DNA Methylation and Complete Transcriptional Silencing of Cancer Genes Persist After Depletion of EZH2 Kelly M

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DNA Methylation and Complete Transcriptional Silencing of Cancer Genes Persist after Depletion of EZH2 Kelly M. McGarvey,1,2 Eriko Greene,1,2 Jill A. Fahrner,1,2 Thomas Jenuwein,3 and Stephen B. Baylin1

1The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University; 2The Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and 3Research Institute of Molecular Pathology, The Vienna Biocenter, Vienna, Austria

Abstract decrease in global DNA methylation and gene re-expression after Recent work suggests a link between the polycomb group introduction of a dominant-negative H3K27R mutant in ovarian protein EZH2 and mediation of gene silencing in association cancer cells, although a reduction in promoter DNA methylation is with maintenance of DNA methylation. However,we show that not observed (13). Importantly, the effects of a lysine-to-arginine whereas basally expressed target cancer genes with minimal substitution may be less specific than a discrete loss of H3K27me3, DNA methylation have increased transcription during EZH2 as evidenced by a greater reduction in global DNA methylation in knockdown,densely DNA hypermethylated and silenced genes the K27R mutant compared with EZH2 knockdown (13). Another retain their methylation and remain transcriptionally silent. recent study used small interferingRNA (siRNA) to reduce EZH2 These results suggest that EZH2 can modulate transcription of levels in an osteosarcoma cell line and proposed that EZH2 directly controls both initiation and maintenance of DNA methylation (14). basally expressed genes but not silent genes that are densely However, we now present findings suggesting that although EZH2 DNA methylated. [Cancer Res 2007;67(11):5097–102] may function to hold genes in a basally low transcription state in the relative absence of DNA methylation, this protein and the Introduction H3K27me3 mark that it catalyzes are not solely responsible for The polycomb group (PcG) proteins in the EED-EZH2 complex maintenance of transcriptional repression at heavily DNA hyper- are involved in gene silencing in multiple settings including methylated tumor suppressor genes. X-chromosome inactivation and developmental gene silencing (1). This silencingis associated with a specific lysine methylation pattern where histone 3 Lysine 27 is trimethylated (H3K27me3) by Materials and Methods the histone methyltransferase EZH2 (2). The role of PcG proteins Cell culture. SW480 and U2OS cells were maintained in McCoy’s 5A and H3K27me3 has particularly been stressed for maintenance of modified medium, and RKO cells were maintained in MEM. All media embryonic stem cell fate through controlling a key subset of genes (Invitrogen) were supplemented with 10% fetal bovine serum (Gemini Bio- that contains promoter CpG islands and for which expression must Products) and 1% penicillin/streptomycin (Invitrogen) and grown at 37jC be delayed until it is required for proper cell commitment and in 5% CO2 atmosphere. specification of cell lineages (3–6). This above role for PcG proteins Transient siRNA transfection. RKO or U2OS cells were transfected with in embryonic stem cells has recently assumed great relevance for either a non-targeting control or EZH2 targeting siRNA (Dharmacon D-001210-01 and D-004218-01) usingLipofectAMINE 2000 (Invitrogen). human cancer in that several groups, including our own, have Cells were transfected at a 25 nmol/L siRNA concentration once and then shown that many genes that become DNA hypermethylated for transfected again 24 h later and continued to be passaged and transfected promoter CpG islands and silenced in adult cancers are marked at every other day for up to 8 days. their promoters, even in the absence of DNA methylation, by PcG Stable siRNA knockdown. A non-targeting or EZH2 targeting shRNA components in embryonic stem cells (7–9). was cloned into the pSuper vector (OligoEngine), and RKO cells were EZH2 is overexpressed in several types of cancer, and the levels transfected with LipofectAMINE 2000 (Invitrogen). The transfectants were of expression correlate with cancer aggressiveness (1, 10, 11). selected with Puromycin, and individual clones were isolated. Recently, we have shown that trimethylation of H3K27 and EZH2 Western blot analysis. Nuclear extracts were isolated usingthe NE-PER bindingare enriched alongmultiple DNA hypermethylated and kit (Pierce). Western blot analysis was conducted usingantibodies against silenced gene promoters in colon and breast cancer cells (12). All of EZH2 (Upstate 07-400), glyceraldehyde-3-phosphate dehydrogenase (Abcam), h-actin (Sigma), H3K9me2, or H3K27me3 (15). these above data support a link between EZH2-mediated H3K27 Reverse transcription-PCR and methylation-specific PCR. Reverse trimethylation and aberrant DNA methylation. However, experi- transcription-PCR (RT-PCR) and methylation-specific PCR (MSP) were done mental evidence connectingthe actual functional link between as previously described (16). Primer sequences are available upon request. these PcG components and aberrant epigenetic cancer gene Chromatin immunoprecipitation. Chromatin immunoprecipitation silencingis limited. To this effect, there is evidence of a modest analysis was done as previously described (16). Antibodies against EZH2 (Upstate), monomethyl, dimethyl, or trimethyl H3K27; trimethyl H3K9 (15); RNA Polymerase (Pol) II (Epiquick); or IgG (Santa Cruz) were used. PCR amplification and analysis. Previously designed primers targeting Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). the promoter region of the hMLH1 (16), MYT1 (17), WNT1 (17), and K.M. McGarvey and E. Greene contributed equally to this work. p16INK4a (18) genes were used to analyze promoter occupancy. Primers were Requests for reprints: Stephen B. Baylin, The Sidney Kimmel Comprehensive purchased from Integrated DNA Technologies. All PCR reactions were done Cancer Center at Johns Hopkins, Suite 541, 1650 Orleans Street, Baltimore, MD 21231. in a total volume of 25 AL, using2 AL of either immunoprecipitated (bound) Phone: 410-955-8506; Fax: 410-614-9884; E-mail: [email protected]. I2007 American Association for Cancer Research. DNA, a 1:100 dilution of non-immunoprecipitated (input) DNA, or a no doi:10.1158/0008-5472.CAN-06-2029 antibody or IgG control; 10 AL of PCR product were size fractionated by www.aacrjournals.org 5097 Cancer Res 2007; 67: (11). June 1, 2007

Figure 1. Transient EZH2 knockdown depletes H3K27me3 globally and at the hMLH1 promoter but does not induce DNA demethylation or gene re-expression. A, Western blot analysis of EZH2, H3K27me3, and H3K9me2 in RKO cells treated with a non-targeting control (NT) or EZH2-targeted siRNA. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) serves as the loading control. B, chromatin immunoprecipitation analysis of the hMLH1 promoter showing occupancy by EZH2 or monomethylation (mono), dimethylation (di), and trimethylation (tri) of H3K27 in RKO cells treated with non-targeting or EZH2 siRNA. A representative PCR analysis done on the immunoprecipitated, mock (No ab), and a 1:100 dilution of non-immunoprecipitated (input) DNA (left). Band intensities were quantified for chromatin immunoprecipitation replicates (right). Columns, mean; bars, SE. C, RT-PCR analysis of hMLH1 expression in RKO cells treated with either non-targeting or EZH2 siRNA. RNA from untreated SW480 and RKO colorectal cancer cells served as the positive and negative controls for hMLH1 expression, respectively, and GAPDH served as a positive control for the PCR. D, MSP analysis of DNA from RKO cells treated with either non-targeting or EZH2 siRNA. Methylation was detected by the presence of a PCR product amplified by methylation-specific primers (lane M). Lack of DNA methylation is indicated by PCR products amplified by unmethylated-specific primers (lane U). Untreated RKO and SW480 serve as positive controls for the methylated and unmethylated PCR reactions, respectively.

PAGE and were quantified using Kodak Digital Science 1D Image Analysis with shakingat 37 jC. Plasmid DNA was isolated usingQIAprep Spin software. Enrichment was calculated by takingthe ratio between the net Miniprep kit followingthe manufacturer’s instructions (Qiagen).Plasmids intensity of the gene promoter PCR products from each primer set for the were screened for inserts by EcoRI digestion and sequenced using the M13 bound, immunoprecipitated sample and the net intensity of the PCR universal reverse primer (Invitrogen). product for the non-immunoprecipitated input sample. Values for enrichment were calculated as the average from at least two independent chromatin immunoprecipitation experiments and multiple independent Results PCR analyses (three PCR reactions for each primer set used per EZH2 knockdown is not sufficient for loss of DNA independent chromatin immunoprecipitation). methylation or gene re-expression of the tumor suppressor Bisulfite sequencing. Genomic DNA was extracted from RKO and U2OS gene hMLH1. We have previously shown that EZH2 is localized to cells that were treated with non-targeting or EZH2 siRNA; 1 Agof DNA was INK4a the hMLH1 promoter when it is silent, and DNA hypermethylated, bisulfite modified as previously described (19), and MYT1, WNT1, p16 , in RKO colorectal cancer cells (12). EZH2 was not found at the and hMLH1 promoters were analyzed (primer sequences are available upon unmethylated, active hMLH1 promoter in SW480 colorectal cancer request). All PCRs were done usingJumpStart Red Taq DNA Polymerase. PCR products were run on 1% agarose gels, and bands were excised using cells or at the demethylated, reactivated hMLH1 promoter in RKO ¶ QIAquick Gel Extraction kit followingthe manufacturer’s instructions cells treated with 5-aza-2 deoxycytidine (12). To first examine the (Qiagen). Purified bands were cloned using TOPO-TA cloning kit following role of EZH2 in hypermethylated tumor suppressor gene silencing, the manufacturer’s instructions (Invitrogen). Colonies were selected and we used siRNA to transiently knockdown EZH2 in RKO cells. grown overnight in Luria-Bertani medium containing ampicillin (100 Ag/mL) Knockdown of EZH2 was associated with a global decrease in

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H3K27 trimethylation, with no effect on dimethylation of H3K9 the MYT1 and WNT1 gene promoters (14). To address the dis- (Fig. 1A). EZH2 knockdown additionally caused depletion of the crepancies between our observation with the hMLH1 gene and this protein at the hMLH1 gene promoter (Fig. 1B). Chromatin recent data suggesting that EZH2 is necessary for gene silencing, immunoprecipitation analysis also showed that at the promoter, and to understand the role that EZH2 has in a cell and gene H3K27me3 sharply decreased after EZH2 knockdown, whereas specific manner, we compared the U2OS and RKO cells. H3K27me1 actually increased, perhaps suggesting that H3K27me1 Interestingly, RT-PCR showed that although MYT1 and WNT1 is a substrate for EZH2 (Fig. 1B). Importantly, even with the loss of were completely silent in RKO colorectal cancer cells, they were EZH2 and the H3K27me3 mark at the hMLH1 promoter, there was basally expressed in U2OS cells (Fig. 2A). We also observed that the no gene re-expression or loss of DNA methylation associated with tumor suppressor gene p16INK4a was completely silent in both cell these changes (Fig. 1C and D). lines and could thus serve as an excellent control gene for our To test whether the transient knockdown allowed enough time studies (Fig. 2A). for gene re-expression or for sufficient depletion of the protein to To determine if chromatin status was responsible for the fully affect its catalyzed mark, we also stably knocked down EZH2 differences in basal expression of MYT1 and WNT1 in the two cell in RKO colorectal cancer cells usinga retroviral pSuper vector with lines, we did chromatin immunoprecipitation at the MYT1, WNT1, a short hairpin loop targeting EZH2. Again, knockdown of EZH2 and p16INK4a gene promoters in both cell lines. We first found caused a global decrease of EZH2 and H3K27me3 (Fig. 3A; data not that whereas EZH2 is equally enriched at all three gene shown) and a significant decrease in EZH2 and H3K27me3 at the promoters in both cell lines, H3K27me3 is distinctly less hMLH1 promoter (Supplementary Fig. S1) similar to the transient abundant at these promoters in U2OS cells compared with RKO approach. Despite long-term EZH2 depletion at the hMLH1 cells (Fig. 3B and C). Thus, the chromatin marks are consistent promoter, gene re-expression or DNA methylation changes were among all three genes. This suggests that the lower H3K27me3 not observed (Fig. 3C; Supplementary Fig. S2). level is not responsible for active transcription of MYT1 and Comparison of EZH2 target genes shows differing basal WNT1 in U2OS cells because p16INK4a shares this chromatin states in two cell lines. Vire et al. recently reported that signature but is silent. We also examined these gene promoters knockdown of EZH2 in U2OS cells led to activation of the MYT1 for RNA Pol II association, and consistent with the differences gene and loss of DNA methylation at some CpG sites in both in basal transcription state, RNA Pol II is strongly associated

Figure 2. Comparison of MYT1, WNT1, and p16INK4a in RKO versus U2OS cells. A, RT-PCR analysis of MYT1, WNT1, and p16INK4a in RKO and U2OS cells. GAPDH was included as a loading control, and RKO cells treated with 5-aza-2¶-deoxycytidine (Aza) were used as a positive control. cDNA was made in the presence of (+) and absence of (À) reverse transcriptase (RT). B, chromatin immunoprecipitation analysis was done on untreated RKO (R) and U2OS (U) cells using antibodies against H3K9me3, H3K27me3, EZH2, and RNA Pol II. PCR analysis of the chromatin immunoprecipitation products was done at MYT1, WNT1 and p16INK4a promoter regions. C, chromatin immunoprecipitation band intensities were quantified as in Fig. 1. www.aacrjournals.org 5099 Cancer Res 2007; 67: (11). June 1, 2007

promoters enriched in both of these silencingmarks (12). We examined the level of H3K9me3 at each of the promoters in both cell lines and found that MYT1, WNT1, and p16INK4a all had equal levels of H3K9me3 in both RKO and U2OS, suggesting that it was not the H3K9me3 mark that contributed to the different expression status of these genes. EZH2 knockdown results in increased expression of unmethylated and basally expressing genes but not of completely silenced and hypermethylated tumor suppressor genes. To further probe the role of EZH2 for the genes under study, we next examined the effect of EZH2 knockdown in both cell lines. Although we were able to significantly decrease levels of EZH2 in both cell lines (Fig. 3A), varyingdownstream effects were observed. U2OS cells were collected after 3 days of EZH2 knockdown, and the associated depletion of EZH2 caused an increase in expression of MYT1 and WNT1, consistent with the Vire et al. data, but did not re-express fully silenced and hypermethylated p16INK4a in these same cells (Fig. 3B). Alternatively, expression of MYT1 and WNT1 were unaffected by EZH2 knockdown in RKO cells (Fig. 3C). Thus, although all three genes share a similar chromatin pattern in the U2OS cells, only the basally expressed genes show any expression changes after EZH2 knockdown. Finally, we employed bisulfite sequencingto examine whether the DNA methylation status, either before or after EZH2 knockdown, might account for the different basal expression states of the genes and differing effects of decreasing EZH2 levels. Contrary to data from the previously published work where they showed 60% and 80% DNA methylation of selected sites in the MYT1 and WNT1 promoters, respectively, we find that both the promoters show minimal basal DNA methylation in U2OS cells (Fig. 4A and B). In sharp contrast, these promoters are fully methylated in RKO cells, correlatingwith their fully repressed transcription state (Fig. 4A and B). Examination of the silenced p16INK4a gene reveals that it is also fully DNA methylated in U2OS (Fig. 4C). In addition, contrary to previously published work, we failed to observe a significant reduction in DNA methylation at the MYT1, WNT1,orp16INK4a promoters in either cell line after EZH2 knockdown (Fig. 4A–C).

Discussion In light of the present data, from examination of hMLH1, INK4a Figure 3. Depletion of EZH2 in U2OS and RKO cells. A, Western blot analysis. p16 , and especially MYT1 and WNT1 in two cancer cell lines, Protein extracts were probed for EZH2 and h-actin in U2OS and RKO cells we conclude that EZH2 is not required for the maintenance of treated with non-targeting control or EZH2 siRNA. B, RT-PCR analysis of MYT1, WNT1, and p16INK4a in U2OS cells treated with non-targeting or EZH2 dense promoter DNA methylation and the transcriptional silencing siRNA. C, RT-PCR analysis of MYT1, WNT1, p16INK4a , and hMLH1 in RKO of genes associated with this promoter change. This conclusion is cells treated with non-targeting or EZH2 shRNA. RKO cells treated with particularly compellingfrom the comparative studies of MYT1 and 5-aza-2¶-deoxycytidine were used as a positive control. cDNA was made in the presence of (+) and absence of (À) reverse transcriptase. WNT1 genes in U2OS versus RKO cells. When these genes are not truly regulated by DNA methylation, and when basal expression is evident, as in U2OS cells, depletion of EZH2 can induce increased with MYT1 and WNT1 but not p16INK4a in the U2OS cells (Fig. 2B transcription. Additionally, we now show that reduction of EZH2 and C) and not with any of the three genes in the RKO cells does not largely affect levels of promoter DNA methylation. It is (Fig. 2B and C). important to note that our data do not rule out, as suggested by We additionally examined whether other repressive chromatin Vire et al., a role for EZH2 and its H3K27me3 mark in initial marks might associate with the genes being examined in the two recruitment of DNA methylation (14). cell lines. MYT1 and WNT1 were originally identified as EZH2 Our data indicate that when EZH2 functions to mediate acute target genes in the SW480 colorectal cancer cell line (17). In the gene changes, it may act as a modulator when the promoter is not SW480 cell line, the promoters of MYT1 and WNT1 were reported associated with dense DNA methylation, as in the case of MYT1 to associate with H3K27me3 but were lackinganother silencing and WNT1 in U2OS cells. Our results for the DNA methylation state mark, H3K9me3 (17). We found that all DNA hypermethylated of these genes in U2OS cells are contrary to previously published tumor suppressor genes we have previously examined had data for reasons that are not clear (14). However, our findings

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. EZH2 Is Not Required for Maintenance of DNA Methylation showingminimal DNA methylation of these genesare based on Our hypotheses are consistent with our recent findings for extensive sequencingof multiple individual alleles for their multiple genes in teratocarcinoma cells, where we found simulta- promoter regions and contrasts sharply with their densely neous enrichment for EZH2, H3K27me3, and the additional methylated state for these same regions in RKO cells. repressive H3K9me3 chromatin mark, in the absence of DNA

Figure 4. EZH2 depletion does not reduce promoter DNA methylation. A, bisulfite sequencing of MYT1 promoter in U2OS and RKO cells treated with non-targeting control or EZH2 siRNA. B, bisulfite sequencing of WNT1 promoter in U2OS and RKO cells treated with non-targeting or EZH2 siRNA. C, bisulfite sequencing of p16INK4a promoter in U2OS cells treated with non-targeting or EZH2 siRNA. Each horizontal line represents an individually sequenced TA-cloned allele, with each circle representing a CpG dinucleotide as distributed in the proximal promoter region with sites numbered along the X-axis. ., methylated cytosine residues; o, unmethylated cytosine residues.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Research methylation (7). In the teratocarcinoma cells, such genes can be Acknowledgments induced by differentiation cues to increase expression (7). Thus, all of our findings stress the dominant role of dense CpG island DNA Received 6/2/2006; revised 3/8/2007; accepted 4/19/2007. Grant support: National Institute of Environmental Health Sciences grant ES11858 methylation over a variety of associated repressive chromatin (K. McGarvey, E. Greene, J. Fahrner, and S. Baylin). T. Jenuwein is sponsored by the states for tightly maintaining the heritable silencing states of genes. IMP through Boehringer Ingelheim, the European Union (NoE network The Epigenome LSHG-CT-2004-503433), and the Austrian GEN-AU initiative financed by funds from This seems to be an important distinction in the control of certain the Austrian Federal Ministry for Education, Science and Culture. tumor suppressor genes in normal cells, such as in embryonic stem The costs of publication of this article were defrayed in part by the payment of page cells, where the gene promoters are associated with PcG proteins, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. versus the added feature of aberrant DNA hypermethylation- We thank Joyce Ohm, Angela Ting, and Helai Mohammad for supplying primers associated tight transcriptional silencing in adult cancer cells. and James Herman for technical advice.

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Kelly M. McGarvey, Eriko Greene, Jill A. Fahrner, et al.

Cancer Res 2007;67:5097-5102.

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