distinct roles for histone methyltransferases g9a and glp in

Distinct Roles for Histone Methyltransferases G9a and GLPin Cancer Germ-Line Antigen Gene Regulation in HumanCancer Cells and Murine Embryonic Stem Cells

Petra A. Link,1 Omkaram Gangisetty,1 Smitha R. James,1 Anna Woloszynska-Read,1

Makoto Tachibana,2 Yoichi Shinkai,2 and Adam R. Karpf1

1Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York and2Institute for Virus Research, Kyoto University, Kyoto, Japan

AbstractThe H3K9me2 histone methyltransferases G9a and GLPrepress Mage-a class cancer germ-line (CG) antigen geneexpression in murine embryonic stem (ES) cells, but the roleof these enzymes in CG antigen gene regulation in humancancer cells is unknown. Here we show that whereasindependent or dual knockdown of G9a and GLP in humancancer cells leads to reduced global and CG antigenpromoter–associated H3K9me2 levels, it does not activateCG antigen gene expression. Moreover, CG antigen generepression is maintained following pharmacologic targetingof G9a or treatment of G9a knockdown cells with thehistone deacetylase inhibitor trichostatin A. However, G9aknockdown cells display increased sensitivity to CG antigengene activation mediated by the DNA methyltransferaseinhibitor decitabine. To account for these findings, weexamined DNA methylation at CG antigen gene promotersin both cell types. We found robust DNA hypomethylationin G9a/GLP targeted murine ES cells but a lack of DNAmethylation changes in G9a/GLP targeted human cancercells; intriguingly, this distinction also extended to markersof global DNA methylation. These data reveal that G9a/GLPis required for DNA methylation of CG antigen genes andgenomic DNA in murine ES cells, but not human cancercells, and implicate DNA methylation status as the keyepigenetic mechanism involved in CG antigen generepression. (Mol Cancer Res 2009;7(6):851–62)

IntroductionCancer germ-line (CG) antigens (also known as cancer-testis

antigens) are targets of tumor vaccines currently undergoingclinical evaluation worldwide (1). The rationale for vaccine-

based targeting of CG antigens in cancer includes both theinherent immunogenicity of these antigens and their restrictedexpression in normal human tissues as well as widespread ex-pression in human tumors (1, 2). CG antigen genes seem to beprincipally regulated at the transcriptional level by epigeneticsignals. Lines of evidence for this are as follows: (a) PromoterDNA hypomethylation correlates with CG antigen gene expres-sion in human tumor and normal tissues (3, 4); (b) treatment ofnonexpressing cancer cells with epigenetic modulatory drugs,including DNA methyltransferase (DNMT) and histone deace-tylase (HDAC) inhibitors, induces CG antigen gene expression(5-7); and (c) specific histone H3 modification patterns areassociated with CG antigen gene expression status in humancancer cells (8-10). More detailed elucidation of the epigeneticmechanisms regulating CG antigen gene expression in humancancer cells and tumors should help facilitate the clinical deve-lopment of CG antigen-targeted therapeutic approaches (2).

The enzymes that mediate histone H3 lysine 9 (H3K9)methylation have been identified, and these include classSuv39 enzymes, which target heterochromatic loci and catalyzeH3K9 trimethylation (H3K9me3), and G9a and GLP (alsoknown as EuHMTaseI), which target euchromatic loci and cata-lyze H3K9 dimethylation (H3K9me2; refs. 11-14). Similar toDNA methyltransferase I (DNMT1) knockout, G9a or GLPknockout causes embryonic lethality in mice, implicating a cru-cial role for epigenetic repression in early mammalian develop-ment (12, 13, 15). Gene expression profiling of G9a knockoutmurine embryonic stem (ES) cell lines identified Mage-a classCG antigen genes as targets of epigenetic repression by G9a,and subsequent experiments showed a similar role for GLP(12, 13). H3K9me2 levels are reduced both genome-wide andat Mage-a promoters in G9a or GLP knockout ES cells (12,13). These data suggest that H3K9me2 may play a primaryrole in mediating CG antigen gene repression in murine EScells, which seems to conflict with the prevailing view thatDNA methylation is the primary mediator of CG antigen genesilencing in human cancer (1, 4, 8).

We previously established the human colon adenocarcinomacell lines HCT116 and RKO as useful models to study themechanisms of CG antigen gene repression in human cancer(5, 8). CG antigen genes are transcriptionally silenced inHCT116 and RKO cells by DNA hypermethylation and areactivated by pharmacologic or genetic targeting of DNMTenzymes in these cells (5, 8). Moreover, CG antigen gene in-duction in DNMT-deficient HCT116 cells coincides with bothDNA hypomethylation and remodeling of histone code patterns

Received 10/22/08; revised 2/8/09; accepted 2/17/09; published OnlineFirst 6/16/09.Grant support: NIH grant RO1CA11674, The Ralph Wilson BiomedicalResearch Foundation, and Phi Beta Psi (A.R. Karpf), and National CancerInstitute Center grant CA16056 (to Roswell Park Cancer Institute).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).Requests for reprints: Adam R. Karpf, Department of Pharmacology andTherapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets,Buffalo, NY 14263. Phone: 716-845-8305; Fax: 716-845-8857. E-mail:[email protected] ©2009 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-08-0497

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at CG antigen gene promoters (8). The fact that CG antigengenes are infrequently expressed in human colorectal canceralso supports the relevance of this experimental model (1).

In the current study, we examined the role of G9a and GLPin regulating CG antigen expression in human cancer cells, us-ing HCT116 and RKO colorectal cancer cells as models. Wehypothesized that G9a/GLP may repress CG antigen genes innonexpressing human cancer cell lines because H3K9me2 andDNA methylation signals are often interdependent and G9apropagates epigenetic repression at the mammalian replicationfork via its interaction with DNMT1 (16). Our data reveal thatin human cancer cells, unlike mouse ES cells, G9a and GLP aredispensable for CG antigen gene repression, despite playing arole in the maintenance of H3K9 methylation in both cell types.Most importantly, we find that CG antigen genes are DNAhypomethylated in G9a or GLP null murine ES cells, but their

DNA methylation status is unchanged in human cancer cellsfollowing G9a and/or GLP targeting. These data reinforce thenotion that epigenetic control processes are regulated in distinctways in ES cells and cells of somatic origin, including cancercells. Furthermore, they implicate DNA methylation as the pri-mary repression mechanism for CG antigen gene expression.

ResultsCG Antigen Gene Expression and Histone Modificationsin G9a Knockdown Human Cancer Cells

To assess the role of G9a in CG antigen gene repression inhuman cancer cells, we initially developed an effective tran-sient siRNA G9a targeting strategy in RKO cells (Fig. 1A).Low siRNA transfection efficiency prevented implementationof this strategy in HCT116 cells (data not shown). Reverse tran-scription-PCR (RT-PCR) analysis revealed that efficient G9aknockdown did not induce expression of three representativeCG antigen genes, MAGE-A1, XAGE-1, and NY-ESO-1 (8), inRKO cells (Fig. 1B). Because sustained knockdown might berequired to affect CG antigen gene expression, and to validatethese findings in another cell type, we established clonal G9astable knockdown cell lines in both HCT116 and RKO cells(Fig. 1C). Interestingly, during derivation of the these cell lines,we observed that cancer cells expressing G9a shRNA showedsubstantially reduced cell clonogenicity, particularly in RKOcells (Fig. 1D; Table 1). We also observed reduced cell viability

FIGURE 1. Transient and stable G9a knockdown in human cancer cells. A. Transient siRNA knockdown of G9a in RKO cells. Control and G9a-specificsiRNAs were administered at either 5 or 50 nmol/L; cells were harvested at day 5; and G9a expression was measured by Western blot. α-Tubulin expressionwas measured as control for protein input, and band densitometry was done as described in Materials and Methods. The sample key is shown at the bottom ofB. B. End-point RT-PCR analysis of MAGE-A1, NY-ESO-1, and XAGE-1 for the experiment shown in A. GAPDH expression was analyzed to control forcDNA input, and amplification of cDNA from RKO cells treated with 5 μmol/L 5-aza-2′-deoxycytidine (decitabine; DAC) served as a positive control for CGantigen gene expression. CG antigen gene amplification was done for 35 cycles, whereas GAPDH was done for 25 cycles. The sample key is shown at thebottom. LF2000, Lipofectamine-only transfection control. C. Western blot analysis of G9a protein expression in control shRNA (shCon) and G9a shRNA(shG9a) stable RKO and HCT116 cell lines. α-Tubulin expression was measured as control for protein input, and band densitometry was done as described inMaterials and Methods. D. Cells stably expressing shG9a have reduced clonogenicity. Representative images of crystal violet-stained colonies of control andG9a knockdown stable RKO cells.

Table 1. Quantification of Colony Number in Control andG9a Knockdown Stable RKO and HCT116 Cell Lines

Cell Line Average No. of Colonies %Change

shCon (n = 2) shG9a (n = 2)

RKO 1,521 894 ↓41.2HCT116 643 598 ↓7.0

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following siRNA-mediated G9a knockdown in RKO (data notshown). These data suggest that G9a contributes to cell growthand/or survival in colorectal cancer cells, consistent with pre-vious reports using mice and prostate cancer cells (12, 17).The role of G9a in cell survival may explain why it was diffi-cult to achieve complete G9a knockdown in human cancer cells(Fig. 1A and C).

To explore the phenotype of stable G9a knockdown humancancer cells, we used Western blot analysis to measure globalH3K9me2 levels and H3K27me2 levels because these twomodifications have been previously linked to G9a function(18, 19). Notably, H3K9me2 levels were reduced in both celltypes whereas H3K27me2 levels were not significantly altered(Fig. 2A). A lack of activity of G9a toward H3K27me2 isconsistent with the results of recent studies of G9a functionin different model systems (20-23). To assess whether G9aknockdown altered histone modifications at CG antigen genepromoters, we used quantitative chromatin immunoprecipi-tation (ChIP)-PCR to determine H3K9me2 levels at theMAGE-A1 and XAGE-1 promoters as described previously(8). Similar to the effect on global levels, H3K9me2 levelsare reduced at both MAGE-A1 and XAGE-1 in G9a stableknockdown HCT116 and RKO cells (Fig. 2B). In contrast,H3K27me2 levels are unchanged at these promoters(Fig. 2C). Whereas reduced H3K9me2 suggested that geneexpression might be altered, CG antigen genes remained re-pressed in both G9a knockdown cell lines (Fig. 2D). This ob-servation was repeatable as independently derived stable G9aknockdown RKO and HCT116 cell clones also did not expressCG-X genes (data not shown). Stable G9a knockdown cellsalso showed slight increases in the global level of the activehistone mark H3K9ac (Fig. 3A). However, consistent with a

lack of CG antigen expression in the stable G9a knockdowncells, ChIP analyses revealed that the levels of H3K9ac andH3K4me2 at CG gene promoters, which are associated withCG gene expression (8), were not increased (Fig. 3B-E).

Because G9a knockdown was robust but not complete inG9a shRNA knockdown clones, we further targeted G9a instable G9a knockdown RKO cells using transient siRNA trans-fection (Fig. 4A). Whereas G9a siRNA transfection resulted infurther diminishment of G9a protein, CG-X genes remainedrepressed (Fig. 4A and B).

Dual Targeting of G9a and EuHMTaseI/GLP in HumanCancer Cells

GLP (EuHMTaseI) has similar substrate specificity as G9aand has a nonredundant and essential role in mammalian devel-opment (13). Despite this nonredundancy in mouse develop-ment, it remained plausible that GLP could compensate forG9a loss in human cancer cells with regard to CG antigen generepression. To test this possibility, we used a siRNA approach.Treatment of RKO cells with GLP siRNA resulted in effectiveGLP knockdown and, notably, also reduced G9a protein levels(Fig. 4C). This may be explained by the fact that GLP and G9aform an intracellular complex that stabilizes the G9a protein(13). Consistent with this idea, GLP knockdown did not affectG9a mRNA expression (data not shown). Despite effective re-duction of both G9a and GLP in RKO cells, CG antigen genesremained repressed (Fig. 4D). Furthermore, we did not observean additional decrease of H3K9me2 levels at CG antigen genepromoters following GLP knockdown in stable G9a knock-down cells (data not shown). These data suggest that redundan-cy between G9a and GLP does not account for the repression ofCG antigen genes in cancer cells sustaining G9a knockdown.

FIGURE 2. Characteriza-tion of stable G9a knockdownhuman cancer cells. A. West-ern blot analysis of H3K9me2and H3K27me2 levels in con-t r o l s h R NA – a n d G 9 ashRNA–expressing stable celll ines. Coomassie stainingconfirmed equivalent proteininput, and band densitometrywas done as described inMaterials and Methods. Band C. Quantitative ChIP-PCR analysis of H3K9me2(B) and H3K27me2 (C) levelsat the MAGE-A1 and XAGE-15′ CpG island regions. Bars,1 SD. D. RT-PCR analysisof MAGE-A1, XAGE-1, andNY-ESO-1 expression. PCRconditions and controls arethe same as described inFig. 1B, and the sample keyis shown at the bottom.

Regulation of CG Antigen Genes by G9a/GLP


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Pharmacologic Targeting of G9a in Human Cancer CellsRecently, a novel small-molecule inhibitor of G9a was iden-

tified by high-throughput screening (23). This agent, BIX-01294, was shown to selectively target G9a and, to a lesserextent, GLP, and to reduce H3K9me2 levels in mammaliancells at low micromolar concentrations (23). Notably, BIX-01294 treatment was also shown to activate Mage-A2 expres-sion in wild-type mouse ES cells (23). Here, we sought tocomplement the genetic knockdown approach described aboveby examining the effect of BIX-01294 treatment on CG antigengene expression in human cancer cells. BIX-01294 treatmentreduced global H3K9me2 levels in both control and G9a stableknockdown HCT116 and RKO cells, validating the activity ofthis agent in human cancer cells (Fig. 5A and B). However,BIX-01294 treatment failed to induce CG-X gene expressionin either cell type (Fig. 5C and D). These data provide addi-tional evidence that G9a is not required for CG antigen generepression in human cancer cells.

Effect of Combined G9a Knockdown and HDAC InhibitorTreatment on CG Antigen Gene Expression in HumanCancer Cells

The observation that G9a knockdown results in reducedH3K9me2 at CG antigen promoters without altering H3K9acraised the question of whether forced induction of H3K9ac inG9a knockdown cells could elicit CG antigen expression. Totest this, we used the classic HDAC inhibitor trichostatin A(TSA) to treat control and stable G9a knockdown cells. Asanticipated, TSA treatment markedly increased global H3

acetylation and H3K9 acetylation levels in both control and sta-ble G9a knockdown cells (Fig. 6A). Moreover, TSA inducedH3K9ac levels at CG antigen promoters (Fig. 6B and C). De-spite this effect, MAGE-A1 remained silent in both RKO andHCT116 cells, whereas XAGE-1 remained silent in RKO(Fig. 6D). For XAGE-1 expression in HCT116, TSA treatmentled to a moderate level of induction, suggesting that histoneacetylation regulates this gene in certain cellular contexts(Fig. 6D). Taken together, however, these data suggest thatremodeling the histone code at CG antigen gene promotersis generally insufficient to provoke CG antigen gene expres-sion. A possible explanation for this phenomenon is that CGantigen gene promoters are densely DNA methylated in thesecell types (8).

G9a Knockdown Sensitizes Human Cancer Cells to 5-Aza-2 ′-Deoxycytidine (Decitabine)–Mediated CGAntigen Gene Activation

Whereas the results above indicate that inhibition of G9a/GLP is not sufficient to provoke CG antigen gene expressionin human cancer cells, either alone or in combination withTSA, it remained plausible that G9a targeting could potentiateCG antigen gene activation by DNMT inhibitors. A similar re-lationship exists between HDAC inhibitors and DNMT inhibi-tors in that HDAC inhibition can facilitate the activation ofmethylation silenced genes by the DNMT inhibitor 5-aza-2′-deoxycytidine (24). We thus tested the response of controland G9a knockdown human cancer cells to treatment with deci-tabine. Notably, stable G9a knockdown HCT116 cells treated

FIGURE 3. H3K9ac and H3K4me2 in G9a knockdown human cancer cells. A. Western blot analysis of H3K9ac and H3K4me2 levels in control shRNA–and G9a shRNA–expressing stable RKO and HCT116 cell lines. Coomassie staining confirmed equivalent protein input, and band densitometry was done asdescribed in Materials and Methods. B and C. Quantitative ChIP-PCR analysis of H3K9ac levels at the MAGE-A1 and XAGE-1 5′ CpG island regions in G9atargeted RKO (B) and HCT116 (C) cell lines. D and E. Quantitative ChIP-PCR analysis of H3K4me2 levels at the MAGE-A1 and XAGE-1 5′ CpG islandregions in G9a targeted RKO (D) and HCT116 (E) cell lines. RKO cells treated with decitabine (DAC) served as a positive control for both histone modifica-tions in RKO cells, whereas DNMT1−/−, DNMT3b−/− HCT116 cells (DKO) served as a positive control for both histone modifications in HCT116. Bars, 1 SD.

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with decitabine showed significantly increased MAGE-A1 andXAGE-1 gene expression and moderately increased NY-ESO-1expression as compared with control cells treated with decita-bine alone (Fig. 7A-C). In contrast to HCT116, an enhancedCG antigen expression effect was only marginal in decita-bine-treated G9a stable knockdown RKO cells (data notshown). These data show that G9a knockdown can synergizewith DNMT inhibition to promote CG antigen gene expression,but that this effect is, in part, cell context dependent.

CG Antigen Gene Regulation and DNA Methylation inG9a and GLP Knockout Mouse ES Cells

In contrast to our data using human cancer cells, G9a andGLP knockout mouse ES cells show robust expression ofMage-a class CG antigen genes (12, 13). Because loss ofH3K9me2 globally and at CG antigen gene promoters is ob-served in both mouse ES cells and human cancer cells respond-ing to G9a and/or GLP targeting (refs. 12, 13, 17; Fig. 2),reduction of this repressive epigenetic histone mark does notseem to explain the differential response. Of note, previousstudies have observed altered DNA methylation in G9a knock-out mouse ES cells (18, 25, 26). We hypothesized that differ-ences in promoter DNA methylation status at CG antigen genesmight explain the distinct response of mouse ES and humancancer cells to G9a/GLP targeting. To test this, we initially usedG9a and GLP knockout murine ES cell lines (Fig. 8A). Usingpan-Mage-a primers that amplify murine Mage-a2, Mage-a5,Mage-a6, and Mage-a8, we observed that G9a and GLP knock-

out ES cells express high levels of Mage-a genes (Fig. 8B). Ofthe murine Mage-a gene family members, only Mage-a2 andMage-a8 contain classic 5′ CpG islands (27), and thus we fo-cused further attention on these two genes. Both Mage-a2 andMage-a8 were induced in G9a and GLP knockout ES cells(Fig. 8B). We next used methylation-specific PCR to determinethe methylation status of the 5′ CpG region of Mage-a2 in wild-type, G9a knockout, and GLP knockout ES cells. As shown inFig. 8C, Mage-a2 was hypermethylated in wild-type ES cellsand becomes dramatically hypomethylated in GLP and G9aknockout cells. Moreover, reintroduction of either GLP orG9a into the corresponding knockout cell line led to partial re-methylation of Mage-a2 (Fig. 8C). This remethylation correlat-ed with repression of Mage-a2 (12, 13). Similar to the Mage-a2result, bisulfite pyrosequencing analysis indicated that Mage-a8was dramatically hypomethylated in G9a and GLP null ES cellsand remethylated following reintroduction of the relevant pro-tein (Fig. 8D). To determine if the effect on DNA methylationin G9a and GLP knockout ES cells extended to markers ofglobal DNA hypomethylation, we used two different measuresof global DNA methylation status: (a) 5-methyl-2′-deoxycyti-dine (5mdC) levels and (b) bisulfite pyrosequencing of the mu-rine repetitive element B1 (28). Both knockout cell typesdisplay sizable reduction of 5mdC levels, which was partiallyrestored following reintroduction of the corresponding proteins(Fig. 8E). A similar phenotype was observed for methylation ofthe repetitive B1 element (Fig. 8F). These data reveal that G9aand GLP are required for DNA methylation of CG antigen

FIGURE 4. CG-X antigen genes are repressed in RKO cells following dual shRNA/siRNA G9a knockdown or dual G9a/GLP knockdown. A. RKO cellsstably expressing control shRNA or G9a shRNA were mock transfected or transfected with a G9a-specific siRNA. Cells were harvested 5 d after transfectionand G9a protein levels were measured by Western blot. α-Tubulin expression was measured as control for protein input, and band densitometry was done asdescribed in Materials and Methods. The sample key is shown at the bottom of B. B. RT-PCR analysis of CG antigen gene expression. PCR conditions andcontrols are the same as described in Fig. 1B, and the sample key is shown at the bottom.C. RKO cells stably expressing control shRNA or G9a shRNA weremock transfected or transfected with a GLP-specific siRNA. Five days after transfection, G9a and GLP protein levels were measured by Western blot. α-Tubulin expression was measured as control for protein input, and band densitometry was done as described in Materials and Methods. The sample key isshown at the bottom of D. D. RT-PCR analysis of CG antigen gene expression. PCR conditions and controls are the same as described in Fig. 1B, and thesample key is shown at the bottom.



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genes as well as other regions of the genome in murine EScells, and provide a plausible explanation for the activation ofCG antigen genes in this cell type.

CG Antigen Gene and Global Genomic DNA Methylationin G9a Knockdown Human Cancer Cells

We next examined DNA methylation in stable G9a and GLPknockdown human cancer cells. We used quantitative bisulfitepyrosequencing to determine the methylation level of the 5′CpG island promoters of MAGE-A1, NY-ESO-1, and XAGE-1. As expected, in both the RKO and HCT116 cell lines, stableG9a knockdown did not alter CG antigen promoter methylationlevels (Fig. 9A and B). Standard sodium bisulfite sequencingand methylation-specific PCR analyses confirmed the pyrose-quencing data (data not shown). In addition, stable G9a knock-down in HCT116 and RKO cells did not lead to altered globalDNA methylation, as assessed either by 5mdC levels or bypyrosequencing of the human LINE-1 repetitive DNA element(Fig. 9C and D). Consistent with the findings using stable G9aknockdown cells, transient knockdown of G9a or GLP in thestable G9a knockdown RKO cell line or combined stableG9a knockdown and TSA treatment in either HCT116 orRKO also did not alter CG antigen or global DNA methylation(data not shown).

DiscussionWe previously reported that disruption of DNMT enzymes

in human cancer cells results in reduced H3K9me2 levels atCG antigen gene promoters, correlating with increased gene ex-pression (8). In addition, it was previously shown that murineES cells deficient for G9a or GLP express Mage-a class CGantigen genes, coincident with reduced H3K9me2 levels at

their promoters (12, 13). These data suggested that H3K9me2and/or G9a/GLP may play a primary role in CG antigen generepression in human cancer cells. However, our current dataargue against this model. First, either transient or stable G9aknockdown in human cancer cell lines does not induce CG an-tigen gene expression, despite loss of H3K9me2 both globallythroughout the genome and specifically at CG antigen promoterregions. Second, simultaneous knockdown of G9a and GLPand combined genetic and pharmacologic inhibition of G9aalso failed to induce CG gene expression. Third, combinedG9a knockdown and HDAC inhibitor treatment generallyfailed to induce CG gene expression. Moreover, we previouslyfound that unlike double DNMT1/3b knockout cells, singleDNMT1−/− or DNMT3b−/− HCT116 cells show reducedH3K9me2 at CG-X gene promoters in the absence of DNAmethylation changes or expression of CG genes (8). Collective-ly, these data suggest that G9a/GLP knockdown and/or alteredhistone modification states are not sufficient alone to reactivatethe expression of CG antigen genes in human cancer cells.Moreover, our data argue that promoter DNA hypomethylationis the chief epigenetic signal leading to CG antigen expressionin human cancer cells. In agreement with our findings for CGantigen genes, it was recently reported that G9a knockdown inhuman prostate cancer cells leads to the up-regulation of veryfew genes in a genome-wide microarray screen and does notcause DNA hypomethylation of methylation-silenced tumorsuppressor genes (17).

Based on our data from human cancer cells, we reexaminedthe mechanism of CG antigen gene activation in G9a- or GLP-deficient murine ES cells (12, 13). We hypothesized that G9a orGLP loss could lead to CG gene promoter DNA hypomethy-lation in murine ES cells, but not in human cancer cells, thus

FIGURE 5. Combined genetic and pharma-cologic targeting of G9a in human cancer cells.A and B. RKO (A) and HCT116 (B) cells weretreated with DMSO (vehicle) or BIX-01294; cellswere harvested 48 h later; and H3K9me2 levelswere determined by Western blot analyses.Coomassie staining confirmed equal protein in-put, and band densitometry was done as de-scribed in Materials and Methods. The samplekey is shown at the bottom of the figure. C andD. RT-PCR analysis of CG antigen gene ex-pression in the experiment described in A andB. PCR conditions and controls are the sameas described in Fig. 1B, and the sample key isshown below the figure.

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explaining the differential outcomes in the two systems. Ourdata validated this hypothesis. In both G9a and GLP null EScells, the Mage-a2 and Mage-a8 promoters were severely hy-pomethylated coincident with gene activation. That this effectwas specific was validated by experiments that showed that re-introduction of wild-type G9a or GLP led to remethylation andsilencing of these genes. Interestingly, G9a and GLP null EScells also displayed both a global loss of methylation at thenon–long terminal repeat (LTR) short interspersed repetitiveelement (SINE) B1 and a reduction of total genomic 5mdClevels. In stark contrast, in human cancer cells in which G9aand/or GLP was down-regulated by RNA interference, bothCG promoter and global DNA methylation levels (either ofthe LINE-1 element or 5mdC levels) were unaltered. Thiswas true despite significant reduction of both global and pro-moter-specific H3K9me2 levels in these cells. These data em-phasize a primary regulatory role for DNA methylation in CGantigen gene regulation. The apparently tight dependence ofCG gene silencing on DNA methylation may be accountedfor by direct inhibition of Ets transcription factor binding, therecruitment of methylated DNA binding proteins at CG antigengene promoter regions, or other as yet undefined mechanisms(5, 29, 30).

Our finding of DNA hypomethylation in G9a/GLP null mu-rine ES cells is consistent with other reports (18, 25, 26). DNAhypomethylation in G9a null ES cells was initially described

for the Prader-Willi imprinting center, and it was later shownthat methylation of the Oct3/4 gene during differentiation isblocked in G9a null ES cells (25, 26). In addition, a recentstudy reported that 32 of 1,300 (∼2.5%) genomic loci are hy-pomethylated in G9a−/− ES cells, implying a more widespreadeffect (18). Here we show that G9a or GLP null ES cells dis-play significant reductions in 5mdC levels and hypomethyla-tion of the murine B1 SINE repetitive DNA element.Similarly, Dong et al. (31) recently reported that murine LTRand non-LTR LINE elements are DNA hypomethylated inG9a null ES cells. Together, these data suggest that G9a orGLP loss disrupts maintenance and/or de novo DNA methyla-tion. Loss of DNA methylation in G9a and GLP knockout EScells does not seem to result from decreased expression ofDnmt genes or proteins (31, 32). Instead, the mechanistic basisfor the DNA hypomethylation effect may reflect either reducedrecruitment of Dnmt1 to target sites during DNA replication orreduced Dnmt3a and/or Dnmt3b targeting to nonreplicatingchromatin (16, 31, 32). Intriguingly, recent data suggest thatDNA hypomethylation in G9a or GLP null murine ES cellsmay result from a loss of a function unrelated to their histonemethyltransferase activity (31, 32).

It is important to note that although H3K9me2 may not besufficient for CG antigen gene repression in human cancercells, it seems to play a secondary role. Notably, we found thatcertain G9a knockdown human colorectal cancer cells show an

FIGURE 6. Effect of combined G9a knockdown and HDAC inhibitor treatment on CG antigen gene expression. RKO and HCT116 stable control shRNAand stable G9a knockdown cells were treated with the HDAC inhibitor TSA at 600 nmol/L concentration for 24 h before harvesting. A. Histone protein extractswere prepared, as described in Materials and Methods, and used for Western blot analysis of acetylated histone H3 or acetylated histone H3K9 levels.Coomassie staining confirmed equivalent protein loading. B and C. Acetylated H3K9 levels at the MAGE-A1 (B) and XAGE-1 (C) promoters were measuredby quantitative ChIP-PCR. D. MAGE-A1 and XAGE-1 mRNA expression was measured by end-point RT-PCR. n and p, negative (no template) and positive(decitabine-treated cells) PCR controls, respectively.



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increased sensitivity to decitabine-mediated CG antigen geneinduction. Additionally, it was recently shown that G9a andDNMT1 knockdown cooperatively facilitate induction of theDNA methylation–silenced tumor suppressor gene MASPINin breast cancer cells (33). In ES cells, unlike human cancercells (or other somatic cells), it seems that either H3K9 meth-ylation or DNA methylation is sufficient for transcriptionalrepression (17, 32). This may reflect a “dual silencing” mecha-nism to maintain silent states during cellular differentiation,which is uniquely important in ES cells (17, 32).

We show here that murine ES cells are more susceptible tothe loss of DNA methylation at CG antigen genes followingG9a or GLP loss than are human cancer cells. One possible ex-planation for the differential effect in the two cell types is basedon the structure of CG antigen genes in the two species. In hu-mans, CG antigen genes have promoter regions of relativelyhigh CpG density, whereas murine Mage-a class CG genesgenerally have CpG poor promoter regions (ref. 8 and datanot shown). To mitigate this issue, our study focused on thetwo murine Mage-a genes (Mage-a2 and Mage-a8) that haveCpG dense promoter regions. Therefore, it is unlikely that theCpG density of the studied CG gene promoters accounts forthe different results observed in the two cell types. Of note,the orthologous human genes MAGE-A2 and MAGE-A8 werenot activated in human cancer cells sustaining G9a knockdown(data not shown). Moreover, the effects of G9a or GLP loss onglobal DNA methylation (e.g., 5mdC levels) in the two celltypes are also distinct, despite widespread conservation of themouse and human genomes. A second possible explanation forthe differential effect relates to the two cell types under study.We speculate that the dramatic loss of DNA methylation in G9aor GLP null ES cells reflects a greater level of “epigenetic plas-ticity” in this cell type than is seen in somatic cells (includingcancer cells). ES cells may require higher epigenetic plasticitybecause the ultimate fate of these cells is epigenetic reprogram-ming along specific cell lineages (34). One example of the epi-genetic plasticity of ES cells is the bivalent chromatin domainsconsisting of H3K4me3 and H3K27me3 often found at thepromoters of developmentally regulated genes in ES cells(35). Also consistent with this explanation, G9a knockout em-bryos (somatic cells) do not display DNA hypomethylation attarget loci, despite loss of H3K9 methylation at these regions(32). Finally, studies of the effect of DNA methylation on chro-matin structure and histone modifications suggest that there areimportant distinctions between epigenetic regulation in ES cellsand somatic cells (36).

In summary, our data emphasize a primary regulatory rolefor DNA methylation in CG antigen gene regulation in humancancer. There has been increasing interest in the utilization ofepigenetic modulatory drugs to promote CG antigen gene ex-pression to augment CG antigen vaccine efficacy in humancancer patients (2, 37). Our data suggest that these approachesmay need to include direct inhibition of DNMT enzymes as arequired step to promote CG antigen gene expression. Howev-er, it remains plausible that agents that modify histone modifi-cation status, including HDAC or H3K9 methyltransferaseinhibitors, may prove useful to enhance the effect of DNMTinhibitors in this context.

Materials and MethodsCell Culture and Drug Treatments

Colon adenocarcinoma cell lines RKO and HCT116(American Type Culture Collection) were cultured as describedpreviously (8). Mouse ES cells [wild-type (TT2), G9a−/−(22-10), GLP−/− (CD-10)] and G9a and GLP reintroductioncell lines were described previously (12, 13). Control or stableG9a knockdown HCT116 or RKO cell lines (described below)were treated with 5-aza-2′-deoxycytidine (decitabine; Sigma)

FIGURE 7. Stable G9a knockdown HCT116 cells show increased sen-sitivity to decitabine-mediated CG antigen gene activation. HCT116 celllines stably expressing control or G9a-specific shRNAs were treated withthe indicated concentrations of decitabine (5-aza-CdR) for 48 h; RNA ex-tracts were harvested; and MAGE-A1 (A), XAGE-1 (B), and NY-ESO-1(C) expression was measured using quantitative RT-PCR. Expression da-ta from all three genes were normalized to GAPDH as a cDNA input con-trol. Columns, mean expression values of triplicate data points; bars, 1 SD.

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dissolved in PBS and were harvested 48 h posttreatment. Inother experiments, cells were treated for 24 h with 600 nmol/LTSA (Sigma) solubilized in DMSO. The small-molecule G9ainhibitor BIX-01294 (23) was solubilized in DMSO and usedto treat control or stable G9a knockdown HCT116 or RKO celllines for 48 h.

Transient and Stable RNA Interference KnockdownExperiments

For transient knockdown of G9a or GLP (EuHMTaseI),RKO cells were transfected with 5 to 50 nmol/L siRNA (Am-bion) using Lipofectamine 2000 (Invitrogen). As a negativecontrol, RKO cells were transfected with 5 to 50 nmol/L ofsiControl Non-Targeting siRNA #2 (Dharmacon). siRNA trans-fections occurred on day 0 and day 2, and cells were harvestedon day 5. For stable knockdown, a shRNA specific for humanG9a was designed using the shRNA insert design tool (Am-bion). The shRNA duplex was ligated into the pSilencer 3.1-H1 neo vector (Ambion). After sequence verification, theG9a shRNA plasmid or a control shRNA vector (Ambion)was transfected into RKO and HCT116 cells using

Lipofectamine 2000 (Invitrogen). Antibiotic selection wasdone using 1.5 mg/mL (RKO) or 400 μg/mL (HCT116)G418 (Mediatech) and was continued until all mock-transfectedcontrol cells were killed. G9a shRNA– and control shRNA–expressing cell clones were picked and expanded while beingmaintained in normal media supplemented with G418 at onehalf of the antibiotic kill concentration. In combinationshRNA/siRNA experiments, control or stable G9a knockdownRKO cells were transfected with various siRNAs usingLipofectamine 2000 on day 0 and day 2, and cells were har-vested on day 5. siRNA and shRNA sequences are shown inSupplementary Table S1.

Colony Formation AssaysEquivalent numbers of RKO and HCT116 cells were trans-

fected with control or G9a shRNA plasmids, and antibiotic se-lection was done on cells grown in 15-cm tissue culture plates.For colony counts, cells were washed in PBS, fixed in 10%formaldehyde, and stained with 0.1% crystal violet in metha-nol. Colonies were counted manually.

FIGURE 8. CG antigen gene expression and DNA methylation in G9a and GLP knockout mouse ES cells. A. Western blot analysis of G9a and GLPprotein expression. α-Tubulin expression was measured as a control for protein input. B. RT-PCR analysis of Mage-a, Mage-a2, and Mage-a8 expression.Gapdh was amplified as a control for cDNA input. All reactions used 30 PCR cycles. Specific amplification of Mage-a2 and Mage-a8 was confirmed by DNAsequencing of the PCR amplicons. C. Methylation-specific PCR analysis of Mage-a2 5′ region methylation. Results are shown for control DNAs, wild-type EScells, and G9a and GLP knockout ES cells before and after reintroduction of the knocked out protein. U and M lanes correspond to PCR amplificationsspecific for unmethylated and methylated DNA, respectively. U and M control DNAs are described in Materials and Methods. D. Bisulfite pyrosequencinganalysis of Mage-a8 5′ region methylation. Samples are as described in C. E. Liquid chromatography mass spectrometry analysis of total genomic 5mdC.Samples are same as described in C. F. Bisulfite pyrosequencing analysis of B1 repetitive element methylation. Samples are as described in C. Statisticalanalysis shown in D to F used one-tailed t test (GraphPad Prism) and the relevant P values are indicated in the figure.



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RT-PCR and Quantitative RT-PCRTotal RNA was extracted using Trizol (Invitrogen), and

cDNA was generated using the First Strand cDNA SynthesisKit (Fermentas). RT-PCR and quantitative RT-PCR amplifica-tions of human MAGE-A1, NY-ESO-1, XAGE-1, and GAPDHwere done as described previously (8). Primer sequences formurine Mage-a, Mage-a2, Mage-a8, and Gapdh were designedusing Primer 3 (38) and are shown in Supplementary Table S1.Murine pan Mage-a primers were adapted from a previousstudy (39) and used to amplify mouse Mage-a2, Mage-a5,Mage-a6, and Mage-a8. Primers were obtained from IDT.

Western Blot Analyses of G9a and GLPWestern blots of whole-cell protein extracts were done as

described previously (8). G9a was detected using the D141-3primary antibody (MBL International Corp.) at 1:100 dilutionfollowed by protein A-horseradish peroxidase (AmershamBiosciences) at 1:1,000 dilution. Alternatively, G9a was de-tected using a polyclonal antibody (kind gift from Dr. SriharsaPradhan, New England Biolabs, Ipswich, MA) at 1:1,000 dilu-tion followed by antirabbit IgG (Santa Cruz Biotechnology) at1:10,000 dilution. GLP (EuHMTaseI) was detected using pri-mary antibody D220-3 (MBL International) at 1 μg/mL, incombination with protein A-horseradish peroxidase at 1:1,000dilution. α-Tubulin was detected as described previously (5).Proteins were detected using Western Lightning Chemilumi-nescence Reagent (Perkin-Elmer Life Sciences) and the Versa-doc imaging system (Bio-Rad).

Measurement of Histone ModificationsGlobal levels of histone H3 lysine 9 dimethylation

(H3K9me2), lysine 27 dimethylation (H3K27me2), and lysine9 acetylation (H3K9ac) were measured by Western blotting of

acid-soluble nuclear protein extracts. Extracts were preparedaccording to the Upstate Acid Extraction protocol (Upstate Bio-technology) and dialysis was accomplished using extra-strength10,000 MWCO Slide-A-Lyzer Dialysis Cassettes (Pierce). Ex-tracts were fractionated by SDS-PAGE, and Coomassie stainingwas used to confirm equivalent histone protein loading. Prima-ry antibodies for Western blot and ChIP analysis specific forH3K9me2, H3K27me2, H3K9ac, and H3K4me2 were obtainedfrom Upstate Biotechnology, and secondary antibodies wereobtained from Amersham Biosciences. Western blot detectionwas done as described above. Quantitative ChIP assays wereused to detect histone code modifications at the MAGE-A1and XAGE-1 promoters as described previously (8).

Densitometry Analysis of Western BlotsProtein densitometry was used to quantify the extent of siR-

NA and shRNA knockdowns and histone modification levelson Western blots. Chemiluminescent imaging of Western blotswas done with VersaDoc (Bio-Rad) and signal quantificationwas done with Quantity One software (Bio-Rad), using back-ground signal correction. G9a and GLP levels in total proteinextracts were normalized by the level of α-tubulin in sequen-tially probed blots. Histone modification levels in acid-solubleprotein extracts were not normalized; however, Coomassiestaining was used to visually confirm equivalent protein input.All Western blots were loaded with equivalent amounts of pro-tein per lane.

DNA Methylation Analysis of CG Antigen GenesDNA methylation levels of human MAGE-A1, NY-ESO-1,

and XAGE-1 were determined using quantitative sodium bisul-fite pyrosequencing (40). NY-ESO-1 pyrosequencing primerswere described previously (41). Pyrosequencing assays specific

FIGURE 9. DNA methyla-tion in stable G9a knockdownhuman cancer cells. A andB. Bisulfite pyrosequencinganalysis of MAGE-A1, NY-ESO-1, and XAGE-1 5′ pro-moter CpG is land regionmethylation in RKO (A) andHCT116 (B) control and stableG9a knockdown cell lines. Cand D. Global DNA methyla-tion analyses of RKO (C) andHCT116 (D) control and stableG9a knockdown cell lines.Two measurements of globalDNA methylation were done:bisulfite pyrosequencing ana-lysis of LINE-1 repetitive ele-ment methylation and liquidchromatography mass spec-trometry determination of total5mdC levels.

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for the 5′ CpG island or CpG-rich regions of human MAGE-A1and XAGE-1 were designed using the Pyrosequencing AssayDesign Software (Biotage) and the primers are shown inSupplementary Table S1. All primers were obtained fromIDT. Genomic DNA samples were isolated using the Puregenekit (Gentra Systems), and sodium bisulfite conversion and quan-titative pyrosequencing were accomplished as described previ-ously (10, 41). Of the murine Mage-a genes, only Mage-a2(region: −179 to +54, 234 bp, 51% GC, obs/exp = 0.6) andMage-a8 (region: −127 to +92, 220 bp, 52% GC, obs/exp = 0.6)contain 5′ CpG islands, meeting the classic definition (ref. 27;data not shown), and thus were selected for further analysis.Mage-a2 methylation was determined by methylation-specificPCR (42) using the primers shown in Supplementary Table S1.Mage-a8 methylation was determined by bisulfite pyrosequen-cing as described above, using the primers shown in Supplemen-tary Table S1. Controls included DNA methylated in vitro withSssI (New England Biolabs), and unmethylated DNA was gen-erated as described previously (43).

Global Genomic DNA Methylation AnalysesGlobal genomic DNA methylation was measured by deter-

mination of 5mdC levels using a quantitative liquid chromato-graphy mass spectrometry method (44) or by sodium bisulfitepyrosequencing of the murine SINE B1 or human LINE-1 re-petitive elements as described previously (28, 41). Primerswere obtained from IDT.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsWe thank Dr. E. Michael August of Boehringer Ingelheim (Ridgefield, CT) forkindly providing BIX-01294.

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2009;7:851-862. Published OnlineFirst June 16, 2009.Mol Cancer Res Petra A. Link, Omkaram Gangisetty, Smitha R. James, et al. Cancer Cells and Murine Embryonic Stem Cellsin Cancer Germ-Line Antigen Gene Regulation in Human Distinct Roles for Histone Methyltransferases G9a and GLP

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