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. Author manuscript; available in PMC: 2016 Jan 28.
Published in final edited form as: Cell Rep. 2016 Jan 7;14(3):506–519. doi: 10.1016/j.celrep.2015.12.053

KDM4C and ATF4 Cooperate in Transcriptional Control of Amino Acid Metabolism

Erhu Zhao 1,2,#, Jane Ding 2,#, Yingfeng Xia 2,8, Mengling Liu 2,8, Bingwei Ye 2, Jeong-Hyeon Choi 2,3, Chunhong Yan 2,4, Zheng Dong 5, Shuang Huang 7, Yunhong Zha 8, Liqun Yang 1, Hongjuan Cui 1,*, Han-Fei Ding 2,4,6,*
PMCID: PMC4731315  NIHMSID: NIHMS747035  PMID: 26774480

SUMMARY

The histone lysine demethylase KDM4C is often overexpressed in cancers primarily through gene amplification. The molecular mechanisms of KDM4C action in tumorigenesis are not well defined. Here we report that KDM4C transcriptionally activates amino acid biosynthesis and transport, leading to a significant increase in intracellular amino acid levels. Examination of the serine-glycine synthesis pathway reveals that KDM4C epigenetically activates the pathway genes under steady-state and serine deprivation conditions by removing the repressive histone modification H3 lysine 9 (H3K9) trimethylation. This action of KDM4C requires ATF4, a transcriptional master regulator of amino acid metabolism and stress responses. KDM4C activates ATF4 transcription and interacts with ATF4 to target serine pathway genes for transcriptional activation. We further present evidence for KDM4C in transcriptional coordination of amino acid metabolism and cell proliferation. These findings suggest a molecular mechanism linking KDM4C-mediated H3K9 demethylation and ATF4-mediated transactivation in reprogramming amino acid metabolism for cancer cell proliferation.

INTRODUCTION

Histone lysine methyltransferases (KMTs) and demethylases (KDMs) have a central role in regulation of transcription by controlling the state of histone lysine methylation. KMTs use S-adenosylmethionine (SAM) as the methyl group donor, while KDM1 and KDM2-KDM8 family members require flavin adenine dinucleotide (FAD) and α-ketoglutarate (α-KG) for demethylation, respectively (Black et al., 2012; Mosammaparast and Shi, 2010). The dependence of KMTs and KDMs on metabolic coenzymes suggests that their activities are sensitive to changes in cell metabolism, a model supported by a compelling body of evidence from recent studies (Gut and Verdin, 2013; Kaelin and McKnight, 2013; Katada et al., 2012; Lu and Thompson, 2012; Lu et al., 2012; Shyh-Chang et al., 2013; Teperino et al., 2010). This notion also suggests that, based on the principle of feedback control, KMTs and KDMs must reciprocally influence cell metabolism through transcriptional regulation of metabolic enzymes (Teperino et al., 2010) (Figure S1A).

Cancer cell growth and proliferation require enhanced metabolic capacity for accumulation of biomass and replication of the genomic DNA (Cairns et al., 2011; DeBerardinis et al., 2008; Vander Heiden et al., 2009). Increased activation of the serine-glycine synthesis pathway (herein referred to as the serine pathway) through genetic (Locasale et al., 2011; Possemato et al., 2011) and epigenetic (Ding et al., 2013) mechanisms has been observed in several cancer types. In addition, recent studies have provided evidence for a key role of serine uptake in sustaining the proliferation of cancer cells (Jain et al., 2012; Labuschagne et al., 2014; Maddocks et al., 2013). The serine pathway is composed of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), phosphoserine phosphatase (PSPH), and serine hydroxymethyltransferase (SHMT). This pathway generates biosynthetic precursors essential for the production of proteins, nucleic acids, fatty acids, and the membranes needed for cell proliferation (Amelio et al., 2014; DeBerardinis, 2011; Kalhan and Hanson, 2012; Locasale, 2013) (Figure S1B). More recently, it has been shown that serine-driven one-carbon metabolism is a major pathway of NADPH production in proliferating cells, with oxidation of 5,10-methylene-tetrahydrofolate to 10-formyl-tetrahydrofolate being coupled to reduction of NADP+ to NADPH (Fan et al., 2014). NADPH is required for reductive biosynthesis, such as the synthesis of nucleotides, amino acids and lipids, and has a pivotal role in maintaining the cellular redox balance (Schulze and Harris, 2012). Also, cancer cells can uptake exogenous serine for the production of glycine and one-carbon units through the final step of the serine pathway catalyzed by SHMT (Labuschagne et al., 2014) (Figure S1B). Thus, a better understanding of the function and regulation of the serine pathway might suggest new therapeutic approaches for inhibiting cancer metabolism and blocking cancer growth (Chaneton et al., 2012; Maddocks et al., 2013).

We recently identified a G9A-dependent epigenetic mechanism for transcriptional activation of the serine pathway in cancer cells (Ding et al., 2013). G9A, also known as EHMT2 and KMT1C, is a H3K9 methyltransferase that has a primary role in catalyzing H3K9me1 and H3K9me2 in euchromatin (Shinkai and Tachibana, 2011), with H3K9me1 being associated with active chromatin and H3K9me2 being a repressive mark (Black et al., 2012; Mosammaparast and Shi, 2010). We found that G9A is required for maintaining the serine pathway genes in an active state and for transcriptional activation of this pathway in response to serine deprivation. Moreover, higher G9A expression significantly increases serine and glycine biosynthesis in the cell. These findings provide direct evidence for transcriptional reprograming of cell metabolism by a KMT.

An implication of the G9A study is that H3K9 methylation states control the transcription of serine pathway genes. This led us to hypothesize that KDMs that target H3K9 may also play a role in transcriptional regulation of the serine pathway. Multiple KDMs catalyze the removal of methyl groups at H3K9: KDM3B can remove all methyl groups (me1-3); KDM4 only me2 and me3; and KDM3A and KDM7A-B only me1 and me2 (Black et al., 2012; Mosammaparast and Shi, 2010). Thus, the KDM4 family of demethylases could transcriptionally activate serine pathway genes by removing the repressive marks H3K9me2 and H3K9me3 at their loci. We focused our study on KDM4C, also known as JMJD2C, primarily because of the strong evidence for an important role of KDM4C in cancer development (Berry and Janknecht, 2013; Labbe et al., 2014). The KDM4C gene is located in chromosome 9p24, which is amplified in various cancer types, including lymphoma, breast cancer, esophageal squamous cell carcinoma, lung sarcomatoid carcinoma, and medulloblastoma (Berdel et al., 2012; Cloos et al., 2006; Ehrbrecht et al., 2006; Italiano et al., 2006; Liu et al., 2009; Northcott et al., 2009; Rui et al., 2010; Vinatzer et al., 2008; Wu et al., 2012; Yang et al., 2000). Our study reveals that KDM4C has a general role in transcriptional activation of amino acid biosynthesis and transport, including serine and glycine. These findings provide evidence for the ability of KDMs to reprogram amino acid metabolism in cancer cells.

RESULTS

KDM4C Is Essential for the Expression of Serine Pathway Genes and Cancer Cell Proliferation

As an initial step to assess the role of KDM4C in transcriptional control of the serine pathway, we analyzed the published dataset GSE28332 from a recent chromatin immunoprecipitation and sequencing (ChIP-seq) study of genome-wide KDM4C binding sites (Pedersen et al., 2014). The analysis revealed specific association of KDM4C with the promoters of serine pathway genes, including PHGDH and PSAT1 (Figure 1A), in KYSE150 cells, a human esophageal squamous cell carcinoma cell line. Interestingly, ChIP-seq data from the same study also showed the binding of Kdm4c (Jmjd2c) to the Phgdh and Psat1 promoters in mouse embryonic stem cells but not fibroblasts (Figure S1C), suggesting a specific role of KDM4C in regulation of serine pathway genes in cells with unlimited proliferative potential.

Figure 1. See also Figures S1 and S2. KDM4C is essential for transcriptional activation of the serine pathway and cell proliferation.

Figure 1

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(A) ChIP-seq tag profiles for KDM4C levels at the PHGDH and PSAT1 promoters in KYSE150 cells expressing control (shCtrl) or KDM4C shRNA (shKDM4C). Horizontal bars represent chromatin regions with significant changes in KDM4C levels (shCtrl/shKDM4C). The ChIP-seq dataset GSE28332 was used for the analysis. (B-C) ChIP-qPCR analysis of KDM4C (B), H3K9me2 and H3K9me3 (C) levels at the PHGDH, PSAT1 and HOXC9 promoters in BE(2)-C cells expressing shRNA to GFP (shGFP) or KDM4C (shKDM4C). Error bars represent SD (n = 3) and are representative of two independent experiments.

(D) qRT-PCR analysis of mRNA expression of serine pathway genes and HOXC9 in the indicated cell lines expressing shGFP, shKDM4C or shG9A. Error bars represent SD (n = 3).

(E) Immunoblotting of KDM4C, PHGDH and PSAT1 in SHEP1 and HeLa cells expressing shGFP or shKDM4C. KDM4C, PHGDH and PSAT1 levels were quantified against β-actin.

(F) Growth assay of the indicated cell lines expressing shGFP or shKDM4C. Error bars represent SD (n = 4). Data were analyzed by two-way ANOVA with p values indicated.

(G-H) Soft agar assay of BE(2)-C and SK-N-DZ cells expressing shGFP or shKDM4C. The image (G) is representative of three independent experiments with BE(2)-C cells. Colonies were counted (H), with error bars indicating SD (n = 3).

Unless indicated, all data were analyzed with two-tailed Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

We next determined whether KDM4C is required for transcription of serine pathway genes in human cancer cell lines by silencing KDM4C with small hairpin RNA (shRNA). We identified two independent shRNA constructs (shKDM4C-54 and -58) that were highly effective in knockdown of KDM4C expression (Figures S2A and S2B) but showed no significant effect on the expression of other KDM4 family members (Figure S2B). Immunofluorescence staining showed that KDM4C knockdown resulted in a significant increase in H3K9me3 levels (Figures S2C and S2D). Consistent with the ChIP-seq finding (Figure 1A), ChIP and quantitative PCR (ChIP-qPCR) revealed significant levels of KDM4C at the promoters of PHGDH and PSAT1 in neuroblastoma BE(2)-C cells (Figure 1B, shGFP). KDM4C knockdown markedly decreased KDM4C levels at these promoters (Figure 1B, shKDM4C-58), with a corresponding increase in H3K9me3, but not H3K9me2, levels (Figure 1C). As a control, we observed no significant binding of KDM4C to the promoter of HOXC9 (Figure 1B), a gene involved in neuroblastoma differentiation (Mao et al., 2011; Wang et al., 2013).

In agreement with the ChIP-qPCR results, quantitative reverse transcriptase-PCR (qRT-PCR) showed that KDM4C knockdown significantly repressed the transcription of serine pathway genes, but not that of HOXC9 (Figures 1D, left and middle panels, and S2E), leading to a marked reduction in PHGDH and PSAT1 protein levels (Figure 1E). Also, as reported previously (Ding et al., 2013), G9A knockdown significantly reduced mRNA expression of serine pathway genes (Figure 1D, right panel). Thus, similar to G9A, KDM4C contributes to the maintenance of serine pathway genes in an epigenetically active state for transcription.

In addition, in all the cancer cell lines examined, we observed a pronounced inhibitory effect of KDM4C silencing on cell proliferation in culture (Figures 1F, S2F and S2G) and in soft agar (Figures 1G and 1H). These findings are in line with recent studies from other groups (Cloos et al., 2006; Gregory and Cheung, 2014; Kim et al., 2014; Pedersen et al., 2014), indicating a general role of KDM4C in sustaining proliferation. Supplemental serine and glycine failed to rescue KDM4C knockdown cells (data not shown), suggesting additional defects in these cells that block proliferation.

Ectopic KDM4C Expression Transcriptionally Activates the Serine Pathway and Promotes Cancer Cell Proliferation

As noted earlier, the KDM4C gene is amplified in various cancer types, leading to elevated levels of KDM4C expression. We therefore investigated the effect of high KDM4C expression on serine pathway gene expression using an inducible system. After induction, KDM4C levels in these inducible cell lines were well within the range as those seen in human neuroblastoma cell lines (Figure S3A), indicating that the expression levels are biologically relevant. Induction of KDM4C in BE(2)-C, HeLa, and U2OS cells led to significantly decreased H3K9me3 levels, accompanied by a marked increase in H3K9me1 levels (Figures S3B-E). We observed no significant changes in H3K9me2 levels (Figures S3B-E). Moreover, ChIP-qPCR revealed significantly higher levels of KDM4C associated with the promoters of PHGDH and PSAT1 following KDM4C induction (Figure 2A), with a corresponding reduction in H3K9me3 levels (Figure 2B) and a significant increase in the ratio of H3K9me1 to H3K9me3 (Figure 2C).

Figure 2. See also Figure S3. KDM4C transcriptionally activates the serine pathway and promotes proliferation and tumorigenicity.

Figure 2

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(A-C) ChIP-qPCR analysis of the levels of KDM4C (A), H3K9me2 and H3K9me3 (B), and the ratio H3K9me1 to H3K9me3 (C) at the PHGDH, PSAT1 and HOXC9 promoters in BE(2)-C cells with inducible KDM4C expression. Error bars represent SD (n = 3) and are representative of two independent experiments.

(D) qRT-PCR analysis of mRNA levels of serine pathway genes and HOXC9 in BE(2)-C and HeLa cells with inducible KDM4C expression. Error bars represent SD (n = 3). (E-F) Immunoblotting of KDM4C, PHGDH and PSAT1 in the indicated cell lines with inducible KDM4C expression (E) and in HeLa cells transfected with plasmids expressing GFP, wild-type KDM4C or catalytic inactive mutant KDM4C S198M (F). KDM4C, PHGDH and PSAT1 levels were quantified against β-actin (E) or α-tubulin (F).

(G) Growth assay of the indicated cell lines with inducible KDM4C expression. Error bars represent SD (n = 4). Data were analyzed by two-way ANOVA with p values indicated.

(H-I) Xenograft assay of BE(2)-C cells with inducible expression of GFP or KDM4C. Tumor weight (I) was analyzed by scatter plot with horizontal lines indicating the mean. (J) Immunoblotting of PHGDH in HeLa_tetoff_KDM4C cells expressing shGFP or shPHGDH. β-actin levels are shown as loading control.

(K) Growth assay of HeLa cells with or without KDM4C induction and PHGDH knockdown (n = 4). Data were analyzed by two-way ANOVA with p values indicated. ***p < 0.001.

In agreement with the ChIP-qPCR results, KDM4C overexpression alone was sufficient to transcriptionally activate serine pathway genes (Figure 2D), leading to a significant increase in PHGDH and PSAT1 protein levels (Figure 2E). Importantly, the ability of KDM4C to transcriptionally activate serine pathway genes depends on its demethylase activity, since overexpression of KDM4C-Ser198Met (S198M), a catalytic inactive KDM4C mutant (Kupershmit et al., 2014), was unable to upregulate PHGDH and PSAT1 (Figure 2F). Together, these results indicate that KDM4C directly targets serine pathway genes for transcriptional activation by conferring an epigenetically active state with decreased H3K9me3 and increased H3K9me1 levels.

In line with recent studies from other groups (Cloos et al., 2006; Gregory and Cheung, 2014; Kim et al., 2014; Pedersen et al., 2014), KDM4C induction in various cancer cell lines markedly enhanced cell proliferation (Figure 2G) and tumorigenicity (Figures 2H and 2I). Knockdown of PHGDH expression by shRNA largely abolished the proliferation-enhancing activity of KDM4C (Figures 2J and 2K). These findings are consistent with the model that the serine pathway is a key downstream target of KDM4C in promoting cell proliferation.

KDM4C Has a General Role in Transcriptional Activation of Amino Acid Biosynthesis and Transport

To better understand the molecular mechanism of KDM4C action, we performed microarray gene expression profiling of BE(2)-C cells before and after KDM4C induction. A total of 401 KDM4C-responsive genes (≥ ±1.40 fold, P < 0.05) were identified, with 250 genes being upregulated and 151 downregulated (Table S1). Consistent with the data presented above, Gene Ontology (GO) analysis revealed that genes upregulated by KDM4C are highly enriched for GO terms associated with biosynthesis and metabolism of the serine family of amino acids (Figure 3A and Table S2). In addition, GO analysis suggests a role of KDM4C in transcriptional activation of amino acid transport, the unfolded protein response and chromatin assembly (Figure 3A and Table S2). PANTHER (Protein ANalysis THrough Evolutionary Relationships) analysis, which examines evolutionary sequence–function relationships (Thomas et al., 2003), further revealed a general role of KDM4C in the control of amino acid transport and biosynthesis (Figure 3B and Table S2). We found no significant effect of KDM4C on other major metabolic pathways including glycolysis and the tricarboxylic acid (TCA) cycle (Tables S1 and S2), suggesting a specific function of KDM4C in regulation of amino acid metabolism.

Figure 3. See also Figure S4 and Tables S1 and S2. KDM4C transcriptionally activates amino acid biosynthesis and transport.

Figure 3

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(A-B) GO and PANTHER analyses of genes upregulated by KDM4C in BE(2)-C cells. Shown are top 10 GO (A) and PANTHER (B) biological process terms based on fold enrichment.

(C-D) qRT-PCR analysis of amino acid biosynthesis (C) and transporter (D) genes induced by KDM4C. Error bars represent SD (n = 3).

(E) GC-MS analysis of amino acid levels in BE(2)-C_tetoff_KDM4C cells with or without doxycycline for 6 days. Error bars represent SD (n = 5).

(F) Immunoblotting of KDM4C, S6K and pS6K (T389) in BE(2)-C and HeLa cells with or without KDM4C induction. α-tubulin levels are shown as loading control.

(G) Growth assay of BE(2)-C cells with or without KDM4C induction and 10 nM Rapamycin (rapa). Error bars represent SD (n = 4).

**p < 0.01, ***p < 0.001.

We confirmed the microarray data by qRT-PCR. In addition to serine pathway genes, KDM4C significantly upregulated mRNA expression of genes encoding asparagine synthetase (ASNS), argininosuccinate synthase 1 (ASS1), cystathionine gamma-lyase (CTH), glutamic-oxaloacetic transaminase 1 (GOT1, also known as aspartate aminotransferase), and glutamic pyruvate transaminase 2 (GPT2) (Figure 3C). These enzymes are involved in the biosynthesis of alanine, arginine, asparagine, aspartic acid, cysteine, and glutamic acid. We also confirmed by qRT-PCR that KDM4C induced mRNA expression of SLC1A4, SLC1A5, SLC3A2, SLC6A9, SLC7A1, SLC7A5, andSLC7A11, but not that of SLC38A2 (Figure 3D). These proteins are involved in the transport of all amino acids except aspartic acid, asparagine, and proline (Hyde et al., 2003). These results, coupled to the observation that KDM4C binds to many of these gene promoters (Figure S4), suggest that KDM4C directly targets these genes for transcriptional activation.

In support of the gene expression data, gas chromatography-mass spectrometry (GC-MS) analysis revealed that KDM4C induction markedly elevated the intracellular levels of 15 amino acids (Figure 3E). In addition, there was a significant increase in the kinase activity of the mechanistic target of rapamycin complex 1 (mTORC1) as demonstrated by increased phosphorylation of the ribosome protein S6 kinase (S6K) (Figure 3F), a key mTORC1 substrate. Importantly, the ability of KDM4C to promote cell proliferation was completely abrogated by rapamycin, an inhibitor of mTORC1 (Figure 3G). It is known that elevated levels of amino acids activate mTORC1, which in turn promotes cell growth and proliferation by stimulating macromolecule biosynthesis and cell cycle progression (Kim and Guan, 2011; Laplante and Sabatini, 2012). These findings link KDM4C to the activation of amino acid-mTORC1 signaling, which may contribute to KDM4C-induced proliferation.

KDM4C Links Amino Acid Metabolism to Cell Cycle Progression

It has been shown recently that KDM4C transcriptionally activates the expression of genes that promote cell cycle progression, such as CCNB1 and MYBL2 (Pedersen et al., 2014) (see also Figure S5). In line with this finding, gene set enrichment analysis (GSEA) of our microarray data revealed that KDM4C induction led to higher expression of a large number of cell cycle genes including those critical for M phase progression (Figures 4A and 4B). Particularly interesting is the observation that most of the Forkhead box protein M1 (FOXM1) pathway genes were transcriptionally activated (Figures 4C and 4D). We confirmed the microarray data by qRT-PCR, which showed that KDM4C significantly increased mRNA expression of CCNB1, CCNE1, CDK2, FOXM1, and MYBL2, but not that of CCND1 (Figure 4E). MYBL2 and FOXM1 are transcription factors that have an essential role in coordinating the expression of cell cycle genes required for S and M phase progression, respectively (Sadasivam and DeCaprio, 2013). These findings, together with those presented above, suggest a KDM4C-dependent epigenetic mechanism in transcriptional coordination of amino acid metabolism and proliferation in cancer cells.

Figure 4. See also Figure S5 and Tables S1. KDM4C induces late cell cycle genes.

Figure 4

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(A-D) GSEA shows significant enrichment of gene sets involved in the cell cycle (A), M phase (B), and the FOXM1 pathway (C-D), with most of the genes being upregulated by KDM4C.

(E) qRT-PCR analysis of mRNA levels of cell cycle genes in BE(2)-C cells with or without KDM4C induction. Error bars represent SD (n = 3). **p < 0.01, ***p < 0.001.

KDM4C Is Required for the Serine Deprivation Response

Our GSEA of the microarray data also showed that KDM4C upregulated most of the genes involved in the amino acid deprivation response, including ASNS, ATF3, DDIT3 (also known as CHOP), and TRIB3 (Figures 5A and S6A), which was confirmed by qRT-PCR (Figure 5B). Moreover, our analysis of the ChIP-seq dataset GSE28332 showed specific association of KDM4C with the promoters of ASNS (Figure S4A), ATF3 and DDIT3 promoters (Figure S6B), suggesting that they are direct target genes of KDM4C.

Figure 5. See also Figure S6 and Tables S1. KDM4C is required for the serine deprivation response.

Figure 5

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(A) GSEA shows significant enrichment of the gene set involved in the amino acid deprivation response, with most of the genes being upregulated by KDM4C.

(B) qRT-PCR analysis of mRNA levels of ASNS, ATF3, DDIT3 and TRIB3 in BE(2)-C and HeLa cells with inducible KDM4C expression. Error bars represent SD (n = 3).

(C-D) qRT-PCR analysis of mRNA levels of serine pathway and amino acid deprivation response genes following serine withdrawal in SHEP1 (C) and HeLa (D) cells expressing shGFP or shKDM4C. Error bars represent SD (n = 3).

(E-F) ChIP-qPCR analysis of KDM4C (E) and H3K9me3 (F) levels at the PHGDH, PSAT1 and HOXC9 promoters in HeLa cells in the presence (Ctrl) or absence of serine for 6 hr. Error bars represent SD (n = 3) and are representative of two independent experiments.

**p < 0.01, ***p < 0.001.

These observations also provided us with an experimental system for investigating whether KDM4C has a physiological function in transcriptional control of amino acid metabolism. It is known that serine deprivation leads to transcriptional activation of the serine pathway (Ding et al., 2013; Maddocks et al., 2013; Ye et al., 2012). As expected, serine removal from the culture media resulted in elevated mRNA expression of serine pathway genes, as well as ASNS, ATF3, DDIT3, and TRIB3 (Figures 5C and 5D, shGFP). Knockdown of KDM4C expression largely abrogated the serine deprivation response at the transcriptional level (Figures 5C and 5D, shKDM4C). Consistent with the qRT-PCR results, serine deprivation markedly increased KDM4C levels at the PHGDH and PSAT1 promoters (Figure 5E), which was accompanied by a significant reduction in H3K9me3 levels (Figure 5F). Together, these results indicate that KDM4C epigenetically activates transcription of serine pathway genes in response to serine deprivation.

ATF4 Is a Direct Transcriptional Target of KDM4C

Activating Transcription Factor 4 (ATF4) has a key role in the amino acid deprivation response (Ameri and Harris, 2008; Kilberg et al., 2009). We found that knockdown of KDM4C expression led to a marked reduction in ATF4 expression at both mRNA and protein levels (Figures 6A and 6B), indicating an essential role of KDM4C in maintaining the basal level of ATF4 expression. Conversely, KDM4C overexpression robustly induced ATF4 at both mRNA and protein levels (Figures 6C and 6D). Examination of the ChIP-seq dataset GSE28332 revealed that KDM4C specifically binds to the ATF4 promoter (Figure S6B, ATF4). The finding was confirmed by ChIP-qPCR assay, which showed that KDM4C overexpression significantly increased the level of KDM4C at the ATF4 promoter (Figure 6E), with a corresponding increase in the ratio of H3K9me1 to H3K9me3 (Figure 6F). Collectively, these results indicate that ATF4 is a direct target gene of KDM4C.

Figure 6. See also Figure S6. KDM4C is required for transcriptional activation of ATF4.

Figure 6

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(A) qRT-PCR analysis of ATF4 mRNA levels in the indicated cancer cell lines with or without KDM4C knockdown. Error bars represent SD (n = 3).

(B) Immunoblotting of ATF4 and KDM4C in HeLa and SHEP1 cells with or without KDM4C knockdown. ATF4 and KDM4C levels were quantified against β-actin.

(C) qRT-PCR analysis of ATF4 mRNA levels in BE(2)-C and HeLa cells with inducible KDM4C expression. Error bars represent SD (n = 3).

(D) Immunoblotting of ATF4 and KDM4C in BE(2)-C and HeLa cells with inducible KDM4C expression. ATF4 and KDM4C levels were quantified against β-actin.

(E-F) ChIP-qPCR analysis of KDM4C levels (E) and the ratio H3K9me1 to H3K9me3 (F) at the ATF4 promoter in BE(2)-C cells with inducible KDM4C expression. Error bars represent SD (n = 3) and are representative of two independent experiments.

(G) ChIP-qPCR analysis of KDM4C and H3K9me3 levels at the ATF4 promoter in HeLa cells with or without serine for 6 hr. Error bars represent SD (n = 3) and are representative of two independent experiments.

(H-I) qRT-PCR analysis of ATF4 mRNA levels in HeLa and SHEP1 cells following serine withdrawal (H) and with or without KDM4C knockdown (I). Error bars represent SD (n = 3).

(J) Immunoblotting of ATF4 and KDM4C in SHEP1 cells following serine withdrawal with or without KDM4C knockdown. ATF4 and KDM4C levels were quantified against β-actin.

*p < 0.05, **p < 0.01, ***p < 0.001.

We next investigated whether KDM4C has a physiological function in transcriptional control of ATF4 expression. It is known that ATF4 expression is markedly increased following amino acid deprivation, as a result of both transactivation of its gene and stabilization of its protein (Ameri and Harris, 2008; Kilberg et al., 2009). We found that serine deprivation significantly increased the level of KDM4C at the ATF4 promoter, accompanied by a significant reduction in the level of H3K9me3 (Figure 6G). As expected, serine deprivation induced ATF4 mRNA expression (Figure 6H). Importantly, the induction was completely abrogated by knockdown of KDM4C expression (Figure 6I), leading to a marked decrease in ATF4 protein levels compared to shGFP control (Figure 6J). Together, these findings identify an essential role of KDM4C both in the maintenance of the basal level of ATF4 expression and in the transcriptional induction of ATF4 in response to serine deprivation.

KDM4C Requires ATF4 for Transcriptional Activation of the Serine Pathway and Amino Acid Transport

Previous studies have shown that PHGDH and PSAT1 are target genes of ATF4 (Adams, 2007; Seo et al., 2009; Ye et al., 2012). It is also known that ATF4 can transcriptionally upregulate amino acid transporters (Adams, 2007). These observations suggest that ATF4 may function downstream of KDM4C in transcriptional regulation of amino acid metabolism. In support of this hypothesis, knockdown of ATF4 expression completely abrogated the ability of KDM4C to promote cell proliferation (Figure 7A) and to upregulate serine pathway and amino acid transporter genes (Figures 7B and 7C). Reciprocally, ATF4 also required KDM4C for maximal induction of serine pathway genes, as KDM4C knockdown significantly inhibited the induction of PHGDH and PSAT1 by ATF4 overexpression (Figure S7). Thus, KDM4C and ATF4 cooperate, and are mutually dependent on each other, in transcriptional regulation of amino acid metabolism.

Figure 7. See also Figure S7. KDM4C requires ATF4 for transcriptional activation of serine pathway genes.

Figure 7

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(A) Growth assay of BE(2)-C_ and HeLa_tetoff_KDM4C cells with or without ATF4 knockdown in the absence of doxycycline (n = 4). Data were analyzed by two-way ANOVA with p values indicated.

(B) qRT-PCR analysis of mRNA levels of ATF4, serine pathway and amino acid transporter genes in HeLa_tetoff_KDM4 cells in the presence or absence of doxycycline for 7 days with or without ATF4 knockdown. Error bars represent SD (n = 3).

(C) Immunoblotting of ATF4, KDM4C and PHGDH in HeLa_tetoff_KDM4C cells in the presence or absence of doxycycline for 7 days with or without ATF4 knockdown. ATF4 and PHGDH levels were quantified against α-tubulin.

(D) ChIP-qPCR analysis of ATF4, KDM4C and H3K9me3 levels at the PHGDH promoter in BE(2)-C_tetoff_KDM4C cells in the absence of doxycycline for 6 days with or without ATF4 knockdown. Error bars represent SD (n = 3) and are representative of two independent experiments.

(E-F) Immunoblot analysis of Co-IP of KDM4C and ATF4 in extracts from BE(2)-C_tetoff_KDM4C cells in the absence of doxycycline for 6 days (E) and from HeLa cells in serine-deficient media for 4 hr (F).

(G) Model for KDM4C-ATF4 cooperation in transactivation of serine pathway genes.

**p < 0.01, ***p < 0.001.

To gain a molecular understanding of the cooperation, we investigated the possibility that KDM4C may require ATF4 for binding to target gene promoters. We examined the PHGDH promoter, which was associated with high levels of KDM4C and ATF4 following KDM4C induction (Figure 7D, shRNA). As expected, ATF4 knockdown significantly reduced the level of ATF4 at the PHGDH promoter (Figure 7D, anti-ATF4_shATF4-73). Importantly, ATF4 knockdown also resulted in a marked reduction in the level of KDM4C at the promoter (Figure 7D, anti-KDM4C_shATF4-73), with a corresponding increase in H3K9me3 levels (Figure 7D, anti-H3K9me3_shATF4-73). Moreover, we found that ATF4 could be co-immunoprecipitated with KDM4C under the conditions of KDM4C overexpression (Figure 7E) and serine deprivation (Figure 7F). Together, these results demonstrate that KDM4C interacts with and depends on ATF4 for targeting serine pathway genes (Figure 7G).

DISCUSSION

In this report, we present evidence for cooperation between the histone H3K9 demethylase KDM4C and the transcription factor ATF4 in transcriptional regulation of amino acid metabolism. In addition, we present evidence suggesting a KDM4C-dependent epigenetic mechanism in coordinating amino acid metabolism and cell cycle progression. Together, these findings provide direct evidence for metabolic reprograming by KDM4C and shed new light on its oncogenic activity in cancer development.

KDM4C in Transcriptional Control of Amino Acid Metabolism

Our study reveals a general role of KDM4C in transcriptional activation of amino acid biosynthesis and transport. KDM4C overexpression transcriptionally activates the serine pathway and several enzymes involved in the biosynthesis of alanine, arginine, asparagine, aspartic acid, cysteine, and glutamic acid. KDM4C can also upregulate the expression of transporters for a majority of amino acids. As a result, cells with KDM4C overexpression show a marked increase in the intracellular levels of 15 amino acids. Given that KDM4C is amplified or overexpressed in a wide range of human cancers, our findings suggest that high KDM4C expression may represent an epigenetic mechanism for activation of amino acid biosynthesis and transport in cancer development.

To gain a molecular understating of KDM4C action in transcriptional control of amino acid metabolism, we examined in detail the effect of KDM4C on transcriptional regulation of the serine pathway. Our study reveals that KDM4C is required both for maintaining basal-level expression of the pathway genes and for their transcriptional induction in response to serine deprivation. KDM4C binds to the promoters of the pathway genes and confers an epigenetically active state by removing the repressive mark H3K9me3. Of note, most of the cell lines examined in this study also express significant levels of KDM4A and KDM4B (data not shown). Whether they have a similar role in serine pathway regulation remains to be defined. However, the observed significant effect of KDM4C knockdown on the pathway gene expression suggests that KDM4C has a non-redundant function in maintaining the transcription state of the serine pathway, at least in the cell lines examined.

We recently reported a G9A-dependent epigenetic program in transcriptional activation of the serine pathway by specifically marking the pathway genes with H3K9me1 (Ding et al., 2013). Our findings with KDM4C provide further evidence in support of the model that H3K9 methylation has a critical role in determining the transcriptional state of serine pathway genes. However, it should be noted that KDM4C and G9A have distinct roles in transcriptional control of amino acid metabolism. In addition to the serine pathway, KDM4C transcriptionally activates several amino acid synthesis enzymes and transporters. This broad role of KDM4C in the control of amino acid metabolism might also be one of the reasons why supplemental serine and glycine failed to rescue the proliferation defect of KDM4C-knockdown cells. By contrast, G9A appears to target specifically the serine pathway, as G9A inhibition or overexpression only affects the intracellular levels of serine and glycine, and supplemental serine can rescue the proliferation defect of G9A inhibition (Ding et al., 2013). The underlying molecular mechanism remains to be determined.

The KDM4C-ATF4 Connection in Transcriptional Control of Amino Acid Metabolism

ATF4 is a transcriptional master regulator of amino acid metabolism and stress responses (Ameri and Harris, 2008; Baird and Wek, 2012; Kilberg et al., 2009). The molecular basis for translational induction of ATF4 in response to amino acid deprivation is well defined. The decrease in cellular amino acid levels results in the accumulation of uncharged tRNA, which then binds and activates the stress kinase General Control Nonderepressible 4 (GCN4, also known as EIF2AK4). GCN4, in turn, phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), which represses global protein synthesis while enhancing the translation and expression of ATF4 (Harding et al., 2003). ATF4 then transcriptionally activates genes involved in amino acid synthesis and transport, leading to alleviation of the nutritional stress.

ATF4 can also be transcriptionally activated in response to amino acid deprivation (Dey et al., 2010; Siu et al., 2002), but the underlying molecular mechanism is not well understood. Our study reveals an essential role of KDM4C in maintaining basal-level expression of ATF4 and in transcriptional activation of ATF4 in response to serine deprivation. KDM4C binds to the ATF4 promoter and confers an epigenetically active state by removing the repressive mark H3K9me3, leading to increased production of ATF4 mRNA and protein.

Our study further demonstrates that the transcriptional induction of ATF4 is essential for KDM4C to activate the serine pathway and amino acid transporters. Moreover, we show that KDM4C interacts with and depends on ATF4 for binding to the promoters of serine pathway genes, thereby providing a molecular mechanism for targeting KDM4C to these genes. In addition to acting through ATF4, KDM4C also contributes directly to the activation of serine pathway genes by generating an epigenetically active state, as evidenced by the observation that even under overexpression conditions, ATF4 still requires KDM4C for maximal levels of gene transcription.

There is evidence suggesting that the ability of KDM4C to activate amino acid biosynthesis and transport contributes to its oncogenic activity in promoting cancer cell proliferation and tumorigenesis since knockdown of PHGDH or ATF4, or inhibition of mTORC1, completely abrogated the proliferation-enhancing activity of KDM4C. Together, our investigation reveals a molecular mechanism linking KDM4C-mediated H3K9 demethylation and ATF4-mediated transcription in reprogramming amino acid metabolism in cancer cells.

KDM4C Links Amino Acid Metabolism to Cell Cycle Control

Cell cycle progression duplicates the entire cellular content through anabolic metabolism, ultimately resulting in cell division that produces two daughter cells. Given the critical role of amino acid availability in the control of protein production and cell proliferation, a cell must have effective mechanisms in place for coordinating amino acid metabolism and cell cycle progression. Our findings suggest a KDM4C-dependent epigenetic mechanism for this coordination. It is well documented that high KDM4C expression promotes cell proliferation and upregulate the expression of cell cycle genes (Cloos et al., 2006; Gregory and Cheung, 2014; Kim et al., 2014; Pedersen et al., 2014). We confirmed these observations. In addition, our gene expression profiling revealed that KDM4C transcriptionally activates FOXM1 and a majority of genes in the FOXM1 pathway. FOXM1 is a transcription factor that activates the expression of many mitotic genes and is essential for execution of the mitotic program (Sadasivam and DeCaprio, 2013). High FOXM1 expression is a common feature of many types of cancers and is generally thought to be a key mechanism underlying the high expression of mitotic genes that is frequently observed in cancers of poor prognosis (Sadasivam and DeCaprio, 2013). Given that KDM4C is amplified or overexpressed in a wide range of human cancers, high KDM4C expression may represent an epigenetic mechanism for the aberrant activation of the FOXM1-dependent transcriptional program in cancer development. Thus, KDM4C coordinates amino acid metabolism and cell cycle progression by epigenetically activating the transcription of genes involved in both cellular processes.

EXPERIMENTAL PROCEDURES

Cell culture and reagents

All cell lines used in this study were originally obtained from ATCC. Cells were cultured in DMEM (HyClone SH30022, Thermo Scientific) or DME/F-12 1:1 (HyClone SH30023) supplemented with 10% FBS (Atlanta Biologicals S11050). For serine deprivation assays, cells were cultured in MEM (Gibco 10095) supplemented with 1x MEM vitamins (Gibco 11120), 25 mM D-glucose (Sigma-Aldrich G8769), 0.4 mM serine (Sigma-Aldrich S4311) and 10% dialyzed FBS (Gibco 26400), and then transferred to the same media without serine. Phase contrast images were captured using an Axio Observer microscope and AxioVision software (Carl Zeiss MicroImaging).

Immunoblotting

Proteins (20-40 μg) were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with primary antibodies as detailed in Supplemental Experimental Procedures. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies. Proteins were visualized using a SuperSignal West Pico chemiluminescence kit (Pierce) and quantified with ImageJ (version 1.49o). Films were exposed for various times for protein quantification within a linear range of detection. For visualization and quantification with the Odyssey system, goat anti-mouse IRDye 800 or 680 and anti-rabbit IRDye 800 or 680 (LI-COR Biosciences) were used as secondary antibodies.

Microarray

Total RNA was isolated using Trizol (Invitrogen) from three independent samples of BE(2)-C_tetoff_KDM4C cells cultured in the presence or absence of doxycycline for 6 days. Affymetrix microarray was performed using the Human Gene 2.0 ST microarray chip. Data were normalized, significance determined by ANOVA, and fold change calculated with the Partek Genomics Suite. GO and PANTHER analyses were performed with DAVID (Huang et al., 2008) for all differentially expressed genes (≥ ±1.4 fold, P < 0.05), and GSEA was performed as described (Subramanian et al., 2005).

qRT-PCR

Total RNA was isolated from three independent samples using Trizol. Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen). qRT-PCR was performed using a RT2 SYBR green/Fluorescein PCR master mix (SABiosciences) on an iQ5 real-time PCR system (Bio-Rad) with primers against various genes (Table S3). All samples were normalized to β2 microglobulin (B2M) mRNA levels.

ChIP-qPCR

ChIP was performed as described (Ding et al., 2013). For qPCR, two independent ChIP samples were analyzed, and each sample was assayed in triplicate using primers that cover the promoter regions of ATF4, PHGDH, PSAT1, and HOXC9. Data were presented as percentage of the input chromatin (bound/input × 100). For ChIP against H3K9 methylation marks, data were normalized to histone H3 content obtained by anti-histone H3 ChIP. ChIP antibodies and primers are described in Supplemental Experimental Procedures.

Metabolite analysis

GC-MS metabolomic analysis was performed as described previously (Ding et al., 2013). BE(2)-C_tetoff_KDM4C cells were cultured in the presence or absence of doxycycline for 6 days and collected for metabolite extraction by 80% methanol at −20oC. Five biological replicate samples (~5 × 106 cells/sample) were analyzed for each condition.

Statistics

Quantitative data are presented as mean ± SD and were analyzed for statistical significance by unpaired, two-tailed Student's t-test or two-way ANOVA using GraphPad Prism 6.0h for Mac.

Supplementary Material

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ACKNOWLEDGMENTS

We thank Dr. Nabieh Ayoub of the Israel Institute of Technology for providing pEGFPKDM4C-wt and pEGFP-KDM4C-S198M, and Drs. LesleyAnn Hawthorn, Sam Chang, and Eiko Kitamura of the Georgia Regents University Cancer Center Genomics Core for assistance in microarray gene expression profiling. The work was supported by a grant from the National Basic Research Program of China (number 2012CB114603) to H.C., and grants from NIH (R01 CA190429) and DoD (W81XWH-12-1-0613) to H.-F.D. Y.Z. was supported in part by a grant from the National Natural Science Foundation of China (number 81201981).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

E.Z. and J.D. independently performed the experiments with assistance from Y.X., M.L. and B.Y. J-H.C., Y.Z. and H-F.D. analyzed the microarray and ChIP-seq data. C.Y., Z.D., S.H. and L.Y. provided reagents and helped designed the study. E.Z., J.D., H.C. and H-F.D. designed the study, analyzed the data, and wrote the paper.

ACCESSION NUMBER

The NCBI Gene Expression Omnibus (GEO) accession number for the microarray data reported in this paper is GSE65966.

SUPPLEMENTAL INFORMATION

Supplemental Information includes 7 figures, 3 tables, and Supplemental Experimental Procedures.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS747035-supplement-1.docx (69.5KB, docx)
2
NIHMS747035-supplement-2.pdf (10.1MB, pdf)
3
NIHMS747035-supplement-3.xlsx (100.7KB, xlsx)
4
NIHMS747035-supplement-4.xlsx (52.1KB, xlsx)

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