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. Author manuscript; available in PMC: 2012 Jun 10.
Published in final edited form as: Mol Cell. 2011 Jun 10;42(5):700–712. doi: 10.1016/j.molcel.2011.03.029

MCM Proteins Are Negative Regulators of Hypoxia-Inducible Factor 1

Maimon E Hubbi 1,2, Weibo Luo 2,3, Jin H Baek 2,3, Gregg L Semenza 2,3,4,*
PMCID: PMC3131976  NIHMSID: NIHMS296693  PMID: 21658608

SUMMARY

MCM proteins are components of a DNA helicase that plays an essential role in DNA replication and cell proliferation. However, MCM proteins are present in excess relative to origins of replication, suggesting they may serve other functions. Decreased proliferation is a fundamental physiological response to hypoxia in many cell types and hypoxia-inducible factor 1 (HIF-1) has been implicated in this process. Here, we demonstrate that multiple MCM proteins bind directly to the HIF-1α subunit and synergistically inhibit HIF-1 transcriptional activity via distinct O2-dependent mechanisms. MCM3 inhibits transactivation domain function whereas MCM7 enhances HIF-1α ubiquitination and proteasomal degradation. HIF-1 activity decreases when quiescent cells re-enter the cell cycle and this effect is MCM dependent. Exposure to hypoxia leads to MCM2–7 downregulation in diverse cell types. These studies reveal a function of MCM proteins apart from their DNA helicase activity and establish a direct link between HIF-1 and the cell cycle machinery.

INTRODUCTION

The MCM proteins 2–7 were first identified in Saccharomyces cerevisiae mutants defective in minichromosome maintenance (Tye, 1999; Forsburg et al., 2004). All six paralagous MCM proteins are ATPases that form a hexamer, which functions as a DNA helicase (Labib et al., 2000; Maiorano et al., 2006). The MCM complex is a critical component of the pre-replicative complexes (pre-RCs), which form during G1 phase, a process that is referred to as replication licensing (Blow and Hodgson, 2002; Sclafani and Holzen, 2007). At the G1-S transition, additional proteins are recruited to pre-RCs, leading to the initiation of DNA replication. Subsequent phosphorylation of the pre-RC during S phase by cyclin-dependent kinases (CDKs) leads to dissociation of the MCM complex (Maiorano et al., 2006), which ensures that each origin is fired only once during the cell cycle. The MCM proteins are present in vast (10–100 fold) excess compared to potential origins of replication and the majority of MCM proteins do not co-localize with sites of DNA synthesis in mammalian cells (Hyrien et al., 2003; Takahashi et al., 2005). These observations constitute the ‘MCM paradox’ and have led to speculation that MCM proteins may serve other functions (Forsburg et al., 2004).

Hypoxia-inducible factor 1 (HIF-1) mediates changes in gene expression that are essential for adaptive responses during hypoxia (Iyer et al., 1998; Yu et al., 1999). HIF-1 is a heterodimeric transcription factor consisting of HIF-1α and HIF-1β subunits (Wang et al., 1995). Under hypoxic conditions, ubiquitination and proteasomal degradation of HIF-1α are inhibited (Salceda and Caro, 1997). HIF-1α can then dimerize with HIF-1β via amino terminal basic-helix-loop-helix (bHLH) and PAS domains, bind to hypoxia-response elements (HREs) in target genes, recruit co-activators, and activate gene transcription (Arany et al., 1996; Jiang et al., 1996). Among the hundreds of genes regulated by HIF-1 are those encoding vascular endothelial growth factor (VEGF), which stimulates angiogenesis and O2 delivery (Forsythe et al., 1996), and the glucose transporter GLUT1, which increases flux through the glycolytic pathway under conditions of reduced O2 availability (Ebert et al., 1995; Iyer et al., 1998; Seagroves et al., 2001). The HIF-2α protein shares a high degree of sequence and functional similarity to HIF-1α, although with a more narrow tissue distribution and in some cases distinct physiological functions (Patel et al., 2008).

HIF-1 activity is modulated through O2-sensitive hydroxylation reactions. HIF-1α is hydroxylated at proline residues 402 and 564 (P402/564) located within the O2-dependent degradation domain (ODDD) (Epstein et al., 2001; Ivan et al., 2001; Jaakkola et al., 2001; Yu et al. 2001). Hydroxylation of these residues by prolyl hydroxylase 2 (PHD2) (Berra et al., 2003) is required for binding of the von Hippel Lindau protein (VHL) (Ivan et al., 2001; Jaakkola et al., 2001). VHL, together with the adaptor protein SSAT2 (Baek et al., 2007), recruits a ubiquitin ligase complex that includes Elongin C, Elongin B, RBX1, and Cullin 2, leading to HIF-1α ubiquitination and degradation (Maxwell et al., 1999; Kamura et al., 2000). By contrast, factor inhibiting HIF-1 (FIH-1) binds to the inhibitory domain and interferes with transactivation domain function (Mahon et al., 2001). FIH-1 hydroxylates asparagine-803 (N803) of HIF-1α, which abrogates binding of the p300 and CBP coactivators (Lando et al., 2002a, 2002b). Thus, HIF-1α protein stability and transactivation function are negatively regulated in oxygenated cells by prolyl and asparaginyl hydroxylation, respectively.

A critical adaptive response mediated by HIF-1α is to induce cell cycle arrest in response to hypoxia. In a variety of cell types, increased HIF-1α levels are associated with diminished cell proliferation and HIF-1α loss of function leads to increased cell proliferation (Goda et al., 2003; Koshiji et al., 2004; Gordan et al., 2007; Biswas et al., 2010). Overexpression of HIF-1α, even under non-hypoxic conditions, is sufficient to induce cell cycle arrest (Hackenbeck et al., 2009). However, because HIF-1α is present at varying levels in different cell types at physiological O2 concentrations (Zhong et al., 1999; Talks et al., 2000), it seems likely that mechanisms must also exist to overcome the inhibitory effects of HIF-1α on cell cycle progression in vivo.

In the present study, we demonstrate that MCM proteins directly interact with HIF-1α and negatively regulate HIF-1 activity. MCM7 enhances prolyl hydroxylation-dependent HIF-1α ubiquitination and degradation, whereas MCM3 inhibits HIF-1α transactivation domain function in an asparaginyl hydroxylation-dependent manner. Thus, the MCM proteins negatively regulate HIF-1 by synergistic and O2-dependent mechanisms. We demonstrate that HIF-1 activity decreases when quiescent cells are stimulated to proliferate and that this effect is MCM7-dependent. Our data indicate that MCM proteins modulate HIF-1 activity in proliferating cells, thus establishing direct crosstalk between the oxygen sensing and cell cycle pathways.

RESULTS

HIF-1α Directly Interacts with MCM3 and MCM7

We used a yeast two-hybrid screening strategy to identify proteins that interact with HIF-1α (Baek et al., 2007). The bait used was a fusion protein containing the DNA-binding domain of the yeast transcription factor Gal4 fused to amino acid residues 17–299 of human HIF-1α, which encompass the bHLH and PAS domains (Figure 1A). A total of 23 interacting prey fusion proteins were identified, consisting of the Gal4 transactivation domain and protein sequences encoded by a human thymus cDNA library. One of the prey proteins identified was MCM7. To determine whether MCM7 and HIF-1α interact in human cells, V5-epitope-tagged MCM7 was co-immunoprecipitated with FLAG-tagged full-length HIF-1α (Figure 1B) or FLAG-HIF-1α(1–329) (Figure 1C) in human embryonic kidney 293T cells. Similarly, MCM7 co-immunoprecipitated with HIF-2α (Figure 1D). To verify that the interaction of MCM7 with HIF-1α was direct and to exclude the possibility of multiple binding sites, we performed glutathione-S-transferase (GST) pulldown assays with a panel of bacterially-expressed and purified GST-HIF-1α fusion proteins, which were incubated with in vitro-transcribed and –translated MCM7. MCM7 bound specifically to GST-HIF-1α(1–329) with strongest binding to the PAS-B domain (residues 201–329) (Figure 1E). A deletion mutant of HIF-1α missing both PAS subdomains was unable to interact with MCM7 (Figure 1F), verifying the importance of this region for the interaction of HIF-1α with MCM7.

Figure 1. HIF-1α Interacts Directly with MCM7 and MCM3.

Figure 1

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(A) The location of the basic helix-loop-helix domain (bHLH), Per-Arnt-Sim homology domain (PAS), O2-dependent degradation domain (ODDD), inhibitory domain (ID), and carboxy-terminal transactivation domain (TAD-C) of HIF-1α are shown along with the sites of prolyl (P) and asparaginyl (N) hydroxylation. The bait vector used for yeast two-hybrid screening encoded amino acid residues 17–299 of human HIF-1α.

(B–C) Co-immunoprecipitation (co-IP) assay of 293T cells co-transfected with expression vectors encoding either FLAG-tagged full-length HIF-1α (B) or FLAG-HIF-1α(1–329) (C) and either V5-MCM7 or empty vector. IP was performed with V5 antibody and Western blot (WB) was performed with FLAG or V5 antibody. An aliquot of cell lysate that was reserved prior to IP was also analyzed (Input).

(D) Co-IP assay of lysates prepared from 293T cells that were co-transfected with HIF-2α vector and either V5-MCM7 or empty vector.

(E) GST pulldown assays of in vitro transcribed and translated (IVTT) 35S-labeled MCM7 using GST only (lane 1) or GST fusion protein containing the indicated amino acid residues from HIF-1α. MCM7 was detected by autoradiography.

(F) Co-IP assay of lysates of 293T cells co-transfected with vector encoding full-length FLAG-HIF-1α, a FLAG-HIF-1α(29–389) (ΔPAS) deletion mutant, and either EV or V5-MCM7.

(G) Proteomics screening assay to identify proteins that interact with HIF-1α(531–826).

(H–I) Co-IP assay of lysates from 293T cells co-transfected with FLAG-HIF-1α vector (H) or HIF-2α vector (I) and either V5-MCM3 or empty vector.

(J) Summary of results from GST pulldown assays of IVTT 35S-labeled MCM3 and indicated GST-HIF-1α fusion proteins.

(K) Co-IP assay of 293T cells exposed to 1% O2 for 6 hr and immunoprecipitated with either IgG or HIF-1α antibody. Immunoprecipitates were analyzed by WB with MCM3, MCM7, Cdt1, or HIF-1α antibody as indicated.

In a separate screen for HIF-1α-interacting proteins, we performed a quantitative proteomic screen using stable isotope labeling by amino acids in cell culture (SILAC) (Figure 1G) (Luo et al., 2010). Whole cell lysates were prepared from 293 cells grown in normal (“light”) media with 12C/14N-containing lysine/arginine or “heavy” media supplemented with 13C/15N-labeled lysine/arginine. The lysate from cells grown in light media was incubated with GST, whereas lysate from cells grown in heavy media was incubated with a GST fusion protein containing HIF-1α residues 531–826 immobilized on glutathione-Sepharose beads. Proteins that bound to GST-HIF-1α(531–826), but not to GST, were identified using mass spectroscopy. The screen identified several known HIF-1α-interacting proteins, including VHL, FIH-1, and p300, which validated our approach. One of the interacting proteins identified was MCM3. We verified that V5-MCM3 co-immunoprecipitated with FLAG-HIF-1α (Figure 1H) and with HIF-2α (Figure 1I). Using in vitro GST pulldown assays, we confirmed that this was a direct interaction and localized the binding of MCM3 to the inhibitory domain (Jiang et al., 1997), which comprises residues 575–785 of HIF-1α (Figure 1J and S1A). Thus, despite a high degree of sequence similarity between the two proteins, MCM3 and MCM7 interact with distinct domains of HIF-1α.

We examined binding of HIF-1α to the MCM proteins at their endogenous levels and asked whether HIF-1α might bind to other components of the pre-RC. Endogenous HIF-1α induced by hypoxic exposure co-immunoprecipitated with endogenous MCM3 and MCM7, but not with Cdt1, which functions as another essential component of pre-RCs (Figure 1K). We detected binding of MCM3 and MCM7 to HIF-1α in the soluble fraction of cells (Figure S1B), but did not detect binding of HIF-1α to MCMs in the chromatin fraction. These data indicate that at endogenous levels HIF-1α binds directly to select MCM proteins but not to assembled pre-RCs.

MCM3 and MCM7 Inhibit HIF-1 and HIF-2 Activity

We examined the effect of MCM3 and MCM7 on the transcriptional activity of a HIF-dependent reporter gene. 293 cells were transfected with p2.1, a reporter plasmid that contains a 68-bp HRE from the human ENO1 gene upstream of an SV40 promoter and firefly luciferase-coding sequences (Semenza et al. 1996), and pSV-RL, a control reporter that contains Renilla luciferase coding sequences downstream of the SV40 promoter only. The ratio of firefly to Renilla luciferase is a specific measure of HIF-1 transcriptional activity. MCM7 decreased HIF-1 reporter activity induced by overexpression of FLAG-HIF-1α under non-hypoxic conditions or by exposure of 293 cells to hypoxia (Figure 2A). For loss-of-function studies, we used a short hairpin RNA (shRNA) targeting MCM7. MCM7 knockdown led to a significant increase in HIF activity induced by hypoxia in 293 cells (Figure 2B) and HCT116 cells (Figure 2C), and by FLAG-HIF-1α overexpression in 293 cells (Figure 2D). To confirm that the effect was not due to an off-target effect, we constructed two additional MCM7 shRNAs (Figure 2E). Both shRNAs increased HIF reporter activity in 293 cells, with the magnitude of the effect correlating with the extent of MCM7 knockdown (Figure 2F).

Figure 2. MCM7 and MCM3 Inhibit HIF-1 and HIF-2 Activity.

Figure 2

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(A) Analysis of luciferase activity in 293 cells that were subjected to FLAG-HIF-1α overexpression (at 20% O2) or hypoxia (1% O2). The cells were co-transfected with HIF-1-dependent firefly luciferase reporter gene p2.1; constitutively expressed Renilla luciferase reporter pSV-RL; and either MCM7 expression vector or empty vector (EV). The ratio of firefly:Renilla luciferase activity was determined 24 hr after transfection.

(B–C) HIF reporter assay in 293 (B) or HCT116 (C) cells co-transfected with p2.1, pSV-RL, and vector encoding shRNA against MCM7 (shMCM7) or empty vector (shEV).

(D) HIF reporter assay in 293 cells co-transfected with FLAG-HIF-1α vector and increasing amounts of shMCM7 vector.

(E) Analysis of endogenous MCM7 and β-actin protein levels in 293T cells transfected with EV or vector encoding shRNA targeting MCM7 (MCM7-1 or MCM7-2).

(F) HIF reporter assay in 293 cells co-transfected with shMCM7-1 or shMCM7-2 vector.

(G) HIF reporter assay in 293 cells co-transfected with MCM3 vector or EV and either FLAG-HIF-1α vector at 20% O2 or EV at 1% O2.

(H) HIF reporter assay in 293 cells co-transfected with FLAG-HIF-1α vector and either shEV or vector encoding shRNA against MCM3 (shMCM3-1 or shMCM3-2).

(I–J) HIF reporter assay in 293 (I) or HCT116 (J) cells co-transfected with shEV, shMCM3-1, or shMCM3-2 and cultured at 20% or 1% O2.

(K) Quantitative real-time RT-PCR assay of MCM3, GLUT1, VEGF, and HIF-1α mRNA performed using total RNA isolated from 293T cells transfected with shEV or shMCM3-2 and cultured under non-hypoxic (N) or hypoxic (H) conditions. Results are shown as mean ± SEM.

(L) HIF reporter assay in HCT116 cells co-transfected with vectors encoding HIF-2α and the indicated shRNA against MCM3 or MCM7.

In all panels, mean ± SD are shown except where otherwise indicated. * p<0.01, # p<.05 for indicated comparisons.

Similarly, overexpression of MCM3 led to a decrease in HIF activity induced by exposure to hypoxia or by FLAG-HIF-1α overexpression (Figure 2G). We constructed two MCM3 shRNAs (Figure S2A). MCM3 knockdown increased HIF transcriptional activity induced by FLAG-HIF-1α overexpression (Figure 2H) or exposure to hypoxia in 293 cells (Figure 2I) or HCT116 cells (Figure 2J), but MCM3 knockdown had no effect under non-hypoxic conditions when the HIF-1α protein is not stabilized (Figure 2J). MCM3 knockdown led to increased expression of two HIF target genes, VEGF and GLUT1, under hypoxic conditions but again had no effect under non-hypoxic conditions (Figure 2K). Finally, knockdown of either MCM3 or MCM7 led to a significant increase in HIF-2α-dependent transcriptional activity (Figure 2L), demonstrating that HIF-1α and HIF-2α are both regulated by the MCM proteins.

Importantly, we confirmed that neither overexpression of MCM3 or MCM7 (Figure S2B) nor knockdown of MCM3 or MCM7 (Figure S2C) led to any significant change in the cell cycle profile of the cells. This result excludes the possibility that the effects observed were an indirect result of changes in the cell cycle. These results are consistent with previous studies demonstrating that the MCM proteins are present in vast excess to the amounts required for normal DNA replication.

MCM7 Enhances HIF-1α and HIF-2α Degradation

During the course of our studies, it became apparent that MCM7 overexpression decreased HIF-1α protein levels. Hypoxic cells transfected with MCM7 had reduced levels of endogenous HIF-1α and HIF-2α levels compared to cells transfected with empty vector (Figure 3A). Conversely, knockdown of MCM7 in either 293T cells (Figure 3B) or HCT116 cells (Figure 3C) led to an increase in endogenous HIF-1α and HIF-2α protein levels. Similarly, MCM7 knockdown with either of two shRNAs led to an increase in FLAG-HIF-1α protein stability, with the magnitude of the effect correlating with the extent of MCM7 knockdown (Figure 3D). Importantly, MCM7 had no effect on the stability of a HIF-1α deletion mutant lacking the PAS domain (Figure 3E) and therefore incapable of interacting with it (Figure 1F), demonstrating that this effect is dependent on a direct protein-protein interaction.

Figure 3. MCM7 Downregulates HIF-1α and HIF-2α Protein Levels.

Figure 3

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(A) Analysis of endogenous HIF-1α and HIF-2α levels in 293T cells transfected with EV (−) or vector encoding V5-MCM7 (+) and exposed to 1% O2 for 6 hr.

(B–C) Analysis of HIF-1α and HIF-2α levels in 293T cells (B) or HCT116 (C) cells transfected with either shEV or shMCM7 vector and exposed to either 20% or 1% O2 for 6 hr.

(D) Analysis of FLAG-HIF-1α and endogenous MCM7 levels in 293T cells co-transfected with shMCM7-1 or shMCM7-2 vector.

(E) Effect of MCM7 overexpression on levels of full-length FLAG-HIF-1α or a FLAG-HIF-1α deletion mutant (ΔPAS) that is incapable of interacting with MCM7.

(F–G) Effect of MCM7 overexpression on FLAG-HIF-1α (F) or HIF-2α (G) protein levels in 293T cells treated with the proteasome inhibitor MG132 (20 μM) for 6 hr.

(H) Effect of MCM7 overexpression on wild-type FLAG-HIF-1α or a double mutant containing proline-to-alanine substitutions at residues 402 and 564 (P/A).

(I) HIF reporter assay in 293 cells co-transfected with EV or MCM7 vector and vector encoding the indicated HIF-1α deletion construct. Mean ± SD are shown. * p<0.01 for indicated comparisons.

(J–K) GST pulldown assays using IVTT 35S-labeled MCM7 and GST alone or GST fused to Elongin C (J) or VHL (K).

(L) Co-IP of endogenous MCM7 with VHL antibody in 293T cells.

(M) HCT116 cells were transfected with FLAG-VHL vector and EV or V5-MCM7 vector. Cell lysates were immunoprecipitated with FLAG antibody, and immunoprecipitates were subjected to WB with Elongin C or FLAG antibody.

(N) HCT116 cells were transfected with FLAG-HIF-1α and EV or V5-MCM7 vector, then treated with 20 μM MG132 for 6 hr. Cell lysates were immunoprecipitated with HIF-1α antibody, and immunoprecipitates were analyzed by WB with Elongin C (Elgn C) or HIF-1α antibody.

(O) Ubiquitination assay of FLAG-HIF-1α. Lysates from 293T cells transfected with FLAG-HIF-1α vector were mixed with lysates from cells co-transfected with His-ubiquitin vector and myc-MCM7 vector or EV and incubated at 37°C. IP was performed with FLAG antibody and WB with His or FLAG antibody.

(P) MCM7 interacts with HIF-1α, VHL, and Elongin C, strengthening the interaction between these proteins, and thereby enhancing O2-dependent HIF-1α ubiquitination and proteasomal degradation.

The effect of MCM7 on HIF-1α (Figure 3F) and HIF-2α (Figure 3G) levels was abolished by treatment with the proteasome inhibitor MG132. HIF-1α proteasomal degradation occurs through two main pathways, a hydroxylation-dependent pathway mediated through the VHL ubiquitin ligase and hydroxylation-independent pathways mediated by RACK1, HAF, or CHIP/HSP70 (Liu et al., 2007; Koh et al., 2008; Luo et al., 2010). The effect of MCM7 on HIF-1α was abolished by mutation of P402/564, implicating the hydroxylation-dependent pathway (Figure 3H). Consistent with this hypothesis, MCM7 had no effect in the p2.1 reporter assay on the transcriptional activity of a deletion mutant of HIF-1α that lacked the ODDD due to deletion of residues 330–575 (Figure 3I). Collectively, the data indicate that MCM7 enhances proteasomal degradation of HIF-1α through the prolyl hydroxylation-dependent pathway.

We examined in vitro binding of MCM7 to other proteins implicated in this pathway, including PHD2, SSAT2, VHL, and Elongin C. MCM7 bound directly to bacterially expressed and purified GST-Elongin C (Figure 3J) and GST-VHL (Figure 3K). MCM7 co-immunoprecipitated with overexpressed VHL (Figure S3A) and endogenous VHL co-immunoprecipitated with endogenous MCM7 (Figure 3L). In contrast, binding of MCM7 to PHD2 was not detected and MCM7 had no effect on HIF-1α hydroxylation (Figure S3B).

In co-immunoprecipitation (co-IP) assays, MCM7 overexpression led to an increase in the VHL-Elongin C interaction (Figure 3M) and the HIF-1α-Elongin C interaction (Figure 3N), suggesting that MCM7 stabilizes ubiquitin ligase complex formation. Consistent with this hypothesis, we detected increased HIF-1α ubiquitination upon MCM7 overexpression (Figure 3O). MCM7 did not increase ubiquitination of a FLAG-HIF-1αΔPAS deletion mutant missing the MCM7-interacting domain (Figure S3C), consistent with the lack of effect of MCM7 on the stability of FLAG-HIF-1αΔPAS (Figure 3E). Thus, MCM7 enhances ubiquitination and proteasomal degradation of prolyl-hydroxylated HIF-1α by strengthening the interaction of HIF-1α with components of the VHL-Elongin C ubiquitin ligase complex (Figure 3P).

MCM3 Inhibits HIF-1α Transactivation Domain Function

MCM3 functions as a subunit of the DNA helicase complex along with MCM proteins 2 and 4–7. Phosphorylation of MCM3 by CDK1 at serine-112 regulates MCM complex formation (Lin et al., 2008). In order to test whether the ability of MCM3 to inhibit HIF-1 depended on incorporation into the MCM complex, we generated a mutant form of MCM3 that cannot be phosphorylated at serine-112. Co-IP experiments confirmed that the MCM3(S112A) mutant has greatly reduced binding to endogenous MCM7 (Figure 4A). However, the MCM3(S112A) mutant inhibited HIF-1 transcriptional activity induced by either FLAG-HIF-1α overexpression or hypoxia as effectively as wild-type MCM3 (Figure 4B). These results demonstrate that MCM complex formation is not required for regulation of HIF-1α by MCM3.

Figure 4. MCM3 Inhibits HIF-1α Transactivation Domain Function.

Figure 4

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(A) Co-IP assay of lysates from 293T cells transfected with vector encoding V5-MCM3 that was wild type or contained an S112A mutation. Lysates were analyzed directly (Input) or subjected to IP with V5 antibody and analyzed by WB with MCM7 antibody.

(B) HIF reporter assay in 293 cells co-transfected with EV or vector encoding MCM3 that was wild type (WT) or mutant (S112A); and FLAG-HIF-1α vector (and incubated at 20% O2) or EV (and incubated at 1% O2).

(C) Analysis of FLAG-HIF-1α levels in 293T cells co-transfected with vector encoding V5-MCM3 or EV.

(D) HIF-1α transactivation domain assay in 293 cells co-transfected with the indicated amount (in ng) of vector encoding MCM3 or FIH-1 and exposed to 1% O2 for 24 hr.

(E) HIF-1α transactivation domain assay in 293 cells co-transfected with vector encoding either MCM7 or FIH-1 and exposed to 1% O2 for 24 hr.

(F) HIF-1α transactivation domain assay in 293 cells co-transfected with pG5E1B-Luc reporter plasmid, Gal4-HIF-1α(531–826) vector, and either shEV, shMCM3-1, or shMCM3-2 vector.

(G–H) HIF-dependent reporter assay (G) or transactivation domain assay (H) following 24-hr exposure to 1% O2, 100 μM desferrioxamine (DFX), and overexpression of MCM3 as indicated.

(I) Co-IP assay of MCM3:HIF-1α interaction in cells co-transfected with vector encoding double-mutant (DM) FLAG-HIF-1αP402/564A and V5-MCM3 vector or EV. Cells were exposed to vehicle or DFX for 24 hr.

(J) HIF-dependent reporter assay in cells co-transfected with FLAG-HIF-1α double-mutant (DM) or triple-mutant (P402A/P564A/N803A; TM); and either MCM3 vector or EV.

(K) HIF-1α transactivation domain assay in 293 cells co-transfected with vectors encoding Gal4-HIF-1α(531–826)(N803A) and MCM3 or empty vector.

(L) Co-IP assay of MCM3:HIF-1α interaction in 293T cells co-transfected with vector encoding FLAG-HIF-1α that was either wild type (WT) or mutant (N803A) and vector encoding V5-MCM3 or EV.

In all panels, mean ± SD are shown. * p<0.01, # p<.05 for indicated comparisons.

Unlike MCM7, MCM3 overexpression had no effect on HIF-1α protein stability (Figure 4C). To examine whether MCM3 has any effect on HIF-1α-dependent transactivation, 293 cells were co-transfected with pG5-E1b-Luc, which contains five Gal4 binding sites upstream of the E1b gene promoter and firefly luciferase coding sequences, and an expression vector encoding the Gal4 DNA-binding domain either alone or fused to HIF-1α(531–826), which encompasses the HIF-1α inhibitory and transactivation domains (Jiang et al. 1997; Pugh et al., 1997). In this assay, MCM3 showed a dose-dependent effect on HIF-1α–dependent transactivation and was as potent as FIH-1 in inhibiting transactivation (Figure 4D) whereas MCM7 overexpression had no effect on transactivation domain function (Figure 4E). Conversely, MCM3 knockdown led to an increase in HIF-1α transactivation domain function (Figure 4F). These results establish MCM3 as an inhibitor of HIF-1α transactivation domain function.

HIF-1α transactivation domain function is inhibited by hydroxylation of residue N803. To ascertain whether hydroxylation of HIF-1α is necessary for the effect of MCM3, we utilized the hydroxylase inhibitor desferrioxamine (DFX). The addition of DFX blocked the effect of MCM3 on HIF-1 transcriptional activity, as measured by the p2.1 reporter assay (Figure 4G), and on HIF-1α transactivation domain function (Figure 4H). However, DFX had no effect on MCM3 levels or the MCM3-HIF-1α interaction (Figure 4I), demonstrating that binding of MCM3 is not hydroxylation-dependent. MCM3 inhibited transcriptional activity of double-mutant HIF-1α (DM), which contains P402A and P564A missense mutations, but not triple-mutant HIF-1α (TM), which also contains an N803A mutation (Figure 4J). The N803A mutation abrogated the effect of MCM3 on HIF-1α transactivation domain function (Figure 4K), but did not affect the binding of MCM3 to HIF-1α (Figure 4L). The results establish that MCM3 is an inhibitor of HIF-1α transactivation that functions in a hydroxylation-dependent manner.

Regulation of HIF-1 Activity By Other MCM Proteins

We examined whether other MCM proteins regulate HIF-1. In co-IP assays MCM2 and MCM5 were also found to interact with HIF-1α, whereas no interaction with MCM4 or MCM6 was detected (Figure 5A). Overexpression of MCM2 and MCM5, but not MCM4 or MCM6, inhibited HIF transcriptional activity induced by hypoxia (Figure 5B) or HIF-2α overexpression (Figure 5C). MCM2 or MCM5 knockdown increased transcriptional activity driven by HIF-1α or HIF-2α, whereas MCM6 knockdown had no effect (Figure 5D). Finally, MCM2 and MCM5 knockdown increased HIF activity induced by exposure to hypoxia (Figure 5E). The data demonstrate that, in addition to MCM3 and MCM7, MCM2 and MCM5 are also negative regulators of HIF activity. The lack of any effect of altering MCM6 levels provides further evidence that individual MCM proteins interact with and negatively regulate HIF-1α independent of helicase complex formation. Under the conditions that these experiments were performed, neither overexpression (Figure S2B) nor knockdown (Figure S2C) of any MCM protein had any significant effect on the cell cycle profile.

Figure 5. MCM2, MCM3, MCM5, and MCM7 Regulate HIF Activity.

Figure 5

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(A) HCT116 cells were co-transfected with vector encoding HIF-1α and the indicated V5-tagged MCM protein. Cells were lysed, lysates immunoprecipitated with V5 antibody, and probed with HIF-1α or V5 antibody.

(B–C) HIF reporter assay in HCT116 cells co-transfected with p2.1 reporter and vector encoding the indicated MCM protein and exposed to 1% O2 (B) or co-transfected with vector encoding HIF-2α (C).

(D) HIF reporter assay in HCT116 cells co-transfected with indicated shRNA vectors and either HIF-1α or HIF-2α vector.

(E) HIF reporter assay in HCT116 cells co-transfected with the indicated shRNA vector and exposed to 1% O2.

In all panels, mean ± SD are shown. *p<0.01 for indicated comparisons.

Quiescence Leads to MCM2–7 Downregulation and HIF Upregulation

The phenomenon of contact inhibition (CI) was described in NIH-3T3 mouse fibroblasts over 40 years ago (Holley et al., 1968) and subsequent work established that coordinated downregulation of the MCM proteins is a key step in this process (Stoeber et al., 2001). If the MCM proteins inhibit HIF activity and CI downregulates MCMs, then we would expect increased HIF activity associated with CI. Contact-inhibited cells expressed lower levels of MCM7 (and MCM2) protein and higher levels of HIF-1α protein (Figure 6A) and HIF-1α transactivation domain function (Figure 6B). We observed a large increase in HIF-1α and HIF-2α-dependent reporter activity in contact-inhibited NIH-3T3 cells compared to non-contact inhibited cells (Figure 6C) and increased expression of all five HIF-1 target genes (BNIP3, GLUT1, LDH-A, PDK1, and VEGF) that were analyzed (Figure 6D).

Figure 6. Quiescence Leads to an MCM-dependent Increase in HIF activity.

Figure 6

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(A) Analysis of HIF-1α and MCM levels in confluent NIH-3T3 cells that were contact inhibited (CI) or sub-confluent cells that were non-contact inhibited (NCI) and exposed to 1% O2 for 6 hr.

(B) HIF-1α transactivation domain function in CI or NCI NIH-3T3 cells that were transfected with vector encoding Gal4 (−) or Gal4-HIF-1α(531–826) (+).

(C) HIF reporter assay in CI or NCI NIH-3T3 cells transfected with either HIF-1α or HIF-2α vector.

(D) Quantitative real-time RT-PCR assay was performed using total RNA isolated from NIH-3T3 cells that were CI or NCI and exposed to 1% O2 for 24 hr.

(E) HIF reporter assay in HCT116 cells that were arrested by serum starvation for 48 hr and then stimulated to divide by the addition of serum for the indicated period of time. Cells were co-transfected with vectors encoding FLAG-HIF-1α and either V5-MCM7, shMCM7, or EV. Data are compared to the EV at the same time point.

(F) HIF reporter assay in HCT116 cells that were co-transfected with FLAG-HIF-1α and indicated MCM overexpression or knockdown vectors and either serum-starved or stimulated by the addition of serum for 24 hr. Data are compared to EV at the same time point.

(H) Analysis of hypoxia-induced cell cycle arrest in HCT116 cells with either shEV or shRNA against MCM7. Cells were exposed to 1% or 20% O2 for 24 hr, after which the difference in cell cycle distribution between the 1% and 20% O2 conditions, representing hypoxia-induced cell cycle arrest, was calculated for the shEV and shMCM7 cells. Results are shown as mean ± SEM.

In all panels, mean ± SD are shown except where otherwise indicated. *p<0.01 for indicated comparisons.

HCT116 cells do not show contact inhibition, but proliferation of these cells is arrested in response to serum starvation. HCT116 cells were serum starved for 48 hr after which they were stimulated to proliferate by the addition of serum. A progressive decline in HIF-1 reporter activity was observed starting within 12 hr of serum stimulation and knockdown of MCM7 was sufficient to block this effect (Figure 6E). Knockdown of MCM2 or MCM5 also blunted the decline in HIF-1 reporter activity (Figure 6F). These results demonstrate that cell proliferation leads to a decline in HIF-1 activity in an MCM-dependent manner.

If the MCM proteins function to promote proliferation by inhibiting HIF-1, knockdown of the MCM proteins should lead to greater hypoxia-induced cell cycle arrest. In HCT116 cells, hypoxia led to arrest of the cells in the G1 phase of the cell cycle (Figure 6G), consistent with previous studies (Koshiji et al., 2004, Gordan et al., 2010). Cells expressing shRNA against MCM7 displayed a more pronounced response to hypoxia (Figure 6G), confirming a role for the MCM proteins in modulating proliferation through effects on HIF-1.

Hypoxia Leads to MCM2–7 Downregulation

Previous work suggested regulation of MCM expression by oxygen levels. Hypoxic exposure led to decreased MCM2–7 expression in endothelial cells (Manalo et al., 2005) and HCT116 cells (Pires et al., 2010). Consistent with previous results, we observed decreased MCM2–7 mRNA levels upon hypoxic exposure in HCT116 cells (Figure 7A). To assess whether HIFs may mediate this process, we examined MCM proteins levels in NIH-3T3 cells exposed to 1% O2 or a hydroxylase inhibitor (DFX or dimethyloxalylglycine). Exposure to hypoxia led to coordinated downregulation of the MCM proteins, which was mimicked by chemical induction of HIF-1 (Figure 7B). Similarly, MCM3 mRNA levels were analyzed in response to hypoxia or the chemical inducers of HIF-1, DFX and cobalt chloride, in a variety of cell types. In all cell types examined, MCM3 levels were reduced upon HIF-1 induction (Figure 7C). To demonstrate more directly that HIFs are responsible for downregulation of the MCM proteins, the effect of hypoxia was compared in MDA-MB-231 cells expressing either shEV or shRNAs against both HIF-1α and HIF-2α. MCM3 and MCM7 were downregulated in MDA-MB-231 cells upon exposure to hypoxia, but not when HIF-1α and HIF-2α were knocked down (Figure 7D). These data demonstrate that MCM2–7 downregulation is a general feature of the hypoxic response mediated by the HIF transcription factors.

Figure 7. Hypoxia Downregulates MCM Expression.

Figure 7

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(A) Analysis of MCM2–7 mRNA levels in HCT116 cells exposed to non-hypoxic (N) or hypoxic (H) conditions for 24 hr.

(B) Analysis of MCM protein levels in NIH-3T3 cells exposed to 20% O2 (N), 1% O2 (H), DFX (100 μM), or DMOG (1 mM) for 24 hr.

(C) MCM3 mRNA levels were determined by quantitative real-time RT-PCR in the indicated cell type exposed to 20% or 1% O2, DFX (100 μM), or cobalt chloride (100 μM) for 24 hr.

(D) MCM3 and MCM7 mRNA levels in MDA-MB-231 cells stably transfected with shEV or shRNAs against HIF-1α and HIF-2α and exposed to 20% or 1% O2 for 24 hr.

In all panels, mean ± SD are shown. * p<0.01, # p<0.05 for indicated comparisons.

(E) MCM7 and MCM3 bind to HIF-1α and inhibit protein stability and transactivation domain function, respectively. Downregulation of the MCM proteins in response to quiescence and/or hypoxia leads to an increase in both HIF-1α protein stability and transactivation domain function. The mutual antagonism of MCMs and HIFs represents a homeostatic mechanism linking O2 sensing and cell cycle control.

DISCUSSION

In this study, we have demonstrated that the MCM proteins are negative regulators of HIF-1 transcriptional activity. MCM2, MCM3, MCM5, and MCM7 all interact with HIF-1α and inhibit HIF-1 activity. MCM7 binds to the PAS domain of HIF-1α as well as to VHL and Elongin C. By stabilizing the interaction between these proteins, it enhances ubiquitination and proteasomal degradation of HIF-1α. In contrast, MCM3 inhibits HIF-1α transactivation domain function. MCM3 may promote HIF-1α asparaginyl hydroxylation, inhibit binding of coactivators p300 or CBP to hydroxylated HIF-1α, or inhibit coactivator function. Furthermore, MCM proteins also bind to HIF-2α and regulate its activity.

The MCM proteins inhibit HIF-1 activity in an O2- and hydroxylation-dependent manner: prolyl hydroxylation was necessary for the effect of MCM7 on HIF-1α stability and asparaginyl hydroxylation was necessary for the effect of MCM3 on HIF-1α transactivation. What is the physiological relevance of these effects? HIF-1α induction is required for G1-phase cell cycle arrest following hypoxic exposure in multiple cell types (Goda et al., 2003; Koshiji et al., 2004; Gordan et al., 2007; Hackenbeck et al., 2009). This is an adaptive response when there is an imbalance between O2 supply and demand, which would only be exacerbated by cell proliferation. However, the proliferation of some cell types may be desirable in the presence of physiological levels of O2 and negative regulation of HIF-1α by MCMs provides a mechanism by which this can occur. Consistent with this hypothesis, we have shown that proliferating fibroblasts have lower HIF-1α protein levels, HIF-1 transcriptional activity, and HIF-1 target gene expression compared to quiescent cells. Similarly, HCT116 cells showed a decline in HIF activity when stimulated to proliferate with serum, and this effect was abolished by MCM knockdown. Finally, MCM7 knockdown was sufficient to enhance hypoxia-induced cell cycle arrest whereas there was no effect of MCM knockdown under basal conditions. That the MCMs function through O2-dependent pathways suggests that under conditions of severe hypoxia, the ability of HIF-1α to inhibit cell cycle progression may be dominant over the ability of MCMs to promote cell cycle progression by inhibiting HIF-1α, since hydroxylation is inhibited under conditions of severe hypoxia (Epstein et al., 2001).

In endothelial cells (Manalo et al., 2005) and HCT116 cells (Pires et al., 2010), exposure to hypoxia was associated with downregulation of MCM2–7 expression. We have confirmed and built on these observations by demonstrating that in diverse cell types, including NIH-3T3 mouse fibroblasts, Hep3B hepatocellular carcinoma cells, MDA-MB-231 breast cancer cells, and HCT116 colorectal carcinoma cells, hypoxia led to MCM downregulation in a HIF-dependent fashion. This suggests the presence of a positive feedback loop, whereby an immediate decrease in asparaginyl and prolyl hydroxylation of HIF-1α due to hypoxia is followed by downregulation of the MCM proteins, which further increases HIF activation. The mutual antagonism between HIF and MCMs (Figure 7E) may represent a critical homeostatic mechanism for the regulation of cell proliferation based on O2 availability.

The ‘MCM paradox’ refers to the vast excess of MCM proteins relative to potential origins of replication (Hyrien et al., 2003; Takahashi et al., 2005). Several studies have proposed that the excess of MCM proteins may play an important role under conditions of replication stress (Ge et al., 2007; Ibarra et al., 2008), when latent origins of replication are activated (Woodward et al., 2006). MCM proteins may play roles in additional cellular processes as well. For example, the MCM proteins associate with and enhance the activity of the Stat1 transcription factor by unwinding DNA during transcription (Snyder et al., 2005; Zhang et al., et al 1998). However, the present study demonstrates a role for the MCM proteins that does not involve their function as a hexameric DNA helicase. MCM7 and MCM3 have independent effects on HIF-1α protein stability and transactivation domain function, respectively. Clearly, if MCMs were functioning as an intact helicase to inhibit HIF-1, then one MCM could not have different effects on HIF-1α compared to another. The absence of any effect of MCM4 or MCM6 on HIF activity also provides compelling evidence against a role for the intact MCM helicase complex. Our data raise the possibility that MCMs may play a general role in mediating crosstalk between the cell cycle machinery and transcription factors that transduce physiological signals. However, regardless of any additional roles for the MCM proteins, our results delineate molecular mechanisms that mediate functional interactions between key proteins regulating cell proliferation and oxygen homeostasis.

EXPERIMENTAL PROCEDURES

Tissue Culture

Human embryonic kidney 293, 293T, Hep3B, MDA-MB-231, and NIH-3T3 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. HCT116 cells were cultured in McCoy Medium 5A with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at 37°C in a 5% CO2, 95% air incubator. Hypoxia was induced by exposing cells to 1% O2/5% CO2/balance N2 at 37°C in a modulator incubator chamber (Billups-Rothenberg).

Co-IP and Immunoblot Assays

Cells were lysed in PBS with 0.1% Tween-20, 1 mM DTT, protease inhibitor cocktail, 1 mM Na3VO4, and 10 mM NaF, followed by gentle sonication. For co-IP assays, 30 μl of V5-agarose beads (Sigma) were incubated with 2.5 mg of cell lysate overnight at 4°C. Beads were washed 4 times in lysis buffer. Proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Antibodies used in immunoblot and co-IP assays were: GST (GE Healthcare); V5 (Invitrogen); HA and β-actin (Santa Cruz); p300 (BD Biosciences); and FLAG, MCM2, MCM3, MCM5, MCM7, Cdt1, myc epitope, elongin C, VHL, HIF-1α, and HIF-2α (Novus Biologicals).

RNA Interference Assay

The vector pSR.retro.GFP.Neo.circular.stuffer (Oligo Engine) was used for shRNA expression. Oligonucleotides were annealed and ligated into BglII and HindIII-digested vectors. The plasmid encoding shMCM7 was obtained from Open Biosystems. Specific oligonucleotide sequences used are provided in Supplemental Table 2.

Quantitative Real-time Reverse-transcriptase PCR Assay

Total RNA was extracted from 293T cells by using Trizol (Invitrogen) and was treated with DNase I (Ambion). Total RNA (1 μg) was used for first-strand synthesis with the iScript cDNA Synthesis system (BioRad). Real-time PCR was performed using IQ SYBR Green Supermix and the iCycler Real-Time PCR Detection System (BioRad). Expression of target mRNA relative to 18S rRNA was calculated based on the threshold cycle (CT) for amplification as 2-(ΔCT), where ΔCT = CT,target − CT,18S. RT-PCR primer sequences are provided in Supplemental Table 1.

Statistical Analysis

All data are presented as mean ± SD, except where otherwise indicated. Differences between two conditions were analyzed using Student’s t-test.

Supplementary Material

01

Acknowledgments

We thank: P. Coulombe, C. Dang, Y. Liu, S. Rey, and G. Tomaselli (Johns Hopkins University) for advice and suggestions; I. Orukari for assistance with data processing; and K. Padgett (Novus Biologicals, Inc.) for generously providing antibodies against MCM2, MCM3, MCM5, MCM7, Cdt1, Myc epitope, Elongin C, VHL, HIF-1α, and HIF-2α. This work was supported by Public Health Service contract N01-HV28180 from the NIH and by funds from the Johns Hopkins Institute for Cell Engineering. M.E.H. was supported by a NIH hematology training grant (T32-HL007525) and a Cellular and Molecular Medicine training grant (T32-GM008752). G.L.S. is the C. Michael Armstrong Professor at the Johns Hopkins University School of Medicine.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental information includes Supplemental Experimental Procedures and References, three figures and two tables.

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