PHC3, a Component of the Hprc-H Complex, Associates with E2F6 During G0 and Is Lost in Osteosarcoma Tumors

Oncogene (2007) 26, 1714–1722 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE PHC3, a component of the hPRC-H complex, associates with E2F6 during G0 and is lost in osteosarcoma tumors

AM Deshpande1,4, JD Akunowicz1, XT Reveles2, BB Patel1, EA Saria1, RG Gorlick3, SL Naylor2, RJ Leach2 and MF Hansen1

1Center for Molecular Medicine, University of Connecticut Health Center, Farmington, CT, USA; 2Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA and 3Department of Pediatrics, The Children’s Hospital at Monteﬁore, Bronx, NY, USA

Polyhomeotic-like 3 (PHC3) is a ubiquitously expressed eucaryotes. PcG complexes are responsible for long- member of the polycomb gene family and part of the term silencing of genes by altering chromatin structure human polycomb complex hPRC-H. We found that in through deacetylation of histone tails and by inhibiting normal cells PHC3 associated with both hPRC-H ATP-dependent chromatin remodeling (Shao et al., complex components and with the transcription factor 1999; van der Vlag and Otte, 1999). E2F6. In differentiating and confluent cells, PHC3 and Studies have demonstrated that PcG proteins divide E2F6 showed nuclear colocalization in a punctate pattern into two separate complexes (Ali and Bender, 2004; that resembled the binding of polycomb bodies to Cao and Zhang, 2004). The first complex, which in- heterochromatin. This punctate pattern was not seen in cludes homologues of the Drosophila esc and E(z) genes, proliferating cells suggesting that PHC3 may be part of has histone deacetylase activity (Sewalt et al., 1998; an E2F6-polycomb complex that has been shown to van Lohuizen et al., 1998). The second PcG complex occupy and silence target promoters in G0. Previous loss (dPRC-1 in Drosophila, mPRC1 in mice and hPRC-H of heterozygosity (LoH) analyses had shown that the in humans) contains homologues of the Drosophila Pc, region containing PHC3 underwent frequent LoH in Psc, Ph, RING and Scm genes (Franke et al., 1992; primary human osteosarcoma tumors. When we examined Alkema et al., 1997; Satijn et al., 1997; Shao et al., 1999) normal bone and human osteosarcoma tumors, we found and can mediate silencing of target genes by interfering loss of PHC3 expression in 36 of 56 osteosarcoma tumors. with SWI/Snf chromatin remodeling machinery, block- Sequence analysis revealed that PHC3 was mutated in ing transcriptional initiation and the recruitment of nine of 15 primary osteosarcoma tumors. These findings additional silencing activities (Shao et al., 1999; Francis suggest that loss of PHC3 may favor tumorigenesis by et al., 2001; King et al., 2002; Levine et al., 2002; Dellino potentially disrupting the ability of cells to remain in G0. et al., 2004; Lavigne et al., 2004). Oncogene (2007) 26, 1714–1722. doi:10.1038/sj.onc.1209988; In humans, there are three orthologues of the published online 25 September 2006 Drosophila Ph gene: PHC1, PHC2 and PHC3 (Gunster et al., 1997; Levine et al., 2002; Tonkin et al., 2002). Keywords: tumor suppressor gene; osteosarcoma; poly- Database screening has shown that all three are comb gene; polyhomeotic-like; cell cycle regulation evolutionarily conserved in vertebrates (Isono et al., 2005). Each of the PHC genes encodes a (Cys)4-type zinc coordination domain (Deatrick et al., 1991), and a sterile alpha motif (SAM) domain (Ponting, 1995) at the Introduction C-terminus. The SAM domain is common to several PcG genes and has been shown to be involved in The Polycomb group genes (PcG) encode a diverse set of protein–protein interactions (Kim et al., 2002; Isono regulatory proteins that are involved in maintenance but et al., 2005). not initiation of expression patterns that control We set out to characterize the function of PHC3. As development (Lund and van Lohuizen, 2004; Ringrose expected, we found that PHC3 interacted with members and Paro, 2004). First identified in Drosophila of the hPRC-H complex. We also found that PHC3 (Lewis, 1978), they have been found throughout higher interacted with E2F6, possibly as part of a complex that occupies E2F- and Myc-responsive genes in G0 (Ogawa Correspondence: Dr MF Hansen, Center for Molecular Medicine, et al., 2002). PHC3 maps to a region of human University of Connecticut Health Center, 263 Farmington Avenue, chromosome 3q26 that has been found to undergo Farmington, CT 06030, USA. tumor-specific loss of heterozygosity (LoH) in human E-mail: [email protected] osteosarcomas (Kruzelock et al., 1997). Because of its 4Current address: Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111, USA potential role in regulating gene expression during G0 Received 10 May 2005; revised 24 July 2006; accepted 24 July 2006; and its localization within a region of frequent loss, we published online 25 September 2006 hypothesized that PHC3 might be a tumor suppressor PHC3 acts as a tumor suppressor gene in osteosarcoma AM Deshpande et al 1715 gene. We found that PHC3 expression was present in induction (Figure 2). This punctate pattern continued normal bone but absent in a majority of primary throughout differentiation. osteosarcoma tumors. When we examined DNA from A similar pattern of punctate localization was primary osteosarcomas, we discovered somatic intra- observed in confluent cells. In nonconfluent cells, genic inactivating mutations. These findings were con- PHC3 was diffusely nuclear. As cells became confluent, sistent with the role of PHC3 as a tumor suppressor PHC3 showed an increasingly punctate sublocalization gene in human osteosarcoma. (Figure 3). The punctate pattern of localization of PHC3 was similar to that seen for heterochromatin-associating polycomb bodies. Results PHC3 and E2F6 colocalize in confluent and PHC3 is part ofthe hPRC-H PcG complex but also differentiating cells binds E2F6 To test whether PHC3 was part of an E2F6-containing Co-immunoprecipitation analyses confirmed that Phc3 complex, we examined the colocalization of PHC3 and co-immunoprecipitated with proteins YY1 and M33 as E2F6. Human mesenchymal stem cell (hMSC) cultures previously reported (Levine et al., 2002) (Figure 1). We were analysed for subcellular localization of PHC3 and found that Phc3 also co-immunoprecipitated with E2F6 E2F6 at 50% confluency and at >80% confluency. In and Bmi1. As a negative control, Phc3 did not co- cells that were 50% confluent, both PHC3 and E2F6 immunoprecipitate with CtBP or RB1 (data not shown), were expressed diffusely in the nucleus. In contrast, when which have been shown to be part of a different PcG the cells were >80% confluent, PHC3 and E2F6 were complex (Dahiya et al., 2001), or with the unrelated colocalized in the nucleus in a punctate pattern (Figure 4). protein Sox9. The data confirmed that PHC3 was part Similar patterns of colocalization were seen when the cells of the hPRC-H complex, which has previously been were induced to differentiate along the osteogenic lineage shown to contain PHC3, YY1 and M33 (Levine et al., (data not shown). This punctate pattern of colocalization 2002) and demonstrated that PHC3 may be part of an was similar to that seen for PHC3 alone in differentiating E2F6-containing PcG complex (Trimarchi et al., 2001; and confluent cells (Figures 2 and 3). This strongly Ogawa et al., 2002). suggested that PHC3 and E2F6 were part of a G0-specific DNA-binding complex (Ogawa et al., 2002). Subcellular localization ofPHC3 is dependent on cell cycle stage Absence ofexpression of PHC3 in primary osteosarcoma PcG proteins form distinct nuclear foci called polycomb tumors bodies that associate with heterochromatin (Saurin If PHC3 was part of an E2F6 G0-regulating complex, et al., 1998). PHC1 and PHC2 are both found in these then loss of PHC3 function from this complex might polycomb bodies (Gunster et al., 1997). Analysis of affect cells entering or remaining in G0, favoring PHC3 expression in human osteoblasts (NHOst) tumorigenesis. Significantly, PHC3 maps to a region showed that PHC3 was diffusely localized in the nuclei of human chromosome 3q26.2 that was shown to of proliferating cells. Following induction of differen- undergo frequent LoH in osteosarcomas (Kruzelock tiation, a punctate pattern of nuclear distribution et al., 1997). As LoH is frequently associated with for PHC3 was observed as soon as day 2 following homozygous inactivating mutations that lead to loss of

Figure 1 Co-immunoprecipitation analysis. Total protein lysates from adult mouse bone were immunoprecipitated with antibody against Phc3, followed by Western blot with a second antibody. Western blotting with antibodies against Bmi1, M33, YY1 and E2F6 detected the presence of these proteins complexed with Phc3. No association was detected using an antibody against Sox9. IP, antibody used for immunoprecipitation; WB, antibody used for Western blot detection; TL, total protein lysate; —, negative control.

Oncogene PHC3 acts as a tumor suppressor gene in osteosarcoma AM Deshpande et al 1716

Figure 2 Subcellular localization of PHC3 in differentiating NHOst cells. Cells seeded at low density were induced to terminally differentiate and PHC3 expression was analyzed at 0, 2, 5 and 7 days post-induction. Analyses of uninduced cells were performed at the same times. Induction of differentiation resulted in a shift to a punctate pattern of PHC3 expression with punctate staining becoming more pronounced through day 7 (left panels). Punctate PHC3 expression was not seen in uninduced cells until conﬂuency was achieved at approximately day 7 (right panels). At day 13 post- induction, induced and uninduced NHOst cells were stained using Alizarin Red to demonstrate mineralization, a function of terminal osteoblast differentiation ( Â 100 magniﬁcation).

Figure 3 Immunofluorescence analysis of confluent NHOst cells. expression of tumor suppressor genes (Haber and Cells were analysed for PHC3 localization at 50% confluency and Harlow, 1997), we tested for PHC3 and E2F6 expression at >80% confluency as well as on days 2, 5 and 7 following in human osteosarcoma tumor tissue arrays. Fifty-six confluency. PHC3 was localized in the nucleus in a punctate informative cases of osteosarcoma were examined. As a expression pattern in the confluent cells. positive control, we examined coexpression in a dpc12.5 embryonic mouse. Our analysis showed that Phc3 was expressed in the cells of the embryonic mouse skeleton Somatic intragenic mutations disrupt PHC3 in human and PHC3 expression was absent in 36 of 56 (64%) primary osteosarcoma tumors osteosarcoma tumors. E2F6 was present in all of the As PHC3 expression was absent in a majority of tumors and the developing mouse skeleton. Examples of osteosarcoma tumors, we sequenced matched sets of the analysis are shown in Figure 5. normal and tumor DNAs from 15 patients with

Oncogene PHC3 acts as a tumor suppressor gene in osteosarcoma AM Deshpande et al 1717 A map of the mutations in relation to the conserved domains of the protein is shown in Figure 6d. The mutations showed no evidence for hot spots, suggesting that the mutations likely inactivated the gene product rather than activated a novel function. This is consistent with the concept that loss of function is the critical event for tumor suppressor genes in tumorigenesis (Haber and Harlow, 1997).

Discussion

In mice, Phc1 has a critical role in mediating PcG- dependent bridging of distant chromatin templates (Lavigne et al., 2004). Co-immunoprecipitation of PHC1 and PHC2 proteins from HeLa and U2OScell extracts suggests that the polymerization of SAM domains of the PHC proteins may be involved in mediating PcG-dependent silencing through higher- order chromatin structures (Gunster et al., 1997; Levine et al., 2002). During the initiation of silencing by the PcG Figure 4 Co-immunofluorescence analysis of PHC3 and E2F6 in complexes, specific subunits of the PRC1 complex must proliferating and confluent cells. hMSC cells were analysed for interact with chromatin to recruit the free nucleosome PHC3 and E2F6 localization by immunofluorescence at 50% arrays. Protein interactions have been shown to be confluency and >80% confluency. At 50% confluency (proliferat- mediated through the SAM domain (Ph) and the RING ing cells), both PHC3 and E2F6 are diffusely nuclear. In the >80% confluent cells, both PHC3 (red) and E2F6 (green) colocalize domain (RING and Bmi1), which may also play a (yellow) in the nucleus in a punctate pattern. critical role in recruitment (Levine et al., 2002; Lavigne et al., 2004). Studies have shown that the PRC1 complex osteosarcoma, to see if mutations could be found in the does not significantly alter nucleosomal position or tumors. In one tumor sample, PCR amplification of access of nucleases to the nucleosomal arrays (Dellino exon 2 revealed two bands (Figure 6a). We sequenced et al., 2004; Lavigne et al., 2004). This suggests that the PCR fragments from this tumor. The larger the PRC1 complex can inhibit chromatin remodeling fragment proved to be owing to a tandem duplication by blocking interaction with SWI/Snf without making that resulted in a 196 bp insertion into the coding region the template inaccessible. This may be important, of exon 2 (Figure 6b). The insertion was not present as it would permit cells to reactivate silenced genes if in the matched normal DNA sample from the same needed. patient, suggesting that the mutation arose somatically The PHC orthologues are not completely interchange- in the tumor. It is not clear whether the mutation was able, as PHC1 has a more restricted tissue distribution actually heterozygous in the tumor or a result of than either PHC2 or PHC3 (Tonkin et al., 2002; Isono contamination of the tumor sample with normal cells et al., 2005). Moreover, mutations in PHC1 or PHC2 during DNA extraction. are not equivalent, as the double mutant Phc1/Phc2 has Sequence analysis of the remaining 14 samples a synergistic effect (Takihara et al., 1997; Isono et al., revealed that seven tumors had homozygous 1–4 bp 2005). Thus, PHC3 either cannot substitute for the other insertions/deletions that caused frameshift mutations PHC proteins or mutant forms of PHC1/PHC2 may (Figure 6c). The most proximal mutation was a 1 bp affect the oligomerization of PRC1 complexes contain- deletion in codon 2 that would eliminate the entire ing PHC3 or PHC3 may not be present in sufficient protein. The most distal mutation was a 4 bp deletion at quantities to allow oligomerization of PRC1 complexes codon 799 that would eliminate the SAM domain of the on DNA (Isono et al., 2005). protein. None of the mutations were found in the matched normal DNA, suggesting that all had arisen PHC3 is part ofan hPRC-H complex that associates somatically in the tumors. with E2F6 One tumor had a C- to T-substitution that led to a We found that in addition to members of the hPRC-H TAG stop codon at codon 419 (Figure 6c). This complex, PHC3 co-immunoprecipitated with E2F6. mutation appeared to be heterozygous in the tumor. This suggested that the PHC3 might be part of an The mutation was not found in the DNA from the E2F6-containing complex that contributes to long-term matched normal sample, suggesting that the mutation silencing of genes in quiescent cells (Ogawa et al., arose somatically in the tumor. As in the first tumor, it is 2002). The idea that PHC3 may play a role in regulation not clear whether the wild-type allele was actually of transcription silencing during G0 was supported present in the tumor or whether the presence of the wild- by localization analysis. In proliferating cells PHC3 had type allele was due to contaminating normal cells. a diffuse nuclear localization. During differentiation,

Oncogene PHC3 acts as a tumor suppressor gene in osteosarcoma AM Deshpande et al 1718

Figure 5 Immunoﬂuorescent analysis of PHC3 expression. Representative examples of normal (hMSC and dpc12.5 mouse skeleton) and primary osteosarcoma tumors (OS-1 through OS-6) were tested for PHC3 (red) and E2F6 (green) expression by immunoﬂuorescence imaging. Hematoxylin and eosin (H&E) stains of serial sections from the same mouse skeleton and tumors are also shown. Positive PHC3 staining can be seen in the hMSC cell line and the developing mouse skeleton vertebrae. Osteosarcoma tumors OS-1 through OS-4 are examples of PHC3À tumors whereas OS-5 and OS-6 are examples of PHC3 þ tumors. All H&E-stained photos were taken at Â 100. NC, negative controls where no primary antibody was used for staining mouse skeleton.

Figure 6 Mutational analysis of PHC3. (a) Agarose gel of exon 2 PCR-ampliﬁed tumor DNAs showing two ampliﬁcation products in one tumor sample (arrow). (b) Sequence analysis of the larger fragment from exon 2 of the tumor shown in (a) revealed a 196 bp tandem repeat at the beginning of exon 2 in the tumor DNA, which was absent in DNA from the matched normal tissue. (c) Representative examples of sequence analysis from osteosarcoma and matching normal DNA samples. The majority of alterations were 1–4 bp insertions/deletions that resulted in frameshift and nonsense mutations. Arrows indicate the alterations. One tumor had a C/T transition in codon 419 that resulted in a heterozygous nonsense mutation. (d) Map of the mutations found in osteosarcoma tumors. The exon structure of PHC3 is shown as alternating boxes. Half height boxes show non-coding portions of the mRNA. N-A and N-B, N-terminal homology domains shared by human and mouse PHC3; HD-1, homology domain shared among PHC proteins; Zn, –putative Zn coordination domain; SAM, sterile alpha motif protein interaction domain.

Oncogene PHC3 acts as a tumor suppressor gene in osteosarcoma AM Deshpande et al 1719 PHC3 shifted to a punctate nuclear localization. Similar Punctate colocalization of PHC3 and E2F6 in results were observed when the cells were allowed conﬂuent and/or differentiating cells supported the to become conﬂuent. These data were consistent model of a PHC3-containing hPRC-H complex binding with localization of PHC3 to the polycomb bodies DNA during G0; our data suggested by extension that during G0. this complex was also the E2F6-containing complex.

Oncogene PHC3 acts as a tumor suppressor gene in osteosarcoma AM Deshpande et al 1720 The role of PHC3 in the complex may be to regulate USA). All samples were completely anonymous with no binding to the DNA or compaction of the nucleosome patient identifiers. DNA was isolated from tumors and blood arrays. As with other PHC proteins, it is possible that and/or adjacent normal bone using the Wizard Genomic DNA the SAM or (Cys)4-type zinc coordination domains may purification kit (Promega Corp., Madison, WI, USA). For play an important functional role in PHC3. Loss of immunofluorescent analysis, commercial tumor microarrays (HistoArrays, Imgenex Corp., San Diego, CA, USA) were PHC3 function could lead to inability to maintain long- used. term G0 silencing mediated by the complex and allow cells to continue proliferating. PHC3 antibody A peptide sequence (ERELRDVRIRKMPENSD) found in PHC3 in osteosarcoma tumorigenesis both human and mouse PHC3, but not PHC1 or PHC2, was Thus, several lines of data suggest that loss of PHC3 conjugated to KLH (Research Genetics/Invitrogen, Carlsbad, function would favor tumorigenesis. First, other PcG CA, USA) and used to generate mouse monoclonal antibodies genes have been implicated in tumorigenesis (Leung (Hybridoma Center for Agriculture and Biological Sciences et al., 2004; Pasini et al., 2004; Gil et al., 2005; (HYCABS), Oklahoma State University (http://www.ento. okstate.edu/hycabs/)). The specificity of the anti-PHC3 antibody Raaphorst, 2005; Nowak et al., 2006). PcG complexes was confirmed by Western analysis. The antibody detected a have been shown to regulate control of cellular single protein band of approximately 110–115 kDa, which is proliferation and loss of this control would favor the same size as previously described (Levine et al., 2002) (data tumorigenesis (Dahiya et al., 2001; Pasini et al., 2004; not shown). Bracken et al., 2006). Second, the discovery that the E2F6 complex present Co-immunoprecipitation in HeLa cells cannot occupy target genes (Ogawa et al., Proteins were extracted from snap-frozen mouse femurs and 2002) suggests that the E2F6-containing complex is immunoprecipitation was performed as described previously incomplete in some form. PHC3 was missing from the (Harlow and Lane, 1988). Western analysis was carried out HeLa cell E2F6-containing complex suggesting that in using the appropriate second antibody as described previously its absence the complex cannot bind DNA or regulate (Harlow and Lane, 1988). The antibodies used were Bmi1 (H95), transcription. CtBP (H440), E2F6 (E20), RB1 (IF8), YY1 (H10) and Sox9 The discovery that PHC3 lies within a region of (H90) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). human chromosome 3q26.2 shown to undergo LoH at high frequency in osteosarcomas (Kruzelock et al., 1997) Immunofluorescence and colocalization analysis suggests PHC3 as a possible tumor suppressor. Expres- For confluency analysis, cells were plated on chamber slides at sion analysis showed PHC3 was present in cultured 50% confluency. The cells were fed with growth media (Cambrex Corp., East Rutherford, NJ, USA) following the hMSC, osteoblasts and embryonic mouse skeleton but manufacturer’s protocol. The cells became >80% confluent absent in a significant fraction of osteosarcoma tumors. within 48 h. Confluent cultures continued to be fed with Finally, tumor suppressor genes characteristically appropriate growth media for 7 days. For analysis of have homozygous intragenic tumor-specific inactivating differentiating cells, cells were grown on chamber slides and mutations that result in an absence of functional protein induced to differentiate by feeding with osteogenic differentia- (Haber and Harlow, 1997). When we examined DNA tion media (Cambrex Corp., East Rutherford, NJ, USA) from matched tumor and normal tissues, we found following the manufacturer’s protocol. somatic mutations in nine of the osteosarcoma tumors. Immunofluorescence analysis was performed as described These frameshift and nonsense mutations showed no previously (Harlow and Lane, 1988) using our mouse evidence of clustering, suggesting that the mutations led monoclonal antibody to PHC3. Goat polyclonal antibody to E2F6 (E-20, Santa Cruz Biotechnology Inc., Santa Cruz, CA, to loss of function rather than alteration in function. USA) was used for colocalization assays. All immunofluores- When taken together, these data are consistent with cently labeled samples were viewed on a BX-60 microscope PHC3 as a tumor suppressor gene. In conjunction (Olympus America Inc., Melville, NY, USA) and images with the loss of checkpoints controlled by the RB1 and captured with a digital cooled-CCD camera (Hamamatsu p53 pathways in osteosarcoma (Wang, 2005), the end Corp., Bridgewater, NJ, USA) and OpenLab image analysis result would be loss of the ability of the cell to exit cell software (Improvision Inc., Lexington, MA, USA). Colocali- cycle or remain in quiescence, thus strongly promoting zation was determined using the OpenLab Colocalization tumorigenesis. software module. All photographs were taken at Â 200 magnification, unless otherwise noted. To show terminal differentiation of the osteoblast lineage, cells were stained with Alizarin Red (Sigma-Aldrich, St Louis, Materials and methods MO, USA) as recommended by the manufacturer.

Patient materials and cell lines NHOst and hMSC (Cambrex Corp., East Rutherford, NJ, Tumor microarray analysis USA) were grown according to the manufacturer’s recom- Analysis of tumor arrays was performed as previously mendations. described (Harlow and Lane, 1988) except that deparaffinized All patient tumor and normal DNA samples used in this array slides were first treated with 4 N HCl for 10 min before study were collected with Institutional Review Board- incubation with mouse monoclonal antibody to PHC3 and approved informed consent as part of Phase III trials goat antibody to E2F6 followed by incubation with Alexa- conducted by the Childrens Oncology Group (Arcadia, CA, fluor-labeled secondary antibodies (anti-mouse IgG and anti-

Oncogene PHC3 acts as a tumor suppressor gene in osteosarcoma AM Deshpande et al 1721 goat IgG, Invitrogen/Molecular Probes Inc., Eugene, OR, was amplified by PCR and the product gel-purified using USA) for detection. Five-micrometer-thick sections of paraf- Sephadex-G50 columns. DNA sequencing was performed by fin-embedded 12.5 dpc mouse embryo sections were also used the University of Connecticut Health Center sequencing as positive controls for PHC3 and E2F6 expression. core. Comparative analysis of the sequences was performed Samples were viewed and images captured as described using CodonCode Aligner (Codon Code Corp., Dedham, MA, above. Samples with less than 50 tumor cells in the microscope USA). field were excluded from the analyses. Fluorescent signal intensities for both PHC3 and E2F6 were compared to positive (mouse embryo) control using OpenLab software. Loss of Acknowledgements expression of PHC3 had occurred if the fluorescent intensity of PHC3 in the tumor sample was p15% of the fluorescent We thank Drs A Lichtler, Y-F Huang, D Pathmanathan, intensity of PHC3 in the positive control and if the fluorescent M Sciaudone and A Arnold for their helpful discussions and intensity of E2F6 in the tumor sample was X85% of the P Vachon, A Marren and V DuPaul-McGloin for their fluorescent intensity of E2F6 in the positive control. administrative support. We also thank the Childrens Oncology Group for their assistance. This work was supported by Sequence analysis grants from the National Institutes of Health (CA074802 Primers for each exon were designed from flanking introns and AR049940), the Charlotte Geyer Foundation and the of the PHC3 gene (Supplementary Table 1). Each exon Murray-Heilig Foundation.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene