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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Jan 28;1853(5):881–891. doi: 10.1016/j.bbamcr.2015.01.011

Functional regulation of hypoxia inducible factor-1α by SET9 lysine methyltransferase

Qiong Liu 1, Hao Geng 1, Changhui Xue 1, Tomasz M Beer 1, David Z Qian 1,#
PMCID: PMC4380656  NIHMSID: NIHMS659673  PMID: 25637186

Abstract

HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells.

Keywords: protein interaction, metabolic adaptation, hypoxia, cancer, transcription factor, gene regulation

1. Introduction

The transcription factor hypoxia-inducible factor 1 (HIF-1) plays an essential role in cellular adaptation to hypoxic stress [1]. HIF-1 consists of an oxygen-sensitive α subunit (HIF-1α) and a constitutive β subunit (HIF-1β) [2, 3]. The activity of HIF-1 is implicated in cancer development and therapy resistance [4]. This is because HIF-1controls gene expression programs for cancer hallmarks, including metabolic adaptation, angiogenesis, metastasis and stem cells maintenance [5]. An increase of glucose metabolism represents a major event in metabolic adaptation to hypoxic stress [6]. Notably, HIF-1 controls genes increasing glycolysis, which cancer cells often employ to gain growth advantage over normal cells [7-9]. The activity of HIF-1 primarily depends on HIF-1α, which is stabilized by tumor hypoxia and oncogenic mutations [3]. In many human cancers, the expression level of HIF-1α is associated with poor prognosis and mortality [6]. Thus, HIF-1α is widely considered as an important therapeutic target to treat cancer [10].

In normoxia, the HIF-1α subunit is constantly synthesized, but rapidly degraded by the oxygen-dependent mechanism, mainly through the prolyl hydroxylase (PHD) and the von Hippel-Lindau (VHL) mediated ubiquitin proteasomal degradation [11, 12]. Several oxygen-independent mechanisms were also reported to regulate HIF-1α stability, suggesting HIF-1α stability is also regulated in hypoxia. For example, RACK1 competes with the chaperon HSP90 and promotes HIF-1α degradation [13, 14], HSP70 and the ubiquitin E3 ligase CHIP promotes ubiquitin proteasomal degradation of HIF-1α [15].

Transcription of genomic sequences is dependent on the accessibility of chromatin to transcription machinery including transcriptional factors and cofactors [16]. As transcriptional cofactors, several histone modifying enzymes have been reported to regulate HIF-1 transactivation. The histone acetyltransferase p300 and histone deacytylases (HDACs) regulate HIF-1 transcriptional activity by acetylation / deacetylation [17-22]. The histone methyltransferase G9a and histone demethylase jumonji domain containing protein 1A (JMJD1A) and JMJD2C regulate HIF-1 transactivation through methylation / demethylation of histone and non-histone proteins [23-25]. However, how HIF-1 transcriptional activity and target gene expression were selectively regulated by histone modifying enzymes is unclear.

Genome wide study indicated that HIF-1 preferentially binds to transcriptional active loci, which already presented under normal condition, as characterized by H3K4 methylation [26, 27]. SET9 is a lysine mono-methyltransferase for histone H3K4 and is thus involved in gene activation through inhibiting heterochromatin formation [28-30]. It is currently unclear whether SET9 contribute to HIF-1 target gene expressions in hypoxia.

Here we report that the histone methyltransferase SET9 enriches at HRE sites within glycolytic HIF-1 target gene promoters and stabilizes HIF-1α protein at these sites by inhibiting proteasomal degradation in hypoxia. Therefore SET9 facilitates HIF-1 to upregulate glycolytic genes and promotes the glycolytic adaptation of cancer cells to hypoxic stress. Our study demonstrates that SET9 is a potential molecular target to inhibit HIF-1α and HIF-1 mediated cancer development.

2. Materials and Methods

2.1. Cell Lines and Reagents

Hep3Bc1, a gift from Dr. Gregg Semenza at Johns Hopkins University, was stably transfected with HRE driven firefly lucifearse reporter (p2.1) and constitutive renilla luciferase reporter (pSV-renilla) [31]. Human embryonic kidney 293T (HEK293T), osteosarcoma U2OS, heptoma Hep3Bc1, and renal cell carcinoma RCC10 cells were cultured in DMEM with 10% FBS as described previously [32]. Human prostate cancer cell lines DU145 and C42B were cultured in RPMI medium as described [33]. The hypoxic condition was defined as 1% oxygen, 5% CO2 and 94% nitrogen. MG132 was purchased from Cayman Chemical (Ann Arbor, MI).

2.2 Plasmids and Transfection

Plasmids encoding 3×FLAG-HIF-1α, p2.1, and pSV-renilla were gifts from Dr. Gregg Semenza at Johns Hopkins University. Plasmids encoding HA-HIF-1α, HA-HIF-2α, CHIP, FLAG-SET9 and FLAG-SET9-H297A were from Addgene (Cambridge, MA). Plasmid encoding HA-SET9 was purchased from Sino Biological (Beijing, China). Plasmid transfection was performed using X-tremeGENE 9 transfection reagent from Roche (Indianapolis, IN) following the manufacturer’s instructions. Scramble control (siC) and SET9 siRNA (siSET9) constructs were purchased from Sigma (St. Louis, MO). Transient siRNA knockdown was performed using Dharmafect reagents from Thermo Scientific (Pittsburgh, PA) following the manufacturer’s instructions.

2.3. Stable Cell Lines

The pLKO.1-puro vector based lentiviruses containing HIF-1α, SET9 or non-targeting control shRNA were purchased from Sigma. Establishment of shC, shHIF-1α and shSET9 stable knockdown cell lines was described previously [32, 34]. Stable cell lines were selected and maintained in growth medium supplemented with puromycin.

2.4. Immunoprecipitation (IP) and Western Blotting

Cells were harvested using lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, pH 7.4) and debris were removed by centrifugation at 12000 rpm for 10 min. For HIF-1α IP, cell lysates were incubated with anti-HIF-1α antibody overnight at 4°C, and then incubated with protein A/G agarose (Santa Cruze Biotechnology, Dallas, TX) for 4 hours. The agarose was precipitated and washed with PBS for 3 times, and protein was recovered with 1×Laemmli sample buffer (Bio-Rad, Hercules, CA) at 95 °C for 10 min. For FLAG IP, cell lysates were incubated with anti-FLAG M2 affinity gel (Sigma) for 4 hours at 4°C, and then the gel was washed with PBS for 3 times and the protein was recovered as above. For western blotting, equal amounts of cell lysates (40–60 μg of protein) were resolved with SDS-PAGE, and transferred to membranes. The membrane was probed with primary antibodies, washed, and then incubated with corresponding fluorescent secondary antibodies and washed. The fluorescent signal was captured using LI-COR (Lincoln, NE) Odyssey Imaging System, and fluorescent intensity was quantified using the Odyssey software where indicated. Antibodies used were: HIF-1α (Santa Cruz), HIF-2α (Novus Biologicals, Littleton, CO), SET9 (Millipore, Billerica, MA), β-tubulin (Sigma), FLAG-M2 (Sigma), HA (Abcam, Cambridge, MA), and CHIP (Santa Cruz).

2.5. Quantitative RT-PCR

U2OS cells were transfected with scramble control (siC) or SET9 siRNA (siSET9) for 48 h, during which cells were exposed to 20% or 1% O2 for 24 h prior to harvest. Real-time PCR was performed as previously described [35]. Briefly, total RNA was extracted using TRIzol reagent, reverse transcribed to cDNA, and quantified by real-time PCR using sybrgreen universal master mix (all reagents were from Life Technologies, Grand Island, NY). 18S rRNA was used as internal reference and siC cells in 20% O2 were used as control. Results were calculated using the ΔΔCt method. ΔCt = Ct − Ct (18S rRNA), ΔΔCt = ΔCt − ΔCt (control), fold change to control = 2^(−ΔΔCt). RT-PCR primer pairs for HIF-1α target genes were described previously [32].

2.6. Reporter Gene Assays

Hep3Bc1 cells stably expressing p2.1 and pSV-renilla [31] were cultured in 24-well plates and transfected with siC or siSET9 siRNA for 48 h. HEK293T cells were transfected with p2.1, pSV-renilla, and siC or siSET9 siRNA for 48 h. Luciferase activity was measured using dual-luciferase assay system (Promega, Madison, WI). Firefly luciferase activity was normalized by renilla signals, and expressed as relative HRE activity compared to control cells (siC in 20% O2).

2.7. Chromatin Immunoprecipitation Assay

U2OS cells were exposed to 20% or 1% O2 for 24 h and fixed by 1% formaldehyde. ChIP assay was performed using ChIP-IT express kit from Active Motif (Carlsbad, CA) according to the manufacture’s protocol. The recovered DNA was quantified using Real-time PCR. Primer pairs amplifying the HRE regions of HIF-1α target genes were designed as described [27]. The enrichment fold was calculated based on ΔCt = Ct − Ct (input), ΔΔCt = ΔCt − ΔCt (IgG), fold change = 2^(−ΔΔCt).

2.8. Lactate Assay

U2OS cells were cultured in 24-well plates for 24 h, and then transfected with scramble control siRNA or SET9 siRNA for 24 h. Cells were switched to fresh growth medium and exposed to 20% or 1% O2 for an additional 24 h. Cell culture medium was used for lactate measurement with a lactate assay kit II (Sigma) following the manufacturer’s instructions. Lactate content was normalized by cell number and expressed as fold change over control (siC cells in 20% O2).

2.9. Cell Viability Assay

Cell viability was measured as previously described [33]. U2OS shC and shSET9 cells were seeded in 96-well plates and cultured for 24 h, which was defined as day 0 in hypoxia (1% O2). Cells were then allowed to growth in 20% or 1% O2 for additional 1 or 2 days. The results were expressed as relative viability over Day 0.

2.10. Data Analysis

Results are expressed as mean ± SD. The statistical difference between multiple groups was analyzed using one way ANOVA. Differences between two groups were analyzed by Student’s t test. Experiments were performed in triplicates and were performed at least three times.

3. Results

3.1. SET9 interacts with HIF-1α

To investigate the role of transcriptional co-factors in HIF-1 function, we initially tested whether histone methyltranferases interact with HIF-1α. We identified SET9 as a potential HIF-1α interacting protein. We co-overexpressed HA-SET9 with FLAG-HIF-1α in HEK293T cells and performed co-immunoprecipitation (co-IP) assay using anti-FLAG antibody. HA-SET9 was detected by western blots in the cell lysates immunoprecipitated with anti-FLAG antibody, suggesting that SET9 interacted with HIF-1α (Fig. 1A). Next, we co-overexpressed HA-HIF-1α and FLAG-SET9 in HEK293T cells and treated cells with or without hypoxia (1% O2) before co-IP. We found that HA-HIF-1α was present in cell lysates immunoprecipitated by anti-FLAG antibody, and the signal was higher in hypoxia compared to normoxia, in consistent with higher total HIF-1α levels in hypoxia (Fig. 1B). To confirm these results, U2OS cells were transfected with SET9 and treated with hypoxic mimetic CoCl2. Endogenous HIF-1α was immunoprecipitated using anti-HIF-1α antibody. Western blots showed that SET9 was able to interact with the endogenous HIF-1α (Fig. 1C). We also examined whether SET9 interacts with HIF-2α, the other major hypoxia inducible transcription factor. We co-overexpressed FLAG-SET9 and HA-HIF-2α in HEK293T cells and performed co-IP with anti-FLAG antibody. The results showed that HIF-2α was not co-immunoprecipitated with SET9. Longer exposure was unable to detect HA-HIF-2α band in the IP products either (Fig. 1D), suggesting that SET9 specifically interacts with HIF-1α but not HIF-2α.

Figure 1. SET9 interacts with HIF-1α.

Figure 1

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A Co-immunoprecipitation (Co-IP) of FLAG-HIF-1α and HA-SET9 using anti-FLAG antibody. HEK293T cells were transfected with FLAG-HIF-1α and HA-SET9 for 48 h. Whole cell lysates (WCL) were immunoprecipitated with anti-FLAG antibody or IgG. IP products were probed with anti-FLAG and anti-HA antibodies. B. Co-IP of FLAG-SET9 and HA-HIF-1α using anti-FLAG antibody. HEK293T cells were transfected with FLAG-SET9 and HA-HIF-1α for 48 h and exposed to 20% (N) or 1% O2 (H) for 4 h. WCL were subject to IP with anti-FLAG antibody or IgG, and probed with anti-FLAG or anti-HA antibodies. C. Co-IP of endogenous HIF-1α and FLAG-SET9 using anti-HIF-1α antibody. U2OS cells were transfected with Empty Vector (EV) or FLAG-SET9 for 48 h and treated with CoCl2 for 4 h. Cell lysates were subject to IP with anti-HIF-1α antibody, and then probed with anti-HIF-1α or anti-FLAG antibodies. D. Co-IP of FLAG-SET9 and HA-HIF-2α using FLAG antibody. HEK293T cells were transfected with FLAG-SET9 and HA-HIF-2α for 48 h. WCL were subject to IP with anti-FLAG antibody or IgG, and probed with anti-HA or anti-FLAG antibodies.

3.2. SET9 stabilizes HIF-1α protein in hypoxia

To determine whether SET9 affects HIF-1α protein levels, we overexpressed SET9 in U2OS cells and cultured cells in normoxia or hypoxia. We found that SET9 overexpression in normoxia had no effect on the HIF-1α protein level. The overexpressed Flag-HIF-1α was used as a positive control for western blot detection. (Fig. 2A left). On the other hand, SET9 overexpression in hypoxia significantly increased both the endogenous (Fig. 2A right) and the overexpressed HIF-1α proteins (Fig. 2B). In contrast, when we knocked down SET9 in U2OS and Hep3Bc1 cells using two different siRNA sequences targeting SET9 (Fig 2C and 2D), we found that both SET9 siRNA constructs decreased the endogenous HIF-1α levels in hypoxia, with the first construct (s1) showing higher knockdown efficiency of SET9 and correspondingly more obvious HIF-1α level decrease. Scramble control siRNA (SET9 siRNA -, or C) was used as negative control in all experiments. To further confirm the results, we knocked down SET9 using the first siRNA construct in additional human cell lines including HEK293T, DU145, C42B and U87. The results showed that knockdown of SET9 by siRNA in hypoxia decreased HIF-1α levels (Fig. 2E). This effect appears to be specific to HIF-1α because knockdown of SET9 did not decrease HIF-1β (Fig. 3A) or HIF-2α levels (Fig. 3B). Of note, U2OS cells showed very weak HIF-2α signal even in hypoxia, which is consistent with a previous report [36]. Taken together, these data suggest that SET9 positively regulates HIF-1α in hypoxia.

Figure 2. SET9 positively regulates HIF-1α in hypoxia.

Figure 2

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A. SET9 overexpression increased endogenous HIF-1α protein levels in hypoxia. Left. U2OS cells were transfected with HA-SET9 or FLAG-HIF-1α for 48 h. Right. U2OS cells were transfected with increasing amounts of HA-SET9 plasmid as indicated for 48 h, and exposed to 1% O2 for 4 h. Cell lysates were probed using anti-HIF-1α or anti-HA antibodies. Fluorescent signals of HIF-1α bands were quantified by the Odyssey software, adjusted by β-tubulin signals, and normalized to empty vector transfected cells in 1% O2. The values are shown below the HIF-1α blot. B. SET9 overexpression increased exogenous HIF-1α levels. HEK293T cells were transfected with FLAG-HIF-1α and HA-SET9 for 48 h and exposed to 1% O2 for 4 h. Cell lysates were probed using anti-FLAG or anti-HA antibodies. C and D. Knockdown of SET9 by different siRNA constructs decreased HIF-1α levels in U2OS (C) and Hep3Bc1 (D) cells. Cells were transfected with scramble control siRNA (−) or two constructs of SET9 siRNA (s1 and s2) for 48 h and exposed to 20% or 1% O2 for 4 h. NT, non-transfected. Cell lysates were probed using anti-HIF-1α or anti-SET9 antibodies. E. siRNA knockdown of SET9 decreased HIF-1α levels in multiple cell lines. HEK293T, DU145, C42B and U87 cells were transfected with scramble control siRNA (−) or SET9 siRNA (+) for 48 h and exposed to 20% or 1% O2 for 4 h. Cell lysates were probed using anti-HIF-1α or anti-SET9 antibodies. In all the experiments β-tubulin levels were used as loading controls. Results shown are representative of three independent experiments.

Figure 3. SET9 regulates HIF-1α protein degradation in hypoxia.

Figure 3

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A. SET9 siRNA did not affect HIF-1β levels in U2OS cells. B. SET9 siRNA did not affect HIF-2α levels in U87 cells. C. SET9 siRNA did not affect HIF-1α mRNA levels in U2OS cells. D. SET9 siRNA did not affect p70S6K or S6 phosphorylation in HIF-1α protein translation machinery. E. MG132 rescued SET9 siRNA induced HIF-1α protein level decrease. U2OS cells were treated with MG132 (20 μM) for 4 h. F. SET9 siRNA increased HIF-1α ubiquitination. MG132 (20 μM) and CoCl2 (150 μM) were incubated for 4 h. Cell lysates were immunoprecipitated with anti-HIF-1α antibody or IgG. In all the experiments cells were transfected with scramble control (−) or SET9 siRNA (+) for 48 h and exposed to 20% or 1% O2 for 4 h (24 h for mRNA levels). Cell lysates were probed using indicated antibodies. β-tubulin levels were used as loading controls. Results shown are representative of three independent experiments.

Next, we determined the mechanism by which SET9 increases HIF-1α in hypoxia. We found that SET9 siRNA in hypoxia did not affect HIF-1α mRNA transcription (Fig. 3C). In addition, SET9 siRNA did not affect the phosphorylation of p70S6K or S6 in hypoxia, which are involved in the translation of HIF-1α protein (Fig. 3D). In contrast, we found that the proteasome inhibitor MG132 could rescue SET9 siRNA induced HIF-1α protein decrease in hypoxia (Fig. 3E). Further, we found that SET9 siRNA increased the level of HIF-1α protein polyubiquitination in hypoxic condition (Fig. 3F). Therefore, these data suggest that SET9 protects HIF-1α protein from proteasomal degradation in hypoxia.

To further determine the effect of SET9 on HIF-1α protein stability in hypoxia, we measured the HIF-1α protein degradation kinetics by a cycloheximide-based assay as previously described [32]. We found that SET9 overexpression increased HIF-1α protein stability in hypoxic condition compared to the EV control. A representative western blot of HIF-1α protein is shown in Figure 4A. Densitometry of HIF-1α protein bands in western blots suggested that HIF-1α protein level decreased to basal level after 30 min of CHX treatment in EV transfected cells, while in SET9 overexpressing cells HIF-1α remained at higher level (p<0.05, Fig. 4B). These results suggested that SET9 stabilized HIF-1α protein in hypoxia.

Figure 4. SET9 protects HIF-1α from proteasome degradation in hypoxia.

Figure 4

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A. SET9 increased HIF-1α protein stability. U2OS cells were transfected with empty vector (EV) or SET9 expression plasmid, then treated with CoCl2 (150 μM, 4 h) and the protein synthesis inhibitor cycloheximide (CHX) for indicated time points. B. Fluorescent signals of HIF-1α bands from three independent experiments were quantified by the Odyssey software, adjusted by β-tubulin signals, and normalized to signals in normal condition. *p<0.05. C. SET9 inhibited the recruitment of E3 ligase CHIP to HIF-1α. HEK293T cells were transfected with FLAG-HIF-1α, HA-SET9 and CHIP plasmids as indicated for 48 h, and exposed to 1% O2 and MG132 (20 μM) for 4 h. Cell lysates were immunoprecipitated with anti-FLAG antibody or IgG. D. SET9 rescued CHIP mediated HIF-1α protein degradation. HEK293T cells were transfected with FLAG-HIF-1α, HA-SET9 and CHIP plasmids as indicated for 48 h, and exposed to 1% O2 for 4 h. E. Effect of SET9 methyltransferase-deficient mutant H297A on HIF-1α protein levels. U2OS cells were transfected with FLAG-HIF-1α and 0.5 or 1 μg FLAG tagged wild type SET9 (FLAG-SET9 wt) or SET9 H297A mutant (FLAG-SET9 mt) for 48 h. Cells were exposed to 1% O2 for 4 h prior to harvest. β-tubulin was used as loading controls in all experiments. Results shown are representative of three independent experiments.

The E3 ubiquitin ligase CHIP was reported to mediate ubiquitin-proteasomal degradation of HIF-1α protein in hypoxia by binding to HIF-1α [15]. Thus, we tested the hypothesis that SET9 may stabilize HIF-1α protein by preventing CHIP-mediated HIF-1α degradation. HEK293T cells were co-transfected with plasmids encoding FLAG-HIF-1α plus HA-SET9 and / or CHIP. The protease inhibitor MG132 was added to overcome the effect of SET9 and CHIP on in HIF-1α protein levels. Whole cell lysates were immunoprecipitated with anti-FLAG antibody (Fig. 4C). Both HA-SET9 and CHIP were co-immunoprecipitated with FLAG-HIF-1α. Notably, we found that HA-SET9 reduced the interaction between HIF-1α and CHIP. Next, we co-overexpressed SET9 and/or CHIP with HIF-1α and examined HIF-1α protein levels. The results showed that overexpression of SET9 increased HIF-1α level, while overexpression of CHIP decreased HIF-1α level. Importantly, co-overexpression of both SET9 and CHIP rescued CHIP mediated HIF-1α protein decrease (Fig. 4D). These observations are consistent with the hypothesis that SET9 stabilizes HIF-1α protein by attenuating CHIP-mediated degradation.

Finally, since SET9 is a methyltransferase, we also investigated whether the methyltransferase activity could contribute to the regulation HIF-1α stability. The SET9 H297A mutant is defective in methyltransferase activity [28]. We co-overexpressed HIF-1α with different doses of wild type and mutant SET9 in U2OS cells to examine its ability to stabilize HIF-1α protein. We found that both SET9 wild-type and methyltransferase defective mutant increased HIF-1α protein levels, suggesting that the methyltransferase activity is not essential for SET9 to stabilize HIF-1α.

3.3. SET9 knockdown decreases HIF-1 transactivation and selectively attenuates a subset of HIF-1 target gene expression in hypoxia

Next we examined whether SET9 could regulate HIF-1 transcriptional activity. We performed reporter gene assays in Hep3Bc1 cells, which had been stably co-transfected with a Firefly luciferase gene under the control of a HIF-1 responsive hypoxia response element (HRE) and with a constitutive Renilla luciferase gene [31]. HIF-1 transcriptional activity was determined by the ratio of Firefly/Renilla and normalized to scramble control siRNA (siC) transfected cells in normoxia. We found that HIF-1 transcriptional activity was significantly increased at 1% O2 compared to 20% O2 as expected, and SET9 siRNA significantly reduced the activity in 1% O2 compared to scramble control siRNA (p<0.05, Fig. 5A). To further confirm the results, HEK293T cells were co-transfected with HRE-driven Firefly and constitutive Renilla luciferase plasmids. The reporter activities were measured in normoxia and hypoxia. We found that hypoxia increased HIF-1 transcriptional activity, while SET9 siRNA significantly attenuated the increase (p<0.001, Fig. 5B), suggesting an inhibition of HIF-1.

Figure 5. SET9 knockdown decreases HIF-1 transactivation and a subset of HIF-1 target gene expressions.

Figure 5

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SET9 knockdown decreased hypoxia induced HIF-1 transcriptional activity in A. Hep3Bc1 and B. HEK293T cells. Relative HRE activity was calculated as fold change to controls (siC cells in 20% O2). C. SET9 siRNA decreased HIF-1 regulated glycolytic gene (LDHA, HK2 and PDK1) transcription but not VEGFa, JMJD1A or JMJD2C transcription in U2OS cells. HIF-1 target gene mRNA levels were expressed as fold change over control (siC cells in 20% O2). D. Effect of SET9 overexpression on glycolytic gene transcription in U2OS cells. *p<0.05, **p<0.01, ***p<0.001, ND, not significantly down-regulated.

Next, to determine whether SET9 regulates HIF-1 target gene expression, U2OS cells were transfected with siC or siSET9 siRNAs and exposed to 20% O2 or 1% O2 for 24 h. Real-time qRT-PCR analysis showed that hypoxia induced HIF-1 target gene expression as expected, while SET9 knockdown decreased the expression of a subset of HIF-1 target genes, including HK2, LDHA and PDK1, which are key regulators of the glycolysis pathway [37]. Interestingly several HIF-1α target genes remain unaffected by siSET9, such as VEGFa, JMJD1A and JMJMD2C (Fig. 5C). Next, we overexpressed SET9 and examined HIF-1α target gene levels. The results showed that SET9 overexpression increased LDHA, HK2 and PDK1 mRNA in hypoxia (Fig. 5D). The increase was not statistically different, which could be due to the already high endogenous SET9 level in U2OS cells.

To confirm the differential HIF-1 target gene regulation by SET9, we examined HIF-1 target gene levels in Hep3Bc1 shC and shHIF-1α cells after SET9 knockdown (Fig. 6). Similar to U2OS cells, SET9 siRNA decreased hypoxia induced glycolytic gene expression (HK2, LDHA and PDK1), but not VEGFa, JMJD1A and JMJD2C expression in Hep3Bc1 shC cells. This selective inhibition of HIF-1 target genes by SET9 siRNA was in sharp contrast to the uniform inhibition by HIF-1α-shRNA (shHIF-1α, Fig. 6). Also, SET9 siRNA could not further decrease the glycolytic HIF-1 target genes under the condition of HIF-1α shRNA (Fig. 6). These results suggested that the selective down-regulation of glycolytic genes by SET9 siRNA was HIF-1α dependent.

Figure 6. SET9 knockdown decreases glycolytic gene expressions in Hep3Bc1 cells.

Figure 6

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Hep3Bc1 shC and shHIF-1α cells were transfected with siC or siSET9 for 48 h and exposed to 20% or 1% O2 for 24 h prior to harvest. HIF-1α target gene mRNA levels were determined by RT-PCR. *p<0.05, **p<0.01, ND, not significantly down-regulated.

3.4. SET9 regulates HIF-1α enrichment at a subset of HIF-1 target gene promoters

Next, we sought to investigate the mechanism by which SET9 selectively regulates HIF-1 target gene expressions. Since SET9 was reported to be a histone methyltransferase and could activate gene expression through epigenetic mechanisms, we hypothesized that SET9 binds to chromatins of HREs within HIF-1α target genes that are affected by SET9 siRNA in figure 5 and 6. To test this hypothesis, we preformed chromatin co-immunoprecipitation (ChIP) assays in U2OS cells using anti-SET9 antibody and PCR primers amplifying HREs regions, which are proven HIF-1α binding sites [27] within the HIF-1 target genes in figure 5 and 6. The results showed that, in consistency with gene expression data, SET9 was highly enriched on HREs of HK2, LDHA and PDK1, but not on HREs of VEGFa, JMJD1A and JMJMD2C (Fig. 7A).

Figure 7. SET9 regulates HIF-1α enrichment on chromatins of glycolytic gene HREs.

Figure 7

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A. SET9 specifically enriched on HREs within glycolytic gene promoters. U2OS cells were exposed to 1% O2 for 24 h and chromatin immunoprecipitation (ChIP) was performed using anti-SET9 antibody and primers specific to the HREs within HIF-1α target genes. B. SET9 enhanced H3K4 mono-methylation levels in LDHA HRE region. U2OS cells were transfected with empty vector (EV) or SET9 and exposed to 1% O2 for 24 h. ChIP analysis was performed using H3K4me antibody and primers for LDHA and JMJD2C as in A. C. Knockdown of SET9 specifically reduced HIF-1α binding to HREs in glycolytic genes but not in VEGFa, JMJD1A or JMJD2C. ChIP was performed using anti-HIF-1α antibody and the same primers as in A. *p<0.05, **p<0.01, ND, not significantly down-regulated.

Since mono-methylation of histone H3K4 by SET9 was related to gene activation, we next examined whether SET9 could affect the levels of mono-methylated H3K4 (H3K4me) in chromatins of HREs. ChIP assay was performed in U2OS cells using anti-H3K4me antibody, and primers amplifying HRE regions. To exclude the interference of internal factors on H3K4 mono-methylation, we overexpressed SET9 and calculated the enrichment as fold over empty vector (EV) control. In the two groups of genes distinguished by high or low SET9 enrichment (Fig. 7A), we selected one gene from each group for testing. The results showed that SET9 overexpression increased H3K4me levels in HRE of LDHA, which has high SET9 enrichment, but not that of JMJD2C (Fig. 7B), which has low SET9 enrichment. These results suggest that SET9 selectively binds to HREs of glycolytic gene and enhanced the mono-methylation levels of H3K4 within these sites.

Next we examined whether SET9 could affect HIF-1α enrichment on chromatins of HRE. We performed ChIP assays using anti-HIF-1α antibody in U2OS cells stably transfected by shRNA against non-targeting control (shC) or SET9 (shSET9). The results showed that hypoxia induced enrichment of HIF-1α on HREs of all HIF-1α target genes as reported previously, while SET9 knockdown specifically decreased HIF-1α levels on HREs of HK2, LDHA and PDK1 but not VEGFa, JMJD1A and JMJMD2C (Fig. 7C). Taken together, the above results suggest that SET9 specifically enriches at HREs of selective HIF-1 target genes, thereby promoting transcriptionally active chromatin formation and increasing HIF-1α stability at these sites, together which lead to the upregulation of these genes in hypoxia.

3.5. SET9 regulates HIF-1 dependent glycolytic metabolism and cell viability

Because HK2, LDHA, and PDK1 are key components of the glycolysis pathway, we next determined whether SET9 could affect HIF-1 dependent hypoxic glycolysis in cancer cells by measuring extracellular lactate levels. Consistent with the expression of glycolytic genes, hypoxia induced significant increase of lactate levels in both Hep3Bc1 (Fig. 8A) and U2OS (Fig. 8B) cells, while SET9 siRNA significantly decreased lactate levels in both cell lines in hypoxia (1% O2). To further confirm that SET9 regulates lactate production through HIF-1α, we measured lactate levels in HIF-1α stable knockdown cells (Fig. 8C). The results showed that hypoxia increased lactate levels in U2OS shC cells but failed to induce lactate production in HIF-1α stable knockdown cells (shHIF-1α). SET9 siRNA decreased lactate levels in U2OS shC cells, but could not further decrease the lactate levels in U2OS shHIF-1α cells, suggesting that SET9 regulates extracellular lactate production and hypoxic glycolysis through HIF-1α.

Figure 8. SET9 knockdown decreases lactate production and cell viability in hypoxia.

Figure 8

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SET9 siRNA reduced hypoxia induced lactate production in A. Hep3Bc1 and B. U2OS cells. Lactate levels were expressed as fold change to controls (siC cells in 20% O2). C. SET9 siRNA failed to further reduce lactate levels in HIF-1α knockdown U2OS cells. Lactate levels were expressed as fold change to controls (shC-siC cells in 20% O2). D. SET9 siRNA inhibited cell viability under hypoxia. Relative cell viability was expressed as fold change to day 0 in hypoxia. *p<0.05, **p<0.01, ***p<0.001.

Transition to glycolytic phenotype plays an important role in metabolic adaptation to hypoxic stress. Given that HIF-1α is a key regulator for cancer cell adaptation and survival in hypoxia, we next examined whether SET9 knockdown could affect cell viability in hypoxia. U2OS cells with control shRNA (shC) or SET9 shRNA (shSET9) stable knockdown were cultured in normal oxygen for 24 h and then exposed to 20% or 1% O2 for additional 1 or 2 days (Fig. 8D). The results showed that cell viability was significantly decrease in 1% O2 compared with 20% O2. Stable knockdown of SET9 did not affect cell viability in 20% O2, but significantly decreased cell viability in 1% O2 compared to control shRNA knockdown after 1 or 2 days in hypoxia.

4. Discussion

In the present study, we demonstrate that SET9 promotes HIF-1α protein stability in hypoxia, and selectively regulates a subset of HIF-1 target gene expression which mediates hypoxic glycolysis.

The transcription factor HIF-1 plays an essential role in cellular adaptation to hypoxic stress and is widely considered as a therapeutic target to treat cancer. Our results showed that SET9 was able to interact with HIF-1α and regulate HIF-1α protein stability as well as downstream functions. In hypoxic conditions, HIF-1α is stabilized due to the lack of oxygen-dependent PHD/VHL -HIF-1α protein degradation pathway. Despite the increased stability, HIF-1α protein can still be negatively regulated in hypoxia. For example, HSP70 interacts with HIF-1α and recruits the E3 ubiquitin ligase CHIP to HIF-1α, therefore leading to proteasomal degradation of HIF-1α in hypoxia [15]. Our results showed that SET9 interacts with HIF-1α and stabilizes HIF-1α by inhibiting the E3 ubiquitin ligase CHIP recruitment, suggesting that SET9 competes with HSP70 or CHIP for binding to HIF-1α, therefore protects HIF-1α from exposing to the proteasomal degradation system. The detailed mechanisms are under investigation in our ongoing study. As a methyltransferase, it is also possible that SET9 directly methylates HIF-1α and makes it structurally unfavorable for binding of the components in protein degradation machinery. In addition, SET9 may methylate HSP70 or CHIP and interfere with HIF-1α protein stability. However, our data showed that loss of methyltranferase activity does not abrogate the ability of SET9 to stabilize HIF-1α. More detailed mechanistic investigations are needed to unveil how SET9 stabilizes HIF-1α in hypoxia.

In normal oxygen conditions, only low level of HIF-1α was observed due to oxygen-dependent degradation machinery [38]. Our results showed that SET9 knockdown could not affect HIF-1α levels in normoxia, and overexpression of SET9 failed to increase HIF-1α protein in normoxia. These findings suggest that oxygen-dependent mechanism overrides the role SET9 in HIF-1α protein stability in normal condition, and that SET9 regulates HIF-1α stability in hypoxia, which represents the physiological condition in which HIF-1α plays a functional role [10].

In consistency with the protein level down-regulation, SET9 knockdown decreased hypoxia induced HIF-1 target gene expressions, including HK2, LDHA and PDK1 which are core components of the glycolysis pathway. Interestingly, knockdown of SET9 does not affect several other HIF-1 target gene expressions such as VEGFa, JMJD1A and JMJD2C. This differential regulation of HIF-1 target genes was observed in two cell lines in this study and also in other studies [39-41], however the mechanism was unclear. Since SET9 mainly functions as a histone methyltransferase and activates gene expression through transcriptionally active chromatin structure formation, we speculated that SET9 regulates HIF-1 transactivation on the chromatin level.

Indeed, further study using chromatin immunoprecipitation showed that in consistency with gene expression data, SET9 specifically binds to the chromatin of HRE sites within glycolytic genes promoters, which are known HIF-1α binding sites. In addition, SET9 was able to increase H3K4me levels in glycolytic gene HREs. More importantly, knockdown of SET9 specifically decreased HIF-1α binding level at these glycolytic gene HRE sites (HK2, LDHA and PDK1), whereas SET9 knockdown does not affect HIF-1α levels on HRE sites without SET9 binding (VEGFa, JMJD1A and JDJD2C). These findings suggest that the regulation of HIF-1α by SET9 are likely to occur on chromatin level, and that SET9 serves as a cofactor to specifically regulate HIF-1α protein stability at promoter HREs, therefore promoting the glycolytic adaptation of cancer cells when oxygen is limited. Expansion of the current study to a larger gene profile would help to further address the mechanism. Additional cofactors including gene transcription activation and suppression complexes, such as p300/CBP [21, 42] are likely to be involved in directing SET9 to chromatins of specific glycolytic gene HREs. In addition to our findings, the histone demethylase JMJD2C was reported to be a coactivator for HIF-1α in breast cancer progression [24]. SET9 was reported to methylate estrogen receptor (ER) and facilitate the recruitment of ER to its target genes, thus serving as a coactivator for gene transcription [43]. Taken together, these studies reveal a novel mechanism that histone modifying enzymes coordinate epigenetic signals with transcription factors to achieve gene specific regulation in cancer cell function.

Targeting HIF-1α represents a promising strategy in treating cancer, and increasing number of HIF-1α inhibitors have been developed, although few of them progressed through preclinical stage [31, 44-46]. Due to the large numbers of genes regulated by HIF-1α, it is important to selectively regulate HIF-1α function to minimize side effects [47]. Our study establishes that the histone methyltransferase SET9 selectively regulates HIF-1 mediated glycolytic gene expressions in cancer cells, thus affecting glycolytic metabolism in cancer cells. Future xenograft assays are planned to further confirm our findings. Recently, several SET9 inhibitors with high potency and selectivity have been developed in high throughput screening assays [48]. However, the role of SET9 in disease pathways was poorly understood. Our study provides foundational mechanisms for utilization of SET9 inhibitors to treat cancer and will greatly facilitate the development of SET9 inhibitors for disease treatment.

Highlights.

  • SET9 interacts with HIF-1α protein.

  • SET9 stabilizes HIF-1α protein at glycolytic gene promoter HREs in hypoxia.

  • SET9 specifically regulates HIF-1 responsive hypoxic glycolysis and cell viability.

Acknowledgements

We thank Dr. Gregg Semenza at Johns Hopkins University for providing the Hep3Bc1 cell line, and Dr. Mushui Dai at OHSU for providing expertise in protein polyubiquitination analysis and for providing U2OS cell line. This work was supported by Public Health Service grants R01CA149253 from the National Cancer Institute, W81XWH-10-1-0142 from Department of Defense Prostate Cancer Research Program, and PNW Prostate Cancer SPORE Pilot Award, all to DZ Qian. This work was also supported by a postdoctoral training grant W81XWH-13-1-0314 from Department of Defense Prostate Cancer Research Program to Q Liu.

Abbreviations

HIF

hypoxia-inducible factor

HRE

hypoxia response element

PHD

prolyl hydroxylase

VHL

von Hippel-Lindau

WCL

whole cell lysate

HK2

hexokinase 2

LDHA

lactate dehydrogenase A

PDK1

pyruvate dehydrogenase kinase, isozyme 1

ENO1

enolase 1

JMJD1A

jumonji domain containing protein 1A

JMJD2C

jumonji domain containing protein 2C

CHIP

carboxyl terminus of Hsc70-interacting protein

Footnotes

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