Originally published as MBC in Press, 10.1091/mbc.E06-05-0419 on September 5, 2007
Vol. 18, Issue 11, 4528-4542, November 2007
The N-Terminal Transactivation Domain Confers Target Gene Specificity of Hypoxia-inducible Factors HIF-1
and HIF-2
Cheng-Jun Hu*,
,
Aneesa Sataur
,
Liyi Wang,
Hongqing Chen
, and
M. Celeste Simon*,
*Abramson Family Cancer Research Institute and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and Department of Craniofacial Biology, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045
Submitted May 15, 2006;
Revised August 14, 2007;
Accepted August 27, 2007
Monitoring Editor: William Tansey
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ABSTRACT
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The basic helix-loop-helix-Per-ARNT-Sim–proteins hypoxia-inducible factor (HIF)-1
and HIF-2 are the principal regulators of the hypoxic transcriptional response. Although highly related, they can activate distinct target genes. In this study, the protein domain and molecular mechanism important for HIF target gene specificity are determined. We demonstrate that although HIF-2 is unable to activate multiple endogenous HIF-1–specific target genes (e.g., glycolytic enzymes), HIF-2 still binds to their promoters in vivo and activates reporter genes derived from such targets. In addition, comparative analysis of the N-terminal DNA binding and dimerization domains of HIF-1 and HIF-2 does not reveal any significant differences between the two proteins. Importantly, replacement of the N-terminal transactivation domain (N-TAD) (but not the DNA binding domain, dimerization domain, or C-terminal transactivation domain [C-TAD]) of HIF-2 with the analogous region of HIF-1 is sufficient to convert HIF-2 into a protein with HIF-1 functional specificity. Nevertheless, both the N-TAD and C-TAD are important for optimal HIF transcriptional activity. Additional experiments indicate that the ETS transcription factor ELK is required for HIF-2 to activate specific target genes such as Cited-2, EPO, and PAI-1. These results demonstrate that the HIF- TADs, particularly the N-TADs, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.
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INTRODUCTION
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Low levels of O2, or hypoxia, are encountered by cells within rapidly growing tissues, such as developing embryos or solid tumors (Semenza, 2001
). In response to hypoxic stress, mammalian cells activate hypoxia-inducible transcription factors (HIFs) to enhance the transcription of genes involved in glycolysis, angiogenesis, and cell survival to maintain oxygen homeostasis (Semenza, 2001). Therefore, hypoxic responses are essential for embryonic development and tumor progression (Semenza, 2001; Giaccia et al., 2004). HIF consists of heterodimers of - and 
-subunits (also known as aryl hydrocarbon receptor nuclear translocator [ARNT]). Both subunits are basic-helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) domain containing proteins. The HLH and PAS domains are necessary for / subunit dimerization, whereas basic regions from both subunits mediate binding to the hypoxia response element (HRE) of HIF target genes (CACGTG) (Wang and Semenza, 1995; Kinoshita et al., 2004).
HIF-1/ARNT and HIF-2/ARNT dimers are the primary factors regulating hypoxic transcriptional responses in most mammalian cells. Initial studies suggest that they have similar functions, consistent with the fact that HIF-1 and HIF-2 proteins are closely related (Ema et al., 1997; Flamme et al., 1997; Hogenesch et al., 1997; Tian et al., 1997; Wiesener et al., 1998; O'Rourke et al., 1999) (Figure 1). For example, HIF-1 and HIF-2 exhibit similar functional domain structures, containing DNA binding and dimerization domains at their N termini, and transactivation domains at their C termini (Jiang et al., 1997; Pugh et al., 1997; O'Rourke et al., 1999). The C termini of HIF-1 and HIF-2 are also similarly subdivided into a N-terminal activation domain (N-TAD, overlapping with the oxygen-dependent degradation domain), an inhibitory domain, and a C-terminal transactivation domain (C-TAD) (Jiang et al., 1997; Pugh et al., 1997; O'Rourke et al., 1999). Most importantly, HIF-1 and HIF-2 exhibit significant homology in several regions: they share 83 and 70% sequence identities in their DNA binding and dimerization domains, respectively. Furthermore, amino acids surrounding the two oxygen-sensitive proline residues (30 amino acids [aa] each) are highly conserved between HIF-1 and HIF-2 (70% similarity). In addition, their C-TADs are also very similar (67% similarity). These homologies provide the molecular basis for several common properties of HIF-1 and HIF-2. For example, both use ARNT as a common binding partner, their protein stabilities are similarly regulated in an oxygen-dependent manner, and the transcriptional activity of both C-TADs is regulated by "factor inhibiting HIF" (FIH)-mediated hydroxylation of a conserved asparagine amino acid that blocks the recruitment of transcriptional coactivators p300 and CBP (Ema et al., 1997; Flamme et al., 1997; Hogenesch et al., 1997; Tian et al., 1997; Wiesener et al., 1998; O'Rourke et al., 1999; Mahon et al., 2001; Lando et al., 2002). Based on these properties, it has been suggested that HIF-1 and HIF-2 play related roles in hypoxic responses, and the unique phenotypes observed in Hif- mutant mice are due to their distinct expression patterns during development (Tian et al., 1998; Peng et al., 2000; Compernolle et al., 2002).
More recently, we and others have shown that HIF-1 and HIF-2 actually regulate both unique and common target genes in vivo and in multiple cell lines (Hu et al., 2003; Grabmaier et al., 2004; Warnecke et al., 2004; Rankin et al., 2005; Raval et al., 2005; Wang et al., 2005; Covello et al., 2006; Gruber et al., 2007). For example, HIF-1 specifically regulates glycolytic genes (including phosphoglycerate kinase [PGK] and lactate dehydrogenase A [LDHA]) (Hu et al., 2003; Rankin et al., 2005; Wang et al., 2005), as well as carbonic hydrase-9 (CA IX) (Grabmaier et al., 2004) and BNIP3 (Raval et al., 2005), whereas HIF-2 exclusively regulates the Pou transcription factor Oct-4, cyclin D1, and transforming growth factor (TGF-) (Raval et al., 2005; Covello et al., 2006). Other hypoxia-inducible genes, such as vascular endothelial growth factor (VEGF), facilitated glucose transporter-1 (GLUT-1), adipose differentiation-related protein (ADRP), adrenomedullin (ADM), and N-myc downstream regulated 1 (NDRG-1) are regulated by both HIF-1 and HIF-2 (Hu et al., 2003; Raval et al., 2005; Hu et al., 2006). These results are in agreement with functional studies indicating that HIF-1 and HIF-2 exhibit distinct roles during both development and tumor progression (Maranchie et al., 2002; Kondo et al., 2003; Scortegagna et al., 2003; Covello et al., 2005, 2006; Raval et al., 2005). By generating embryonic stem (ES) cells expressing HIF-2 at the Hif-1 locus, we directly compared HIF-1 and HIF-2 functions in embryos and tumors, and we showed that HIF-2 more potently promotes teratoma growth in nude mice (Covello et al., 2005, 2006).
HIF-1 and HIF-2 exhibit unique target genes; however, the molecular mechanism(s) providing target gene specificity remain unclear. Differences in their DNA binding and dimerization domains could be important for target gene specificity by binding to related but not identical cis-elements; alternatively, unique transcriptional activation and inhibitory domains may be responsible for different functions via interaction with distinct promoter-specific transcription factors. In this study, the relative contributions of DNA binding and transcriptional activation domains of HIF- to target gene specificity were investigated. Binding and activation of HIF-1 target genes by HIF-2 protein was compared with HIF-1 by using both plasmid-based reporters and endogenous genes. A number of deletion and domain swap mutants were constructed and their ability to activate reporter and endogenous target genes was analyzed. The results revealed how target gene specificity is achieved by the closely related HIF-1 and HIF-2 proteins.
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MATERIALS AND METHODS
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Cell Culture
Wild-type (WT), Hif-1–/–, Hif-2–/–, and Hif-1–/– mouse ES cells stably transfected with WT HIF-1 or HIF-2 cDNA have been described previously (Hu et al., 2006). Human embryonic kidney (HEK)293 Tet-on HIF-1DM (double proline mutation) or HIF-2DM cells have also been described previously (Hu et al., 2003). The WT8/HIF-1Flag or -HIF-2Flag cells were established by stable transfection of pcDNA3-HIF-1Flag or pcDNA3-HIF-2Flag plasmid into 786-O/WT8 renal clear cell carcinoma cells and hygromycin selection. BpRc-1 cells (ARNT-deficient) stably transfected with control or ARNT-expressing plasmid were described previously by Arsham et al., 2002. HEK293, Hep3B, and the cells described above were cultured in DMEM containing 4.5 g/ml glucose, 10% fetal calf serum (15% for ES cells), and 25 mM HEPES, under 1.5% O2 for hypoxia or 21% O2 for normoxia experiments.
Knockdown of HIF- and ELK-1 mRNAs by Using Small Interfering RNAs (siRNAs)
Control or siRNAs specific for human HIF-1, HIF-2, and ELK-1 mRNAs were synthesized by QIAGEN (Valencia, CA). Hep3B cells were transfected with siRNAs at 60% confluence using HiPerFect Transfection Reagent according to the manufacturer's protocol. 24 h posttransfection, cells were cultured at 21 or 1.5% O2 for 12 h, and they were collected to analyze HIF- or ELK-1 RNA, protein, or target genes.
Plasmid Construction
pcDNA3 vectors expressing full-length normoxia-stable mouse HIF-1 (P402A/P577A), or HIF-2 (P405A/P530A) proteins were described previously (Hu et al., 2003). To generate normoxia-active HIF- protein, N813 of HIF-1 or N851 of HIF-2 was mutated to alanine by amplifying the full-length plasmid by using mutation-incorporated primers and Pfu enzyme as described previously (Hu and Gupta, 2000). These constructs were called "HIF- triple mutants (TMs)." Full-length pcDNA3 HIF-TM DNA served as a template to generate several deletion mutants that lacked the N-TAD (
N-TAD), or inhibitory domain (IH), or C-TAD (C-TAD) for both HIF-1 and HIF-2. HIF-1/HIF-2 hybrid constructs "122," "211," "112," and "221" TMs were generated by ligation of polymerase chin reaction (PCR) DNA fragments from pcDNA3 HIF-1TM with PCR DNA fragments from pcDNA3 HIF-2TM. Full-length cDNA from pcDNA3 HIF-1TM and HIF-2TM were cloned into the pcDNA 3.1-Flag vector by using BamHI/BstXI and KpnI/BstXI sites, respectively, to produce HIF-1Flag and HIF-2Flag plasmids. HIF-1NFlag or HIF-2NFlag plasmids were generated by PCR-mediated deletion of amino acids 365–836 from full-length HIF-1Flag or 367–874 from full-length HIF-2Flag. Addition of the VP16 transactivation domain to HIF-1N or HIF-2N produced HIF-1N/VP16 or HIF-2N/VP16. All newly constructed plasmids were further analyzed by sequencing to confirm that they encoded the desired HIF- proteins.
RNA Preparation, Northern Blot Analysis, and Quantitative Reverse Transcription-PCR (Q-PCR)
RNA isolation and Northern blot analysis were performed using standard protocols. Human DNA fragments for Northern probes (PGK, LDHA, NDRG-1, ADRP, and -tubulin) have been described previously (Hu et al., 2003). Mixed primer/probes sets for human or mouse HIF-1, HIF-2, VEGF, NDRG-1, ADM, PGK, LDHA, GLUT-1, PAI-1, Cited-2, IGFBP-1, and 18S rRNA (endogenous control) were used to measure the levels of these transcripts by the 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.
Protein Analysis
Nuclear extracts (NEs) and cytoplasmic fractions were prepared in the presence of protease inhibitors as well as 200 µM deferoxamine (DFX) as described previously (Hu et al., 2003). Western blot analysis was performed using standard protocols with the following primary antibodies: anti-HIF-1 monoclonal antibodies (mAbs) (NB 100-105; Novus Biologicals, Littleton, CO, for mouse HIF-1 protein in Figures 3A and 10B), anti-HIF-1 mAb (610959; BD Biosciences Transduction Laboratories, Lexington, KY, for human HIF-1 protein in Figures 2B and 12A), anti-HIF-2 polyclonal antibodies (pAbs) (NB 100-122; Novus Biologicals), anti-ARNT mAb (NB 100-124; Novus Biologicals), anti-Flag M2 mAb (F-3165; Sigma-Aldrich, St. Louis, MO), anti-Myc mAb (clone 9E10; Roche Diagnostics, Indianapolis, IN), and anti-ELK mAb (H00002002-M01; Novus Biologicals).
Chromatin Immunoprecipitation (ChIP) Assays
ChIP assays were performed as described previously (Hu et al., 2006). Anti-HIF-1 mAb (NB 100-105; Novus Biologicals) was used for HIF-1 protein precipitation with mouse IgG2b immunoglobulin at the same concentration as a control. Anti-HIF-2 pAb (NB 100-122; Novus Biologicals) was used for HIF-2 precipitation, whereas rabbit preimmune serum served as a control. DNA from input (1:20 diluted) or immunoprecipitated samples was assayed using regular PCR in the presence of [-32P]dCTP. The PCR products were separated by acrylamide gel electrophoresis and detected by PhosphorImager analysis. Alternatively, DNA from input or immunoprecipitated samples was quantified by SYBR Green-based quantitative (Q)-PCR. All PCR products were compared with input amounts to normalize for variations in the input signal that could arise from variable chromatin preparation. The results were plotted as -fold changes relative to their individual control antibody in individual cells. Primer pairs were pretested to amplify target genomic DNA in a linear manner. The following forward (F) and reverse (R) primers were used to detect HRE-containing LDHA genomic DNA in Hot-PCR: LDHA F, 5'-TGGCCTTTCTTTGGGGTGTCGCAGC-3'; and LDHA R, 5'-GGGGCCCAACCGTACCGCTAGATGC-3'.
The following were the primers to detect HRE-containing PGK and LDHA genomic using SYBR-Green Q-PCR: PGK F, 5'-GGCATTCTGCACGCTTCAA-3'; PGK R, 5'-GAAGAGGAGAACAGCGCGG-3'; LDHA F, 5'-ATCGATGCATTTGGGCTC-3'; and LDHA R, 5'-CAACCCGACATGCTCCTCA-3'.
Transient Transfection
WT HRE-Luciferase reporter, as well as mutant HRE-Luciferase reporter, were described by Hu et al. (2003). The human PGK promoter reporter was constructed by inserting a 910-bp human PGK promoter fragment into the pGL3Basic vector at the BglII/HindIII sites. The control plasmid PGKdHRE contained a 50-bp deletion covering all three HIF binding sites in the PGK HRE. The human GLUT-1 reporter was similarly made, with a 700-bp HRE-containing enhancer and a 1.5-kb GLUT-1 promoter in the pGL3Basic vector. The control GLUT-1dHRE plasmid exhibited a 30-bp GLUT-1 HRE deletion. Transient transfection of cells in a 35-mm dish with HRE-dependent reporters (100 ng) and HIF- expression plasmids (200 ng) was performed with FuGENE 6 (for HEK293 and HEK293 Tet-on; Roche Diagnostics) or Lipofectamine Plus Reagent (for BpRc-1 and Hep3B cells; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. -Galactosidase (-Gal) activity was analyzed to monitor transfection efficiencies in reporter gene experiments.
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RESULTS
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PGK and LDHA Are Exclusively Regulated by HIF-1 in Hep3B Cells
Glycolytic pathway components such as PGK and LDHA are classic hypoxia inducible genes; however, none of the 13 glycolytic genes are induced by hypoxia in 786-O/WT8 renal carcinoma cells, a line that exclusively expresses HIF-2 (Hu et al., 2003). Induction of glycolytic genes is restored in 786-O/WT8 cells upon HIF-1 introduction, indicating that HIF-1, not HIF-2, activates glycolytic gene expression (Hu et al., 2003). Consistent with results from 786-O/WT8 cells, glycolytic genes are induced solely by HIF-1 in HEK293 Tet-on cells where addition of doxycycline induces normoxically stable HIF-1 or HIF-2 protein expression (Hu et al., 2003). Similarly, recombinant adenovirus-mediated expression of HIF-1, but not HIF-2, stimulates glycolytic gene expression in HEK293 cells (Wang et al., 2005). These results indicate that the glycolytic genes are HIF-1-specific targets.
However, it is still unclear whether glycolytic genes are only regulated by HIF-1 in a cell type that expresses functional HIF-1 and HIF-2 proteins endogenously. Hep3B cells were selected to address this question, because Hep3B cells have been shown to express both HIF-1 and HIF-2 proteins, and they exhibit high levels of hypoxic induction of HIF targets such as the glycolytic genes PGK and LDHA (Hu et al., 2003; Warnecke et al., 2004). Hep3B cells were transfected with siRNAs specific to HIF-1, HIF-2, or both HIF- subunit mRNAs. HIF- target gene induction was assessed using real-time Q-PCR on RNAs prepared from siRNA-targeted cells cultured at 21% O2 (N) or 1.5% O2 (H). HIF-1 siRNA reduced HIF-1 mRNA expression to 
10% of the levels in nontargeted cells (Figure 2A, left), whereas HIF-2 siRNA dramatically decreased HIF-2 mRNA expression to 15% of the levels in nontargeted cells (Figure 2A, right). Furthermore, combination of both siRNAs significantly diminished mRNA levels of both subunits (Figure 2A). Consistent with Q-PCR assays of HIF- mRNA, Western blot analysis indicated that HIF-1 or HIF-2 protein levels were greatly reduced in hypoxic Hep3B cells targeted with HIF-1 or HIF-2 siRNA, whereas cells targeted with both siRNAs exhibited a significant reduction (90%) of both HIF- proteins (Figure 2B). However, transfection of control siRNA had no effect on HIF- mRNA (Figure 2A, con) or protein levels (Figure 2B, con), and no cross-reactivity between HIF-1 and HIF-2 siRNAs was observed (Figure 2, A and B).
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Figure 2. HIF-1 and HIF-2 regulate different target genes in Hep3B cells. (A) Q-PCR analysis of HIF-1 (left) and HIF-2 (right) mRNA levels in Hep3B cells 40 h posttransfection of control (con) or HIF- siRNAs (1 for HIF-1 siRNA, 2 for HIF-2 siRNA, 1 + 2 for HIF-1, and HIF-2 siRNA combination) at 21% O2 (N) or 1.5% O2 (H). Data represent means of three independent experiments ± SD, setting HIF- mRNA levels in normoxic Hep3B cells at 100%. (B) Western blot analysis of HIF-1 and HIF-2 protein expression in Hep3B cells (Cell) or cells transfected with control (Con.), HIF-1 (1), HIF-2 (2), or both HIF-1 and HIF-2 (1 + 2) siRNAs at 21 or 1.5% O2 (H). ARNT served as a loading control. (C) Q-PCR analysis of HIF target gene expression in HIF- siRNA–transfected Hep3B cells at 21% O2 (N) or 1.5% O2 (H). Hypoxic induction of VEGF, NDRG-1 and ADM was primarily decreased by HIF-2 siRNA, whereas hypoxic induction of PGK-1, LDHA, and GLUT-1 was lost or greatly reduced in Hep3B cells targeted with HIF-1 siRNA, but not with HIF-2 siRNA.
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In accordance with our previous Northern blot analysis (Hu et al., 2003), Q-PCR assays indicated that several hypoxia-inducible genes exhibited a significant induction in hypoxic Hep3B cells, with NDRG-1 displaying the highest induction at 16-fold (Figure 2C). HIF-2 siRNA significantly reduced hypoxic induction of VEGF, NDRG-1, and ADM expression, whereas HIF-1 siRNA decreased their induction much less than that of HIF-2 siRNA (Figure 2C). Interestingly, the greatest reduction was observed in Hep3B cells targeted with both HIF-1 and HIF-2 siRNAs (Figure 2C). These results confirmed that VEGF, NDRG-1, and ADM were common target genes of HIF-1 and HIF-2, although they were preferentially regulated by HIF-2 in Hep3B cells. Several hypoxia-inducible genes involved in glucose metabolism (PGK, LDHA, and GLUT-1) were also analyzed. In contrast to VEGF, NDRG-1, and ADM, knockdown of HIF-1 seemed to be sufficient to ablate hypoxic induction of PGK, LDHA and GLUT-1 in Hep3B cells (Figure 2C). We concluded that in Hep3B cells exhibiting both HIF-1 and HIF-2 proteins, endogenous HIF-1 specifically regulates the glycolytic genes PGK and LDHA, whereas both HIF-1 and HIF-2 stimulate VEGF, NDRG-1, and ADM expression. However, HIF-2 seems to be the critical HIF- regulator of these genes in the Hep3B hepatocytes.
High Levels of HIF-2 Do Not Induce PGK and LDHA Expression in 786-O/WT8 and Mouse ES Cells
We showed previously that introduction of HIF-1 into 786-O/WT8 cells restores glycolytic gene induction (Hu et al., 2003). It is of interest to see whether overexpressed HIF-2 activates glycolytic gene expression. To test this, we generated 786-O/WT8 clones that expressed high levels of HIF-2 protein by stably transfecting them with WT HIF-2 cDNA (Figure 3A, right). Similarly, 786-O/WT8 cells expressing WT HIF-1 protein were created to serve as a control (Figure 3A, left). Both HIF-1 and HIF-2 cDNAs were Flag-tagged at their C termini, allowing detection of HIF- proteins by using anti-Flag antibody. As shown in Figure 3A, left, parental 786-O/WT8 cells lacked HIF-1 expression, but two independent 786-O/WT8/HIF-1Flag clones expressed HIF-1 protein under hypoxia as detected by anti-Flag and anti-HIF-1 antibodies. Although parental 786-O/WT8 cells exhibited endogenous HIF-2 protein expression under hypoxia (Figure 3A, right), HIF-2 protein levels were increased in 786-O/WT8/HIF-2Flag clones to a level that allowed detection of HIF-2 protein under normoxia (Figure 3A, right). Q-PCR analysis of HIF target genes was performed in parental 786-O/WT8 cells, and cells expressing high levels of either HIF-1 or HIF-2. Consistent with our previous results (Hu et al., 2003), parental 786-O/WT8 cells (expressing HIF-2, but not HIF-1) showed hypoxic induction of ADM and GLUT-1, but not PGK and LDHA. Of note, LDHA exhibited weak HIF-independent hypoxic induction (Figure 3B, WT8) as we showed previously in mouse ES cells (Hu et al., 2006). As expected, 786-O/WT8/HIF-1Flag cells revealed increased hypoxic induction of ADM and GLUT-1 genes, and restored hypoxic induction of PGK and LDHA genes (Figure 3B, HIF-1), as HIF-1 has been shown to regulate all four genes in multiple cell types (Hu et al., 2003; Hu et al., 2006). Interestingly, 786-O/WT8/HIF-2Flag cells exhibited higher levels of hypoxic expression of ADM and GLUT-1 genes than that of 786-O/WT8/HIF-1Flag cells. Higher levels of ADM and GLUT-1 expression in normoxic 786-O/WT8/HIF-2Flag cells likely reflected HIF-2 protein expression in these cells under normoxia (Figure 3B, HIF-2). In contrast to HIF-1, overexpressed HIF-2 protein did not activate PGK and LDHA gene expression in 786-O/WT8/HIF-2Flag cells (LDHA induction was changed from 1.3-fold in parental cells to 1.5-fold in HIF-2–overexpressed cells). These data indicated that overexpressed HIF-2 in 786-O/WT-8 cells has no significant effect on the expression of the HIF-1 unique genes PGK and LDHA, despite that HIF-2 regulates ADM and GLUT-1 efficiently.
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Figure 3. Overexpressed HIF-2 does not activate HIF-1 specific PGK and LDHA gene expression in 786-O/WT8 and mouse embryonic stem (ES) cells. (A) Generation of 786-O/WT8 clones expressing Flag-tagged WT HIF-1 (left) or HIF-2 (right). Anti-Flag or anti-HIF-1 antibodies detected HIF-1 protein in WT8/HIF-1Flag cells, not in parental 786-O/WT8 cells (left). Anti-HIF-2 antibodies detected increased levels of HIF-2 protein in WT8/HIF-2Flag cells in comparison with parental 786-O/WT8 cells (right). Overexpression of HIF-2 in 786-O/WT8/HIF-2Flag clones resulted in expression of HIF-2 protein under normoxia. Asterisk (*) indicates nonspecific bands in HIF-1 or HIF-2 western blots. (B) Q-PCR analysis of HIF- target genes in parental 786-O/WT8 cells (WT8), and 786-O/WT8 cells overexpressing HIF-1 (HIF-1) or HIF-2 protein (HIF-2) cultured under normoxia (N) or hypoxia (H). ADM and GLUT-1 genes were hypoxically induced in parental 786-O/WT8 cells, and they exhibited increased hypoxic induction in 786-O/WT8 cells transfected with either HIF-1 or HIF-2. PGK and LDHA were not hypoxically induced in parental 786-O/WT8 cells that express only HIF-2. Expression of HIF-1 restored hypoxic induction of both PGK and LDHA genes, whereas overexpressed HIF-2 was unable to induce PGK, but weakly induced LDHA expression. Data represent means of three independent experiments ± SD, setting target gene mRNA level in normoxic 786-O/WT8 cells at 100%. (C) Q-PCR analysis of HIF target gene in mouse WT, Hif-1–/– (1KO), and Hif-1–/– ES cells stably transfected with HIF-1 (HIF-1) or HIF-2 (HIF-2) cDNA cultured at 21% O2 (N) or 1.5% O2 (H). All four HIF target genes were hypoxically induced in WT, but not in Hif-1–/– ES cells (1KO). Stable transfection of HIF-1 cDNA into Hif-1–/– ES cells (HIF-1) restored HIF target gene induction. Overexpression of HIF-2 in Hif-1–/– ES cells (HIF-2) rendered HIF-2 functional in ES cells and induced ADM and GLUT-1 expression, but not PGK and LDHA gene expression in ES cells.
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The effect of overexpressed HIF-2 on glycolytic gene expression was further investigated in mouse ES cells (Figure 3C). ADM, GLUT-1, PGK, and LDHA were hypoxically induced in WT ES cells, but not in Hif-1–/– ES cells (Figure 3C). This is consistent with our previous findings that HIF-2 is not functional in mouse ES cells (Hu et al., 2006). As expected, stable reintroduction of WT HIF-1 into Hif-1–/– ES cells restored hypoxic induction of all four genes (Figure 3C, HIF-1). Overexpression of WT HIF-2 into Hif-1–/– ES cells by stable transfection restored hypoxic induction of ADM and GLUT-1 (Figure 3C, HIF-2), but not PGK and LDHA (LDHA induction was changed from 1.5-fold in parental cells to 1.8-fold in HIF-2–overexpressed cells) (Figure 3C, HIF-2). Thus, the HIF-1 target genes PGK and LDHA were not stimulated by overexpressed HIF-2 in both 786-O/WT8 and mouse ES cells, whereas HIF-2 activated the HIF-1/HIF-2 common target genes GLUT-1 and ADM. These data further confirmed target gene specificity between HIF-1 and HIF-2. In addition, endogenous target gene selectivity for PGK and LDHA is also essentially maintained with overexpressed HIF- protein.
HIF-2 Induces PGK Promoter Reporters as Efficiently as HIF-1
Having confirmed HIF target gene specificity in several cell lines, we wanted to use PGK (HIF-1 unique target) and GLUT-1 (HIF-1/HIF-2 common target) as models to investigate which domain of HIF- is important for target gene specificity. Although HIF-1 and HIF-2 proteins share highly conserved N-terminal DNA binding and dimerization domains, they exhibit differences in these regions that could be important for target gene specificity. We hypothesized that HIF-2 is unable to bind to the PGK promoter; therefore, it does not activate PGK expression. To test this hypothesis, we first assessed the ability of HIF-2 to activate a luciferase reporter gene under the control of a synthetic promoter containing three copies of the mouse PGK HRE (WT-HRE) (Arsham et al., 2002). Normoxic-stable HIF-2 stimulated the cotransfected WT-HRE reporter as efficiently as normoxic-stable HIF-1 in HEK293 cells under normoxia (Figure 4A, left). HIF- proteins activated reporter gene expression via HRE binding, because no reporter gene induction was observed for Mut-HRE where HIF binding sites (HBSs) in HREs are mutated (Figure 4A, left). To better control HIF protein levels in reporter gene assays, activation of the WT-HRE reporter was analyzed in HEK293 Tet-on HIF-DM (double proline mutation) cells in which physiological levels of HIF-1 and HIF-2 proteins were expressed upon doxycycline addition (Hu et al., 2003). Interestingly, HIF-1 and HIF-2 exhibited similar levels of WT-HRE reporter induction in HEK293 Tet-on HIF-DM cells (Figure 4A, right). These data are in agreement with previous reports that HIF-2 has similar transactivation capability to that of HIF-1 in PGK reporter gene assays (Wiesener et al., 1998), suggesting HIF-2 can bind to and activate the HRE isolated from PGK.
The WT-HRE reporter is an artificial plasmid constructed by linking three copies of 18 nt PGK HREs in tandem with the thymidine kinase (TK) promoter (Arsham et al., 2002). To test whether HIF-2 induces a more natural PGK promoter, we generated a PGK reporter derived from a human genomic DNA fragment containing the PGK promoter including its HREs (Figure 4B). As stated above, GLUT-1 represents a gene that is regulated by both HIF-1 and HIF-2. Its promoter and HRE-containing enhancer were isolated and used to generate an additional reporter construct (Figure 4B). For controls, HREs were deleted from PGK and GLUT-1 plasmids, resulting in the PGKdHRE-Luc and GLUT-1dHRE-Luc constructs shown (Figure 4B). As expected, both HIF-1 and HIF-2 stimulated the WT GLUT-1 reporter to similar levels in the transient transfection experiments, and no induction was observed for the GLUT-1dHRE construct (Figure 4C, right). Interestingly, HIF-2 protein clearly induced the WT PGK reporter, albeit less efficiently than the same amount of transfected HIF-1 plasmid (Figure 4C, left). The results suggest that reporter gene assays fail to detect different responses to HIF-1 and HIF-2 clearly displayed by endogenous gene assays (Figures 2C and 3, B and C). However, the reporter gene studies suggest that HIF-2 can bind to the PGK HRE in the context of an artificial or more natural episome.
HIF-2 Protein Binds to the HREs of the HIF-1 Unique Genes PGK and LDHA in Mouse ES Cells In Vivo, as Shown by ChIP
Whereas reporter gene studies indicated that HIF-2 has access to PGK HREs in a plasmid, it is still possible that HIF-2 is unable to bind to the chromosomal PGK promoter. ChIP experiments were therefore performed to directly investigate whether HIF-2 could bind to the PGK HRE in vivo. ES cells were selected due to the availability of Hif-1–/– and Hif-2–/– ES cells, which served as useful negative controls for ChIP. In addition, we reported previously that HIF-2 binds to its target genes GLUT-1 and VEGF in WT and Hif-1–/– ES cells (Hu et al., 2006). Anti-HIF-1 or HIF-2 antibodies were used to precipitate cross-linked HIF-1 or HIF-2 protein, and the amount of coprecipitated HRE-containing genomic DNA fragments from PGK and LDHA promoters was assessed by SYBR Green-based Q-PCR (Figure 5A), as well as regular PCR by using 32P-labeled dCTP (Figure 5B). Isotype-matched control antibodies generated similar background signals from WT, Hif-1–/–, Hif-2–/–, and Hif-1–/–/HIF-2Flag ES cells, whereas anti-HIF-1 antibody enriched the HRE-containing PGK and LDHA genomic DNA fragments only from WT and Hif-2–/– ES cells. This enrichment was not observed in HIF-1-deficient ES cells, including Hif-1–/– and Hif-1–/–/HIF-2Flag ES cells (Figure 5A), suggesting that HIF-1 antibodies specifically immunoprecipitated HIF-1–associated genomic DNA fragments, and more importantly that HIF-1 protein interacted with the HREs of PGK and LDHA in hypoxia-treated HIF-1–expressing cells. This was consistent with the fact that HIF-1 protein stimulated PGK and LDHA gene expression in WT and Hif-2–/– ES cells (Figure 3C). ChIP with anti-HIF-2 antibody coprecipitated HRE-containing genomic DNA fragments from PGK and LDHA in HIF-2 expressing hypoxia-treated WT, Hif-1–/–, and Hif-1–/–/HIF-2Flag cells, but not in Hif-2–/– ES cells (Figure 5A). Hif-1–/–/HIF-2Flag cells exhibited more HIF-2 binding to the HREs in comparison with that in Hif-1–/– ES cells, which was consistent with higher levels of HIF-2 protein expression in these cells (Figure 5A). In agreement with SYBR Green Q-PCR detection, primers covering the entire LDHA HRE also detected HIF-1 or HIF-2 binding to the LDHA promoter in cells with appropriate HIF- protein expression (Figure 5B). The ChIP results indicated that although HIF-2 does not activate the expression of the HIF-1 target genes PGK and LDHA, endogenous HIF-2, like HIF-1, binds to PGK and LDHA HREs. The ChIP data are also consistent with reporter gene studies showing that HIF-2 binds to the HREs and activates the PGK reporter. Moreover, these results clearly eliminated the possibility that failure of HIF-2 to induce PGK expression is due to the inability of HIF-2 to bind to endogenous PGK HREs.
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Figure 5. HIF-2 protein binds to the HREs of PGK and LDHA genes in mouse ES cells in vivo, as shown by ChIP. (A) Anti-HIF-1, anti-HIF-2, or control antibodies (mouse IgGb2 for anti-HIF-1 and rabbit preserum for anti-HIF-2) were used to precipitate HIF- protein in cross-linked hypoxic ES cells (WT, HIF-1KO, HIF-2KO, and HIF-1KO ES cells stably transfected with HIF-2 cDNA). Coprecipitated DNA fragments were detected using SYBR Green-based Q-PCR by primers specific for PGK and LDHA HREs. Results represent the average of three independent experiments, and they are plotted as -fold changes relative to their individual control antibodies. (B) ChIP assays detected by regular PCR labeled with [32P]dCTP by using primers spanning LDHA HRE to assess HIF binding. A representative assay from three independent experiments was shown. a-1, a-2, and C indicate anti-HIF-1, anti-HIF-2, or control antibodies for immunoprecipitation, respectively.
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N-Terminal bHLH and PAS Domains of HIF-1 and HIF-2 Exhibit Similar Functions
The ChIP and reporter assays described above demonstrated that HIF-2 binds the promoters of HIF-1 unique genes, suggesting that the N-terminal bHLH and PAS domains of HIF-1 and HIF-2 are functionally interchangeable. To directly compare the N termini of HIF-1 and HIF-2, we deleted the C-terminal transactivation domains from full-length (Figure 6A, HIF-1TM and HIF-2TM) to generate HIF-1N (aa1–aa364) and HIF-2N (aa 1–aa366) mutants of HIF- that contained intact DNA binding and dimerization (bHLH and PAS) domains, but lacked the transactivation domains. HIF-1N/VP16 and HIF-2N/VP16 proteins were also generated by the addition of a potent transactivation domain from VP16 at the C termini of HIF-N (Figure 6A), allowing us to investigate HIF-N–mediated transcriptional activation. The HIF-N and HIF-N/VP16 constructs were Flag-tagged at their C termini to facilitate their detection. We first tested the expression and subcellular localization of the proteins produced from these constructs in normoxic HEK293 cells. Anti-Flag Western blot analysis detected their expression in HEK293 cells transfected with the indicated plasmids (Figure 6B). Although HIF-2N and HIF-2N/VP16 had similar numbers of amino acids to their HIF-1 counterparts, HIF-2N and HIF-2N/VP16 migrated more slowly, likely due to different posttranslational modification between HIF-1 and HIF-2 proteins. All proteins were detected in nuclear fractions under normoxia (Figure 6B), consistent with these constructs containing a nuclear localization signal in their bHLH domains and lacking an oxygen-dependent degradation domain (Kallio et al., 1998). All constructs were then tested for transcriptional activity in transient transfection assays by using a WT-HRE reporter gene in BpRc-1 cells with or without ARNT function. As expected, both full-length normoxia-functional HIF- proteins (HIF-1TM and HIF-2TM) activated the reporter construct in an ARNT-dependent manner (Figure 6C, 1TM and 2TM), whereas HIF-1N and HIF-2N proteins exhibited no transcriptional activities (Figure 6C, 1N and 2N), likely due to the lack of a transcriptional activation domain. Importantly, HIF-1N/VP16 and HIF-2N/VP16 exhibited strong ARNT-dependent transcriptional activities (Figure 6C, 1N/VP and 2N/VP). These results indicated that HIF-N/VP16 and ARNT dimers, not HIF-/VP16 itself, activate WT-HRE reporter gene expression, providing strong evidence that HIF-N proteins dimerize with ARNT and bind to the HREs of HIF target genes.
HIF-1N and HIF-2N proteins lack transcriptional activation domains, but they are capable of interacting with ARNT and binding to HREs. If HIF-1N and HIF-2N are interchangeable, HIF-2N (like HIF-1N) should be able to inhibit full-length HIF-1 protein-mediated induction of HIF-1 unique genes (e.g., PGK), as well as HIF-1/HIF-2 common targets (e.g., GLUT-1) by occupying the HREs in a nonproductive way. As expected, full-length HIF-1 induced both PGK and GLUT-1 reporter genes in HEK293 cells, whereas HIF-1N or HIF-2N exhibited no effect on reporter gene activities (Figure 7A, top and middle). Introduction of increasing amounts of HIF-1N decreased full-length HIF-1–mediated activation of PGK and GLUT-1 reporters proportionally (Figure 7A, top and middle), demonstrating the feasibility of our experimental design. Interestingly, HIF-2N also decreased full-length HIF-1–mediated PGK and GLUT-1 reporter expression in a dose-dependent manner (Figure 7A, top and middle), suggesting that HIF-2N and HIF-1N performed similar functions in these reporter assays. Cotransfection of ARNT did not relieve HIF-N–mediated repressive effects on full-length HIF-1, suggesting that HIF-N repressed full-length HIF-1 through competitive binding to the HREs of reporter genes, but not via competitive interaction with ARNT protein (unpublished data). This was further confirmed by results demonstrating that HIF-1mbHLH and HIF-2mbHLH proteins fail to inhibit full-length HIF-1 function in identical reporter assays (Figure 7A, bottom), because these mutants maintain their ARNT binding capability, but they are unable to bind DNA (Hu et al., 2006). We concluded that HIF-2N behaved identically to HIF-1N in transient reporter gene assays.
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Figure 7. N-terminal DNA binding and dimerization domains of HIF-1 and HIF-2 exhibit similar functions. (A) HIF-2N, like HIF-1N serves as a dominant-negative regulator for full-length HIF-1 protein, and this inhibition depends on its HRE binding activity. HEK293 cells were transfected with reporter constructs along with full-length HIF-1TM, and different amounts of HIF-N (top and middle), or HIF-mbHLH plasmids (bottom). HIF-mbHLH plasmids had four basic amino acids in their DNA binding region mutated, which results in loss of HRE-binding activity, but still maintained their ARNT binding function. Dose-dependent inhibition of HIF-1TM activity was observed similarly for both HIF-1N and HIF-2N, but no inhibition was observed in cells cotransfected with HIF-1mbHLH or HIF-2mbHLH. (B) HIF-2N/VP16 stimulated PGK and GLUT-1 reporter genes as efficiently as HIF-1N/VP16 in transient transfection in HEK 293 cells. (C) Q-PCR analysis of endogenous HIF target gene expression in HEK293 cells transfected with the indicated plasmids. HIF-2N/VP16, like HIF-1N/VP16, stimulated HIF common target genes ADM and GLUT-1, and the HIF-1 target genes PGK and LDHA in HEK 293 cells although full-length HIF-2 did not induce PGK, or weakly induced LDHA expression.
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Next, HIF-1N/VP16 and HIF-2N/VP16 proteins were tested for their ability to stimulate HIF reporter genes (Figure 7B) as well as endogenous target genes in HEK293 cells (Figure 7C). As expected, both HIF-N/VP16 proteins exhibited similar activities in promoting GLUT-1 reporter expression in HEK293 cells (Figure 7B, right). Interestingly, HIF-1N/VP16 and HIF-2N/VP16 proteins also activated the PGK reporter gene to similar levels (Figure 7B, left), although weaker PGK reporter induction by full-length HIF-2 protein was observed (Figure 7B, left). Consistent with stable transfection in 786-O/WT8 and ES cells (Figure 3, B and C), full-length HIF-1 activated all four endogenous HIF target genes in HEK293 cells, whereas HIF-2 induced the expression of ADM and GLUT-1, but not PGK and LDHA (Figure 7C, 1TM and 2TM). However, HIF-2N/VP16 protein (like HIF-1N/VP16) activated the endogenous HIF-1/HIF-2 common target genes ADM and GLUT-1, as well as the HIF-1 unique target genes PGK and LDHA (Figure 7C, 1N/VP and 2N/VP). This was in direct contrast to our finding that full-length HIF-2 was unable to induce PGK and LDHA (Figure 6C, 2TM), suggesting that regions outside of the DNA binding and dimerization domains are responsible for the inability of HIF-2 to regulate PGK and LDHA. The identical repressive functions of HIF-1N and HIF-2N, and similar positive activities of HIF-1N/VP16 and HIF-2N/VP16, suggest that the N termini of HIF-1 and HIF-2 (bHLH and PAS domains) play no critical roles in HIF target gene specificity by using reporter gene and endogenous gene assays.
Both the N-TAD and C-TAD of HIF-1 and HIF-2 Are Required for Maximal Induction of Some HIF-1/HIF-2 Common Targets, whereas the N-TAD of HIF-1 or HIF-2 Plays a Dominant Role in Activating HIF-1– or HIF-2–specific Genes
The above-mentioned results indicate that differences in DNA binding and dimerization domains are not sufficient for HIF- target gene specificity, suggesting that other regions of the protein must be responsible, such as the more diverse transcriptional activation domains. The C-termini of HIF-1 and HIF-2 contain two TADs (N- and C-TADs) and an inhibitory region flanked by the two TADs (Figure 1) (Jiang et al., 1997; Pugh et al., 1997; Maemura et al., 1999; O'Rourke et al., 1999). The N-TAD of HIF-1 is located approximately from residues 360–600 (Jiang et al., 1997) (Pugh et al., 1997), whereas the HIF-2 N-TAD spans aa500 to aa582 (O'Rourke et al., 1999) or aa500 to aa639 (Maemura et al., 1999). The C-TADs located at the C termini of both proteins (aa786–aa826 for HIF-1 and aa828–aa870 for HIF-2) are 69% identical (Jiang et al., 1997; Pugh et al., 1997; Maemura et al., 1999; O'Rourke et al., 1999). Although HIF transcriptional activity depends on these domains, the relative contribution of the N-TAD and C-TAD to HIF function is unclear. To address this question, a number of HIF- deletion mutants (lacking the N-TAD, C-TAD, or inhibitory domain) were generated, and their activities in activating HRE-reporter genes and several endogenous HIF- target genes were analyzed. HIF-1 deletion mutants were derived from a full-length, normoxia-active HIF-1 construct (Figure 8A, left, HIF-1TM Myc tagged) by deleting aa365–aa587 (N-TAD-S, S for small), aa365–aa659 (N-TAD-L, L for large), aa660–aa782 (IH, IH for inhibitory domain), or aa783–aa836 (C-TAD). HIF-2 deletion mutants were generated by deleting aa366–aa541 (-N-TAD-S), aa366–aa618 (-N-TAD-L), aa619-aa820 (-IH), or aa821–aa874 (-C-TAD) from full-length, normoxia-functional HIF-2 (HIF-2TM) (Figure 8A, right). Two versions of N-TAD deletion mutants (small or large) were created to control for a possible inconsistency for HIF-2 N-TAD as reported previously (Maemura et al., 1999; O'Rourke et al., 1999). We initially checked the expression and cellular localization of these deletion constructs by transfecting them into HEK293 cells. All plasmids produced normoxia-stable proteins with the anticipated size, as detected by anti-Myc Western blot analysis (Figure 8B). Although the amount of transfected plasmids remained constant, IHs and C-TADs of HIF-1 and HIF-2 consistently produced more protein than other constructs (Figure 8B). Importantly, all HIF- deletion mutants translocated to the nucleus as they were readily detected in the nuclear fractions of transfected cells (Figure 8B).
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Figure 8. Characterization of HIF- deletion mutants. (A) Schematic of HIF-1 (left) and HIF-2 (right) deletion mutants. Full-length HIF-1 (HIF-1TM) and HIF-2 (HIF-2TM) proteins are normoxia-active due to th | |
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ABSTRACT
INTRODUCTION
