Hypoxic Stress Induces Dimethylated Histone H3 Lysine 9 Through Histone Methyltransferase G9a in Mammalian Cells Haobin Chen,1 Yan Yan,1 Todd L

Research Article

Hypoxic Stress Induces Dimethylated Histone H3 Lysine 9 through Histone Methyltransferase G9a in Mammalian Cells Haobin Chen,1 Yan Yan,1 Todd L. Davidson,1 Yoichi Shinkai,2 and Max Costa1

1Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, New York and 2Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto, Japan

Abstract typically associated with constitutive heterochromatin, whereas Dimethylated histone H3 lysine 9 (H3K9me2) is a critical monomethylated H3K9 (H3K9me1) and dimethylated H3K9 epigenetic mark for gene repression and silencing and plays (H3K9me2) are mainly found in euchromatin and are associated an essential role in embryogenesis and carcinogenesis. Here, with repressed promoter regions (3). we investigated the effects of hypoxic stress on H3K9me2 at Several H3K9-specific methyltransferases have been identified both global and gene-specific level. We found that hypoxia and characterized, including Suv39h1, Suv39h2, G9a, GLP/ increased global H3K9me2 in several mammalian cell lines. EuHMTase 1, and SETDB1/ESET. Among them, G9a and GLP/ This hypoxia-induced H3K9me2 was temporally correlated EuHMTase 1 were found to be responsible for the dimethylation with an increase in histone methyltransferase G9a protein and processes on H3K9 in vivo (4, 5). The genetic ablation of either G9a or GLP/EuHMTase 1 resulted in a striking loss of global enzyme activity. The increase in H3K9me2 was significantly À À mitigated in G9a / mouse embryonic stem cells following H3K9me2 (4, 5). G9a was found to participate in gene silencing hypoxia challenge, indicating that G9a was involved in the processes by interacting with several repressive transcriptional hypoxia-induced H3K9me2. In addition to the activation of factors, including NRSF/REST, CDP/cut, PRDI-BF1/Blimp-1, and G9a, our results also indicated that hypoxia increased SHP (6–9). Recently, SETDB1/ESET has also been found to catalyze H3K9me2 by inhibiting H3K9 demethylation processes. Hyp- the formation of H3K9me2 both in vitro and in vivo (10). oxic mimetics, such as deferoxamine and dimethyloxalylgly- Compared with the methylation processes that act on histone cine, were also found to increase H3K9me2 as well as G9a lysines, the demethylation processes are just being studied recently. protein and activity. Finally, hypoxia increased H3K9me2 in The discovery of H3K4 demethylase, LSD1, provided the first the promoter regions of the Mlh1 and Dhfr genes, and these evidence for the existence of any histone lysine demethylase (11). LSD1 is a flavin-dependent amine oxidase and removes the methyl increases temporally correlated with the repression of these genes. Collectively, these results indicate that G9a plays an groupfrom monomethylated or dimethylated H3K4 by catalyzing important role in the hypoxia-induced H3K9me2, which would the oxidation of amine (11). LSD1 has also been reported to inhibit the expression of several genes that would likely lead to demethylate H3K9me2 when associated with the androgen receptor (12). Recently, Trewick et al. (13) have proposed that the solid tumor progression. (Cancer Res 2006; 66(18): 9009-16) methyl groups on histone lysine could also be removed by an oxidative demethylation mechanism. As proposed, a novel class of Introduction demethylases should possess iron-binding domains (e.g., Jmjc Post-translational modifications of histone NH2-terminal tails, domains) and could catalyze the generation of highly reactive such as acetylation, methylation, ubiquitination, and phosphoryla- oxygen species (ROS) in the presence of iron, 2-oxoglutarate, and tion, are important for chromatin organization and gene tran- oxygen (13). These generated ROS attack the methyl groups on scription (1). To date, histone acetylation and methylation are histone lysines and produce unstable intermediate oxidized among the best-characterized modifications. Histone lysine acety- products that spontaneously release formaldehyde, resulting in lations are generally associated with gene activation, whereas lysine the removal of methyl groups from histone lysines (13). JHDM1 is methylation may have either positive or negative effects on the first discovered demethylase that uses this proposed oxidative transcription depending on the sites (2). For instance, methylations demethylation process to remove the methyl groups from on histone H3 lysine 4 (H3K4), histone H3 lysine 36 (H3K36), and monomethylated and dimethylated H3K36 (14). Recently, JHDM2A histone H3 lysine 79 are often associated with transcriptional and the family of JMJD2 histone demethylases (JMJD2A-D) were activation and elongation, whereas methylations on histone H3 reported to demethylate H3K9 residues (15, 16). Similar to JHDM1, lysine 9 (H3K9) and histone H3 lysine 27 correlate with these newly identified H3K9 demethylases all possess Jmjc domains transcriptional repression (2). Adding to this complexity is the and require the presence of both iron and 2-oxoglutarate to be fact that lysine residues on histone can be monomethylated, active. Importantly, the overexpression or knockdown of these dimethylated, or trimethylated. Trimethylated H3K9 (H3K9me3) is H3K9 demethylases result in the alterations of global H3K9 methylations (15, 16). These discoveries indicate that H3K9 methylations are more dynamically involved in the regulation of Note: H. Chen and Y. Yan contributed equally to this work. chromatin functions than previously thought. Current address for Y. Yan: Faculty of Life Science, Northwestern Polytechnical Aberrant gene silencing is a common phenomenon in many University, 127 Youyi West Road, Xi’an, Shaanxi, 710072, China. Current address for T.L. Davidson: SafeBridge East, LLC, 330 Seventh Avenue, 8th Floor, New York, NY 10001. types of cancers. Hypoxia often occurs in a solid tumor because Requests for reprints: Max Costa, Nelson Institute of Environmental Medicine, normal tissue vasculature can only support tumor growth within a New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: diameter of f2 mm (17). Most studies have focused on the 845-731-3515; Fax: 845-351-2118; E-mail: [email protected]. I2006 American Association for Cancer Research. up-regulation of gene expression during hypoxia, which are doi:10.1158/0008-5472.CAN-06-0101 often mediated through hypoxia-inducible factor (HIF; ref. 18). www.aacrjournals.org 9009 Cancer Res 2006; 66: (18). September 15, 2006

The elevation of gene expression, such as vascular endothelial Immunoblottings were done with GFP antibody (1:200; Santa Cruz growth factor and the glycolytic enzymes, functions either to Biotechnology, Santa Cruz, CA). increase oxygen availability or to adjust intracellular metabolism After immunoblotting, the membranes were stripped as described a and thus improves cell survival under hypoxic stress. However, less previously (20) and reprobed with -tubulin (Sigma) or c-Jun (Santa Cruz Biotechnology) antibodies to assess the loading. The immunoblots for G9a attention has been paid to the down-regulation of gene expression and methylated H3K9s were scanned and subjected to densitometric during hypoxia. In this study, we report that exposure to hypoxia or analysis by Kodak 1D 3.52 (Rochester, NY) for Macintosh, and values were hypoxia mimetics is able to increase global H3K9me2 in several normalized to that obtained in the control sample. mammalian cell lines. Our results indicate that these alterations of Transient transfection and in vitro H3K9 methyltransferase activity global H3K9me2 can be attributed to both an increase in G9a assay. Transient transfection was done in HEK293 cells using Lipofect- protein and enzymatic activity as well as an inhibition of histone AMINE 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s demethylation processes under hypoxic conditions. In addition, an protocol. Twenty-four hours after transfection, the cells were exposed to increase in H3K9me2 at the promoter regions of Mlh1 and Dhfr hypoxia for 6 hours or 100 Amol/L deferoxamine for 24 hours. The cell genes was found to correlate with the repression of these two genes extracts were prepared either for Western blotting or for the in vitro H3K9 during hypoxic stress, indicating that the hypoxia-induced methyltransferase (HKMT) assay as described previously (20). The pellets were used for histone extraction as described earlier. H3K9me2 might play an important role in gene silencing during Northern blots. After treatments, A549 cells in each 100-mm dish were tumor progression. mixed with Trizol (Invitrogen) and total RNA was isolated following the manufacturer’s protocol. RNA (15 Ag) was separated over 1% agarose/ formaldehyde gels and transferred to BrightStar-Plus positively charged Materials and Methods nylon membranes (Ambion, Austin, TX). The Mlh1 and Dhfr probes were Cell culture. Human lung carcinoma A549 cells were grown in Ham’s amplified by PCR using primers that have been described previously F-12K medium (Life Technologies, Inc., Grand Island, NY); human embryonic (22, 23), and the G9a probes were amplified using the following set of kidney (HEK) 293 cells were grown in DMEM (Life Technologies); mouse primers: 5¶-GGAGGCACCCAAGATTGACC-3¶ (sense) and 5¶-CCTGAGCTGT- À À embryonic stem (MES) cell clones wild-type (WT), G9a knockout (G9a / ), CAATGGTGTC-3¶ (antisense). The probes were labeled with [a-32P]dCTP À À and a derivative from G9a / cells with a stably transfected G9a expression using a Prime-a-Gene Labeling System kit (Promega, Madison, WI). À À vector (G9a / + G9a WT) were grown in DMEM supplemented with Membranes were prehybridized in a hybridization solution (Sigma) for 1 Â 103 units/mL murine ESGRO (Chemicon, Temecula, CA); mouse 3 hours and then hybridized overnight with the radiolabeled probes. The embryonic fibroblast HIF-1a proficient (HIF-1a+/+) and deficient membranes were washed, and their radioactivity was visualized by À À (HIF-1a / ) cells were obtained from R. Johnson (University of California autoradiography. The same membranes were reprobed with h-actin probes at San Diego, San Diego, CA; ref. 19) and grown in DMEM. All media were as described previously (24). supplemented with 10% fetal bovine serum (Omega Scientific, Inc., Tarzana, Chromatin immunoprecipitation assays. The chromatin immuno- CA) and 1% penicillin/streptomycin (Life Technologies). Cells were passaged precipitation (ChIP) assays were done as described previously (25, 26). twice to thrice each week and grown to 80% to 90% confluency before any The following sets of primers were used for PCR amplification: Mlh1, treatment. Cells were exposed to hypoxic conditions in a chamber with a 5¶-ACCGCTCGTAGTATTCGTGCTC-3¶ (sense) and 5¶-GTGGATGACGCC- ¶ ¶ ¶ humidified gas mixture of 0.5% O2 and 99.5% N2 at 37jC. The levels of oxygen CAAAAGAAG-3 (antisense); Dhfr,5-AAACTGGAGACCTAAGGGCAGC-3 in chambers were verified using a gas monitor (SKC, Inc., Eighty Four, PA). (sense) and 5¶-ATTCTGATGAGGGGGCTTGTG-3¶ (antisense); and Cap43, Chemicals and expression vectors. Dimethyloxalylglycine was pur- 5¶-GGAGCGGGGAAGGGGTGTGT-3¶ (sense) and 5¶-GGCGGATGGTGAACT- chased from Frontier Scientific, Inc. (Logan, UT). All other reagents were GACGA-3¶ (antisense). To facilitate the detection of PCR product, [32P]dCTP obtained from Sigma (St. Louis, MO) unless otherwise specified. The (0.1 AL; Perkin-Elmer, Wellesley, MA) was added into each PCR mixture. The green fluorescent protein (GFP)–tagged human G9a expression vector, PCR products were separated over 5% Tris-EDTA polyacrylamide gels. The pEGFP-hG9a, and its SET domain deletion mutant expression vector, gels were dried, and the radioactivity was visualized by autoradiography. pEGFP-hG9a(DSET), were kindly provided by M. Walsh (Mt. Sinai School Statistical analysis. The two-tailed Student’s t test was used to of Medicine, New York, NY; ref. 8). determine the significance of difference between treated samples and Western blotting. Histones were prepared from cells as described control. The difference was considered significant at P < 0.05. previously (20). Equal amounts of histones (5 Ag) were separated over 15% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Roche Applied Sciences, Indianapolis, IN). Immuno- Results blottings were done with dimethyl H3K9 (1:2,000; Upstate Biotechnology, Hypoxic stress alters global H3K9 acetylation and methyl- Inc., Lake Placid, NY), monomethyl H3K9 (1:1,000; Upstate Biotechnology), ations. To examine the possible effects of hypoxia on global trimethyl H3K9 (1:400; Abcam, Cambridge, MA), acetylated H3K9 (Ac-H3K9; 1:2,000; Upstate Biotechnology), or acetylated H4 (1:4,000; Upstate Biotech- histone modifications, histones were extracted from lung carcino- nology) antibodies. The primary antibodies were detected by chemical ma A549 cells at selected time intervals, and the acetylation and fluorescence following an enhanced chemiluminescence Western blotting methylation levels on H3K9 were assessed by Western blotting. protocol (Amersham, Piscataway, NJ). After transfer to PVDF membranes, Interestingly, hypoxia was found to decrease Ac-H3K9 and all SDS-polyacrylamide gels were stained with Bio-safe Coomassie blue stain monomethylated H3K9 but increase dimethylated and trimethy- (Bio-Rad, Hercules, CA) to assess the loading of histones. lated H3K9 in a time-dependent manner (Fig. 1A). In parallel The cell extracts for HIF-1a detection were prepared as described samples, intracellular HIF-1a protein levels were found to increase a previously (21). The immunoblottings were done with HIF-1 antibodies in a time-dependent manner during hypoxic stress (Fig. 1A). It was (1:500) from either Transduction Laboratories (BD Biosciences, San Jose, noticed that both isoforms of human HIF-1a that result from CA) or Novus Biologicals (Littleton, CO). In order to detect G9a in nuclei, alternative splicing were present in A549 cells as were previously the nuclear extracts were prepared using a CelLytic NuCLEAR Extraction kit a (Sigma). The nuclear extracts containing 40 Ag protein per sample were found in HeLa cells (27). This increase in HIF-1 protein levels separated over 7.5% SDS-polyacrylamide gels. Immunoblotting was done indicates that a hypoxic response is initiated in these cells under with G9a antibody (1:500; Upstate Biotechnology). To detect overexpressed the low-oxygen condition used in this study. GFP-hG9a or GFP-hG9a(DSET) fusion proteins, the whole-cell lysates The hypoxia-induced H3K9me2 was further studied. As shown containing 75 Ag protein per sample were used for Western blotting. in Fig. 1B, the increase in H3K9me2 occurred following hypoxia

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Figure 1. Hypoxic stress altered global H3K9 modifications. A, A549 cells were exposed to a hypoxic atmosphere of 0.5% O2 and 99.5% N2, and histones were then extracted at selected time intervals. The modifications on H3K9 were detected using Western blotting as described in Materials and Methods. In parallel samples, the intracellular HIF-1a protein levels were measured using Western blotting. After HIF-1a immunoblotting, the same membrane was stripped and restained with a-tubulin antibody to assess protein loading. B, Exposure to hypoxia for 24 hours increased H3K9me2 levels in different cell types. C, A549 cells were incubated with 25 ng/mL TSA for 1 hour and then exposed to hypoxia for 6 hours. Histones were extracted and immunoblotted for H3K9me2 and Ac-H4. Loading of the histones in all SDS-polyacrylamide gels were assessed using Coomassie blue staining. in several other cell lines, including human osteosarcoma and synthesis inhibitor) before hypoxia challenge. As shown in Fig. 2C, HEK293 cells. Because the decrease in Ac-H3K9 was more striking cyclohexamide decreased nuclear G9a protein but did not prevent and appeared at earlier time intervals after hypoxia (Fig. 1A), we its relative increase during hypoxic stress. Collectively, these results investigated whether this decrease in Ac-H3K9 was required for the indicated that hypoxia increased nuclear G9a protein by post- increase in H3K9me2. As shown in Fig. 1C, trichostatin A (TSA), a translational mechanisms. histone deacetylase inhibitor, dramatically increased acetylated Hypoxic stress increases G9a methyltransferase activity. To histone H4 but failed to block the hypoxia-induced H3K9me2. examine whether the increases in nuclear G9a protein coincided Therefore, the increase in H3K9me2 is likely caused by an with its higher methyltransferase activity, an in vitro methyltrans- imbalance between the methylation and demethylation processes ferase assay was done. It has been reported that, in addition to G9a, that act on H3K9 residues during hypoxic stress. many other methyltransferases also exhibit HKMT activity in vitro Hypoxic stress increases nuclear methyltransferase G9a (4). Therefore, GFP-tagged G9a fusion protein was overexpressed in protein by post-translational mechanisms. Among several HEK293 cells to specifically measure G9a activity. In agreement identified histone methyltransferases, G9a plays a dominant role with our hypothesis that hypoxia increases G9a protein levels by in the dimethylation of H3K9 in vivo (4). To examine the role of post-translational mechanisms, hypoxia increased the overex- G9a in the hypoxia-induced H3K9me2, G9a mRNA levels were pressed GFP-hG9a fusion protein levels (Fig. 3). In contrast, hypoxia measured in A549 cells following hypoxia exposure. It was found decreased the overexpressed GFP-hG9a(DSET) protein levels, that hypoxia did not cause any measurable changes of G9a mRNA suggesting that the methyltransferase SET domain was essential at two earlier time intervals (1 and 3 hours) but led to a significant for the increase in G9a protein during hypoxic stress decrease at 6 hours after challenge (Fig. 2A). (Fig. 3). As shown in Fig. 3, overexpression of GFP-hG9a fusion The effects of hypoxia on G9a protein were next examined. As proteins increased H3K9me2 in transfected cells but did not further shown in Fig. 2B, hypoxia increased both isoforms of G9a protein increase the hypoxia-induced H3K9me2. Overexpression of GFP- that result from alternative splicing (4, 28) in the nuclei. These hG9a(DSET) fusion proteins, which has been reported to have increases temporally correlated with the higher levels of H3K9me2 dominant-negative effects against the functions of endogenous G9a in the same cells following hypoxic stress (Fig. 2B). Consistent with (9), partially blocked the increases in H3K9me2 during hypoxic previous reports showing that the overexpressed exogenous G9a stress without changing the basal levels of H3K9me2 (Fig. 3). proteins were exclusively present in the nuclei (4, 28), G9a protein Afterwards, the overexpressed GFP fusion proteins were immuno- was not detected in the cytoplasm from either exposed or precipitated and their HKMT activity was measured by an in vitro unexposed cells by Western blotting (data not shown). To examine assay. Because no methyltransferase activity was detected in the whether hypoxia up-regulates G9a protein by increasing its protein samples transfected with GFP-hG9a(DSET) vectors, the activity translation, cells were pretreated with cyclohexamide (a protein measured in the samples transfected with GFP-hG9a specifically www.aacrjournals.org 9011 Cancer Res 2006; 66: (18). September 15, 2006

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Cancer Research represented the HKMT activity of the overexpressed GFP-hG9a still elevated global H3K9me2 levels compared with untreated cells fusion proteins (Fig. 3). Consistent with the increased protein levels (Fig. 4B). Therefore, these results provided indirect evidence to of GFP-hG9a, hypoxia increased its methyltransferase activity support that, in addition to the increases in G9a protein and (Fig. 3). It was noticed that the increases in GFP-hG9a fusion enzymatic activity, hypoxia also increases H3K9me2 by inhibiting proteins and activity seemed to be less remarkable than the in- histone lysine demethylation processes. creases in H3K9me2 during hypoxic stress, indicating other me- Hypoxia mimetics increase global H3K9me2 levels as well as chanisms likely also contribute to the hypoxia-induced H3K9me2. G9a expression and activity. Similar to hypoxia challenge, Hypoxic stress also increases global H3K9me2 levels by a hypoxia mimetics (deferoxamine and dimethyloxalylglycine) also G9a-independent mechanism. To further study the roles of G9a increased H3K9me2 and nuclear G9a protein in A549 cells (Fig. 5A). in the hypoxia-induced H3K9me2, a MES cell clone homozygous In addition, deferoxamine increased the methyltransferase activity À À for G9a deletion (G9a / ) and its derivative containing a stably of overexpressed GFP-hG9a fusion proteins (Fig. 5B). Similar to À À transfected G9a expression vector (G9a / + G9a WT) were used. the findings in Fig. 4B, deferoxamine was also able to increase the À À Because the H3K9me2 levels in G9a / MES cells were very low global levels of H3K9me2 under methionine-deficient conditions, compared with WT cells (4), proportionately greater amounts of suggesting that the demethylation processes for H3K9 were À À histones extracted from G9a / cells were loaded to facilitate inhibited by deferoxamine. comparisons. Exposure to hypoxia decreased Ac-H3K9, and this Because a common consequence of both deferoxamine and decrease was not affected by the presence of a functional G9a gene hypoxia challenge is the stabilization and transactivation of HIF- (Fig. 4A). Genetic ablation of G9a significantly mitigated the 1a, a mouse embryonic fibroblast cell line with genetic ablation À À hypoxia-induced H3K9me2, but increases of H3K9me2 were still of HIF-1a (HIF-1a / ) was used to examine the role of this À À observed but to a lesser degree in G9a / cells following exposure transcription factor in the hypoxia-induced H3K9me2. As shown in À À to hypoxia (1.8-fold increase in H3K9me2 in G9a / cells and Fig. 5D, a slightly higher basal level of H3K9me2 was observed À À À À 2.8-fold increases in WT and G9a / + G9a WT cells). These results in HIF-1a / cells compared with HIF-1a+/+ cells. However, hypoxia confirm that G9a is involved in the hypoxia-induced H3K9me2 but increased H3K9me2 in both cell lines to similar levels (Fig. 5D), also suggest that other mechanism(s) may contribute to the indicating that HIF-1a was not involved in the increase in hypoxia-induced H3K9me2. These alternative mechanisms may H3K9me2 by hypoxia challenge and by hypoxia mimetics. include an activation of other H3K9 methyltransferase(s) and/or Hypoxia decreases the expression of Mlh1 and Dhfr genes inhibition of histone demethylation. and increases H3K9me2 in their promoters. H3K9me2 is usually To further investigate the G9a-independent mechanism(s) by associated with gene repression and silencing (2). Previously, it has which hypoxia increases H3K9me2, cells were incubated in a been reported that H3K9 methylation is connected with the methionine-deficient medium before hypoxia challenge. Because repression or silencing of Dhfr (dihydrofolate reductase) and Mlh1 methionine is essential for S-adenosylmethionine (SAM) synthesis (involved in mismatch repair) genes (30, 31). It is of our interest to and SAM has a short half-life in cells (29), the withdrawal of study whether the hypoxia-induced H3K9me2 is involved in the methionine from the culture medium should cause a lowered repression of these two genes. As shown in Fig. 6A, hypoxia intracellular SAM pool and a generalized inhibition of methyl decreased the mRNA levels of Mlh1 and Dhfr genes in a time- transfer reactions. As shown in Fig. 4B, incubation of cells in the dependent manner. Next, ChIP assays were used to analyze their methionine-deficient medium for 28 hours dramatically decreased promoter regions following hypoxia challenge. Consistent with the global H3K9me2 levels, indicating that the histone methylation repression of gene expression, the H3K9me2 levels at the promoter processes were attenuated when methionine was removed from the regions of Mlh1 and Dhfr genes were significantly increased medium. Under methionine-deficient conditions, hypoxia challenge following hypoxia challenge (Fig. 6B). We have reported previously

Figure 2. Hypoxic stress increased the protein levels of histone methyltransferase G9a by a post-translational mechanism. A, A549 cells were exposed to hypoxia and their RNA was extracted at selected time intervals. RNA (15 Ag) was subjected to Northern blot analysis with human G9a probes. Afterwards, the membranes were reprobed with human b-actin probes. Loading of the total RNA in the agarose/formaldehyde gels was assessed using ethidium bromide staining. B, A549 cells were exposed to hypoxia, and the nuclear extracts and histones were then prepared at selected time intervals. Western blottings were done for G9a and H3K9me2 detection. The loading of nuclear extracts was assessed by reprobing the same membranes with c-Jun antibody. C, A549 cells were pretreated with 100 Amol/L cyclohexamide (CHX) for 4 hours and then exposed to hypoxia for 6 hours. Western blottings for G9a and c-Jun were done as described earlier.

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suggest that this increase in H3K9me2 is likely linked with the repression of several genes during hypoxic stress. H3K9-specific methyltransferase G9a is identified as one important cause of the hypoxia-induced H3K9me2. This conclusion is based on several lines of evidence, including that hypoxia increased both G9a protein and HKMT activity as well as genetic ablation of G9a significantly mitigated the hypoxia-induced H3K9me2. It was found that H3K9me2 and G9a protein levels were slightly increased in A549 control samples at later time intervals (Fig. 2B). These increases were likely caused by pericellular hypoxia as a result of increased cell densities in nontreated cells, which had already reached 80% to 90% confluence before hypoxia challenge. In support of this hypothesis, several studies reported that HIF-1a protein levels and the expression of HIF-regulated genes were increased in numerous mammalian cell lines when their cell density reached confluency (39, 40). We had also found that the expression of Cap43/Ndrg1, a HIF-regulated gene (24), was elevated in confluent A549 cells (data not shown). Our results indicate that hypoxia increases G9a protein levels by post-translational mechanisms. In support of this notion, it was found that hypoxia did not increase G9a mRNA levels and an inhibition of general protein synthesis failed to block the increase of nuclear G9a protein in the hypoxia-exposed cells. In fact, hypoxia decreased G9a mRNA levels at later time interval, which was likely caused by the general inhibitory effects of lowered Ac-H3K9 and higher H3K9me2 levels on gene transcription. It was noticed that the mRNA levels of G9a began to decrease at 6 hours after hypoxia Figure 3. Hypoxia increased histone methyltransferase G9a activity. HEK293 cells were transiently transfected with pEGFP, pEGFP-hG9a, or pEGFP- challenge (Fig. 2A), whereas the protein levels of G9a and H3K9me2 hG9a(DSET) expression vectors and then exposed to hypoxia for 6 hours. The continued to increase at later time intervals (Fig. 2B). Such a whole-cell extracts and histones were prepared as described in Materials and phenomenon indicates that the inhibition of G9a protein Methods. The expression of fusion proteins in whole-cell lysates was assessed by Western blotting using anti-GFP antibody. The histones from the same degradation has more prominent effects on G9a protein levels samples were immunoblotted for H3K9me2. The overexpressed fusion proteins than the decrease of G9a mRNA levels and thus allows for a were immunoprecipitated using anti-GFP antibody and used for in vitro continual increase of G9a protein at later time intervals. Several HKMT assays. The transfer of 3H-labeled methyl groups catalyzed by the immunoprecipitates was either visualized by autoradiography or measured with a attempts were made to identify the nature of this post-translational scintillation counter. The results from three independent experiments on G9a mechanism. In agreement with previous reports that HIF is not activity measurement were converted to the values relative to that of samples transfected with GFP-hG9a vector, and these relative values were then used for involved in the repression of several genes during hypoxic stress the statistical analysis. *, P < 0.05, statistically significant compared with control (35, 41), genetic ablation of HIF-1a did not affect the hypoxia- samples. induced H3K9me2. Hypoxia has also been known to activate multiple signaling pathways in cells, such as mitogen-activated protein kinases and phosphatidylinositol 3-kinase (42, 43). that hypoxia increased the expression of Cap43/Ndrg1 (a HIF-1- However, several chemical inhibitors of these signaling pathways responsive gene) as early as 4 hours after challenge (24). It was failed to block the hypoxia-induced H3K9me2 in A549 cells (data found that the H3K9me2 levels at the promoter region of Cap43 not shown). At present, the precise mechanism by which hypoxia gene remained unchanged (Fig. 6B). Collectively, these findings increases nuclear G9a protein remains unknown. indicate that the increase in H3K9me2 is likely involved in the In addition to the increases in G9a protein and methyltransfer- repression of genes during hypoxic stress. ase activity, our results also indicate that the inhibition of histone lysine demethylation processes contributes to the hypoxia-induced H3K9me2. It was reported that LSD1 demethylated H3K9me2 when Discussion associated the androgen receptor in human LNCaP prostate tumor Hypoxia is commonly present in many solid tumors and often cells (12). However, this mechanism is not responsible for H3K9 occurs when the growth of tumors outstrips their blood supply demethylation in A549 cells because the androgen receptor gene is (32). In response to hypoxia, tumor cells increase the expression of not expressed in A549 lung cells (data not shown). Moreover, genes related to angiogenesis, glycolysis, and glucose uptake using H3K9me2 was not increased in A549 cells following treatment with the transcription factor HIF (18). Hypoxia also decreases the deprenyl hydrochloride, a monoamine oxidase inhibitor that has expression of several genes, such as cellular adhesion proteins and been shown to abolish LSD1 activity in a previous study (12). A new DNA repair proteins, and leads to genetic instability and metastasis class of H3K9 demethylases have just being identified recently. of tumor cells (33–36). In agreement with these reports, several These H3K9 demethylases, JHDM2A and JMJD2A-D, all possess clinical studies have shown that tumor oxygenation is an important Jmjc domains similar to the one found in the JHDM1 and require marker for cancer patients’ prognosis and future distant disease iron and 2-oxoglutarate to catalyze oxidative demethylations recurrence (37, 38). Here, we report that hypoxia increases (15, 16). JHDM2A demethylates both H3K9me1 and H3K9me2 H3K9me2, an important repressive epigenetic mark. Our results in vivo (16), whereas JMJD2A mainly demethylates H3K9me3 www.aacrjournals.org 9013 Cancer Res 2006; 66: (18). September 15, 2006

Figure 4. Hypoxia increased global H3K9me2levels by both G9a-dependent and G9a-independent mechanisms. A, WT, G9a À/À,andG9a À/À + G9a WT MES cells were exposed to hypoxia for 6 hours. The histones were extracted and immunoblotted for H3K9me2and Ac-H3K9. B, HEK293 cells were replenished with either complete or methionine-deficient DMEM and incubated for 4 hours before hypoxia exposure. After hypoxia challenge for 24 hours, histones were extracted and immunoblotted for H3K9me2. in vivo (15). Because oxygen is essential for the oxidative (Fig. 1A). Interestingly, hypoxia was found to increase both demethylation reactions catalyzed by these demethylases, oxygen H3K9me2 and H3K9me3 in this study. At the time this article deprivation should inhibit the activity of these H3K9 demethy- was written, the iron- and 2-oxoglutarate-dependent histone lase(s). In support of this notion, the hypoxic conditions used in demethylases have been found to remove the methyl groups from this study inhibited HIF-related prolyl hydroxylases that belong to H3K9 and H3K36 residues, but the list of their potential substrates the same enzyme family as these H3K9 demethylases and thus is likely to grow. Hypoxia should also inhibit the demethylases for resulted in the stabilization and accumulation of HIF-1a protein H3K36 or other unrecognized lysine sites. However, because the

Figure 5. Hypoxia mimetics increased global H3K9me2levels as well as G9a expression and activity. A, A549 cells were exposed to hypoxia, 100 Amol/L deferoxamine (DFX), or 1 mmol/L dimethyloxalylglycine (DMOG)for 18 hours. Western blottings for G9a and H3K9me2were done as described earlier. B, HEK293 cells were transiently transfected with pEGFP-hG9a or pEGFP- hG9a(DSET) expression vectors. Twelve hours after transfection, cells were exposed to 100 Amol/L deferoxamine for 18 hours. The methyltransferase activity of the overexpressed proteins was measured as described in Fig. 3. Columns, mean from the relative values of three independent experiments; bars, SD. *, P < 0.05, statistically significant change compared with control samples. C, A549 cells were seeded in F-12K complete medium. After overnight of incubation, the cells were replenished with either complete or methionine-deficient DMEM and incubated for another 4 hours. After exposure to 100 Amol/L deferoxamine for 24 hours, histones were extracted and immunoblotted for H3K9me2. D, HIF-1a +/+ and HIF-1a À/À mouse embryonic fibroblast cells were exposed to hypoxia for 6 hours. Histones were extracted and immunoblotted for H3K9me2. In parallel samples, intracellular HIF-1a protein levels were measured with Western blotting.

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Figure 6. Hypoxia decreased the expression of Dhfr and Mlh1 genes and increased H3K9me2levels in their promoters. A, A549 cells were exposed to hypoxia and RNA was extracted at selected time intervals. RNA (15 Ag) was subjected to Northern blot analysis with human Dhfr or Mlh1 probes. B, A549 cells were exposed to hypoxia for 6 hours, and the ChIP assays were done with either H3K9me2or rabbit anti-mouse IgG (Abcam) antibodies. The specific primers were used for the PCR amplification as indicated. Results of two independent experiments.

demethylation processes for histone lysines are only one arm of an endogenous gene silencing process reported that both H3 their methylation metabolism, the global methylation levels are deacetylation and H3K9 methylation occurred within the same also affected by the effects of hypoxia on the other arm, the time window as gene inactivation, and these changes preceded the methylation processes. In other words, if the expression/activity of establishment of DNA methylation at the promoter region of this specific methyltransferases is decreased during hypoxic stress, the gene (46). Our results are consistent with the gene silencing process global levels of the corresponding histone modification may not described in the second scenario because an increase of H3K9me2 change or only slightly increase, although the demethylases are occurred before or concurrent with the repression of genes during inhibited. The possible effects of hypoxia on other histone lysine hypoxic stress. The persistence of hypoxia may eventually lead to marks are not investigated here and should be examined in future the silencing of genes (such as Mlh1) that indirectly cause other studies. gene mutation and genome instability and transform cells into a Hypoxia was also found to increase H3K9me2 at the promoter more aggressive phenotype. regions of several genes. The increases in H3K9me2, together with In conclusion, this study provides the first evidence to our the hypoxia-induced decrease in histone acetylation, may act in knowledge that hypoxia exposure increases global H3K9me2, which concert to repress gene expressions during hypoxic stress. In plays an important role in gene repression during hypoxia. This agreement with this notion, hypoxia was also found to decrease the increase of global H3K9me2 by hypoxia is likely to be a key event in histone acetylation at the promoter region of Mlh1 gene in several selection for the more aggressive phenotype of tumor cells. mammalian cell lines (35), and genetic ablation of G9a alone did not attenuate the decrease of Mlh1 mRNA levels during hypoxic stress (data not shown). As a repressive epigenetic mark, H3K9me2 Acknowledgments is critical for the establishment of DNA methylation and long-term Received 1/10/2006; revised 7/9/2006; accepted 7/12/2006. Grant support: National Institute of Environmental Health Sciences grants gene silencing (44). In a previous study on transgenes, Mutskov ES00260, ES10344, and T32-ES07324 and National Cancer Institute grant CA16087. et al. (45) reported that both histone deacetylation and loss of The costs of publication of this article were defrayed in part by the payment of page H3K4 methylation were early events of the transgene silencing, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. whereas H3K9 and DNA methylation only appeared in the late We thank Juliana Powell for her secretarial support and Martin Walsh for providing phase of the silencing process. In contrast, a recent study on pEGFP-G9a and pEGFP-G9a (DSET) expression vectors.

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Haobin Chen, Yan Yan, Todd L. Davidson, et al.

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