniguchi. 1990. Expression ofnovel DNA-binding protein with zinc finger structure in various tumor cells.J. Biol. Chem. 265:20021–20026.

46. Van Lohuizen, M., S. Verbeek, B. Scheijen, E. Wientjes, H. van der Gulden,and A. Berns. 1991. Identification of cooperating oncogenes in E(mu)-myctransgenic mice by provirus tagging. Cell 65:737–752.

47. Wagner, A. J., J. M. Kokontis, and N. Hay. 1994. Myc-mediated apoptosisrequires wild-type p53 in a manner independent of cell cycle arrest and theability of p53 to induce p21waf1/cip1. Genes Dev. 8:2817–2830.

48. Zink, B., and R. Paro. 1989. In vivo binding pattern of a transregulator ofhomeotic genes in Drosophila melanogaster. Nature (London) 337:468–471.


View publication statsView publication stats
https://www.researchgate.net/publication/13906168

Interference with the Expression of a Novel Human Polycomb ......Interference with the Expression of...

Documents

Transcript of Interference with the Expression of a Novel Human Polycomb ......Interference with the Expression of...