Priority Report 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 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Research 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