Cell, Vol.

111, 709–720, November 27, 2002, Copyright 2002 by Cell Press

Regulation and Destabilization of HIF-1␣ by ARD1-Mediated Acetylation
Joo-Won Jeong,1,2 Moon-Kyoung Bae,1,2 Mee-Young Ahn,2 Se-Hee Kim,1 Tae-Kwon Sohn,1,2 Myung-Ho Bae,1,2 Mi-Ae Yoo,2 Eun Joo Song,3 Kong-Joo Lee,3 and Kyu-Won Kim1,4 1 Research Institute of Pharmaceutical Sciences College of Pharmacy Seoul National University Seoul 151-742 Korea 2 Department of Molecular Biology Pusan National University Busan 609-735 Korea 3 Center for Cell Signaling Research Division of Molecular Life Sciences and College of Pharmacy Ewha Womans University Seoul 120-750 Korea quitin-proteasome pathway (Salceda and Caro, 1997; Huang et al., 1998; Maxwell et al., 1999; Berra et al., 2001). The association of pVHL and HIF-1␣ under normoxic conditions is triggered by the posttranslational hydroxylation of prolines (Pro402, Pro564) within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain (Ivan et al., 2001; Jaakkola et al., 2001; Masson et al., 2001) through the function of specific HIF-prolyl hydroxylases (HIF-PHs) (Bruick and McKnight, 2001; Epstein et al., 2001). Reports show that p53 directly interacts with HIF1␣ and limits hypoxia-induced expression of HIF-1␣ by promoting Mdm2-mediated ubiquitination and proteasomal degradation under hypoxic conditions (Ohh et al., 2000; Ravi et al., 2000). Furthermore, the degradation of HIF-1␣ by p53 in a hypoxic condition is inhibited by direct interaction with the Jun activation domain binding protein-1 (Jab1) and the ODD domain by blocking the interaction with p53 (Bae et al., 2002). Phosphorylation and dephosphorylation in the signaling pathway can result in HIF-1 activation (Richard et al., 1999). During hypoxia, p42/p44 MAPK activity induces posttranslational phosphorylation of HIF-1␣ and promotes the transcriptional activity of HIF-1 (Richard et al., 1999). According to a recent report, the hydroxylation of asparagine (Asn803), located in the C-terminal transactivation domain (C-TAD) of HIF-1␣, inhibits interaction with the p300/CBP coactivator and reduces the transcriptional activity of HIF-1 during normoxic conditions (Hewitson et al., 2002; Lando et al., 2002a, 2002b). Thus, the modulation of HIF-1␣ stability and its activation involve multiple proteins and several posttranslational modifications. Until recently, studies of posttranslational modifications of HIF-1␣ were restricted to hydroxylation, ubiquitination, and phosphorylation. Other posttranslational modifications of HIF-1␣ have not been defined. In the present study, we used the yeast two-hybrid system to identify proteins that interact with the ODD domain of HIF-1. One of the HIF-1␣-interacting clones, mouse ARD1, shares 57% identity with the ARD1 acetyltransferase sequence of Saccharomyces cerevisiae (Tribioli et al., 1994). ARD1 is required for the expression of protein N-acetyltransferase (NAT) activity in lower eukaryotes and bacteria (Tribioli et al., 1994; Park and Szostal, 1992; Ingram et al., 2000). However, its function in mammalian cells has not been defined. Acetylation is a posttranslational modification of proteins, with histones the best-known example (Kouzarides, 2000). Several enzymes are reported as histone acetyltransferases; these include PCAF, p300/CBP, SRC1, and MOZ (Kouzarides, 1999). Recently, these acetyltransferases have been shown to modify other proteins besides histones, including general transcription factors, e.g., E2F1, MyoD, GATA-1, and p53 (Kouzarides, 2000; Ogryzko et al., 1998; Boyes et al., 1998). Similar to phosphorylation, the posttranslational modification of general transcription factors by acetylation can regulate their DNA binding activity and interaction with other proteins such as coactivator proteins and tran-

Summary Hypoxia-inducible factor 1 (HIF-1) plays a central role in cellular adaptation to changes in oxygen availability. Recently, prolyl hydroxylation was identified as a key regulatory event that targets the HIF-1␣ subunit for proteasomal degradation via the pVHL ubiquitination complex. In this report, we reveal an important function for ARD1 in mammalian cells as a protein acetyltransferase by direct binding to HIF-1␣ to regulate its stability. We present further evidence showing that ARD1-mediated acetylation enhances interaction of HIF-1␣ with pVHL and HIF-1␣ ubiquitination, suggesting that the acetylation of HIF-1␣ by ARD1 is critical to proteasomal degradation. Therefore, we have concluded that the role of ARD1 in the acetylation of HIF-1␣ provides a key regulatory mechanism underlying HIF-1␣ stability. Introduction Eukaryotic cells sense oxygen and adapt to hypoxia by regulating a number of genes. In mammalian cells, the transcriptional complex HIF-1 plays an essential role in cellular and systemic oxygen homeostasis (Iyer et al., 1998; Semenza, 2000). HIF-1 stimulates the transcription of genes, such as erythropoietin and VEGF, whose protein products function to either increase oxygen availability by promoting erythropoiesis and angiogenesis, or activates genes involved in glucose transport and metabolism (Semenza, 1999). HIF-1 is composed of two subunits: the hypoxia-regulated ␣ subunit, HIF-1␣, and the oxygen-insensitive HIF1␤ subunit (Wang et al., 1995). Under normoxia, the HIF1␣ subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)-mediated ubi4

Correspondence: qwonkim@plaza.snu.ac.kr

Arrowheads at left indicate nonspecific bands. 2. 1–827 aa . prolyl hydroxylation. 1–400 aa. cerevisiae ARD1 (M11621). The presence of GFP-HIF1␣ in the immunoprecipitates was examined using anti-HIF-1␣ antibody (top).. In this report. 401–827) and the full-length HIF-1␣ were pulled down by interaction with the GSTfused ARD1 protein. including the ODD domain.Cell 710 Figure 1. 401–827 aa. However. whereas the amino acid 1–400 and 604–827 regions of HIF-1␣ showed no interactions. normoxia. respectively (middle. and ubiquitination. except for histone acetyltransferases. N. We synthesized 35 S-labeled deletion constructs of HIF-1␣ (Figure 1A) in vitro. 2001). studies of protein acetyltransferases. 1998) and prey vectors. has 96% homology to the human homolog ARDH (NM_003491) and 57% homology to S. 401–603 aa. None of the HIF-1␣ constructs used in this assay bound GST alone. Barlev et al. Immunoprecipitated materials and total cell extracts were analyzed by Western blots with antiFlag antibody and anti-HIF-1␣ antibody. 190 Hisϩ/Lacϩ double-positive clones were isolated and 64 clones were further selected by ␤-galactosidase assays in another yeast strain. In vitro translated proteins were mixed with GST-ARD1 bound beads (middle) or GST bound beads (right). and a GST pull-down assay was performed. After 24 hr posttransfection. hypoxia. (B) HIF-1␣ and its deletion derivatives were translated by programmed reticulocyte lysate in the presence of [35S]methionine. (C) HEK293 cells were transfected with expression vector for CMV-ARD1 and/or GFPHIF-1␣ as indicated. then tested for interactions with bacterially expressed glutathione-S-transferase (GST) or GST-fused ARD1. called mouse ARD1 in GenBank databases. the hisϪ yeast strain HF7C was cotransformed with a bait vector. and 6. bottom). To perform the two-hybrid assay. 604–827 aa. Gray boxes represent the ODD domain. . scriptional regulator proteins (Bannister and Miska. ARD1 Interacts with HIF-1␣ In Vitro and In Vivo (A) Structures of HIF-1␣ and its derivatives are schematically shown. DNA sequencing and database searches revealed that the nucleotide sequences of 13 clones (5 clones from a 17 day embryonic mouse cDNA library and 8 clones from mouse T cell cDNA library) correspond to a 700 bp mouse cDNA of a mouse homolog for ARD1 N-acetyltransferase (GenBank accession number NM_019870). 5. we established the function of ARD1 as a protein acetyltransferase in mammalian cells. Full-length HIF-1␣. we show that the acetylation of HIF-1␣ by ARD1 regulates the stability of HIF-1␣ in response to O2 concentration and further demonstrate that the acetylation of HIF-1␣ by ARD1 can be coordinated with other posttranslational modifications. 21% O2. The results shown in Figure 1B indicated that three deletion constructs. The interaction of ARD1 with HIF-1␣ under normoxia was set to 100% (right). immunoprecipitation. We screened 6 ϫ 106 yeast transformants for histidine auxotrophy and ␤-galactosidase expression. 1. The numbers at the bottom indicate the derivatives of HIF-1␣ shown in (A). IP. 3.. 4. Total cell lysates were immunoprecipitated with antiFLAG antibody. To overexpress the ARD1 and HIF-1␣ proteins in HEK 293 cells. Direct interaction of ARD1 with HIF-1␣ in vitro was examined using GST pull-down assays. 401–603. and their substrates are in an early stage. The bait did not show any self-activity of transcriptional activation for the reporters. From these transformants. allowing expression of mouse embryonic and T cell lymphoma cDNA sequences in-frame with GAL4 transactivation domain. H. with HIF1␣ a possible target protein for the enzyme. (HIF-1␣ 1–603. 2000. 1% O2. The relative interactions from three independent assays were quantified by densitometry. Interaction between ARD1 and HIF-1␣ in vivo was confirmed by coimmunoprecipitation assays. SFY 526. Results Identification of ARD1 for HIF-1␣-Interacting Protein We used the yeast two-hybrid system to identify candidate proteins that interact with HIF-1␣ in vivo. containing a GAL1UAS-LacZ reporter gene. the cells were incubated under hypoxia and the cell extracts were prepared. 1–603 aa. This gene. Moreover. encoding the GAL4 DNA binding domain in-frame with HIF-1␣ residues 401–603 (ODD domain) (Huang et al. 10% of the material used in the GST pull-down experiment is shown (left).

As shown in Figure 3A. Furthermore. The expression under normoxia was set to 100% (right). to HT1080-ARD1 cells and checked the level of HIF-1␣ protein. The mean and standard deviation based on three (mutated EpoHRE) or six (wild-type EpoHRE) independent transfections are shown. the HIF-1␣ protein level decreased by ARD1 was restored to the level of hypoxia-treated HT1080 cells (Figure 3B). in spite of the higher GFP-HIF-1␣ expression level under this condition (Figure 1C). the overexpressed ARD1 downregulated the protein level of HIF-1␣ induced by hypoxia. The VEGF mRNA expression level was measured in four independent experiments followed by densitometry. pBOS-hHIF-1␣ (0. but it did not affect mRNA level of HIF-1␣.1 ␮g). Furthermore. HT1080 cells overexpressing the ARD1 protein were designated as HT1080-ARD1 cells. (B) HT1080 cells were stably transfected with the indicated expression vector and left untreated or exposed to 1% O2 for 16 hr. this interaction was decreased when cells were exposed to 1% O2. however.ARD1 Acetylates and Destabilizes HIF-1␣ 711 Figure 2. The downregulation of the HIF-1␣ protein level by ARD1 under normoxic conditions was undetectable be- . suggesting that the reduction of the HIF-1␣ protein level by ARD1 was not due to the transcriptional reduction of the HIF-1␣ gene. Transfected cells were incubated for 24 hr at 21% O2 and then incubated at 21% O2 or 1% O2 for an additional 16 hr. ARNT. ARD1 interacted with HIF-1␣ under both normoxic and hypoxic conditions. ARD1 Downregulates HIF-1␣ Protein Stability Since the ARD1 protein directly binds to the ODD domain. Anti-VEGF and anti␣-tubulin Western blots from total protein extracts (bottom two) were performed. we cotransfected the Flag-tagged ARD1 expression vector and the GFP-HIF-1␣ expression vector into HEK 293 cells. As shown in Figure 2B. and mutated EpoHRE-Luc reporters. the increased expressions of VEGF mRNA and protein by hypoxia were downregulated in HT1080-ARD1 cells. we examined the effect of ARD1 on HIF-1␣ protein stability. reporter activity was markedly increased in cells exposed to 1% O2 (Figure 2A). However. we added MG132. When the Flag-ARD1 protein was immunoprecipitated from transfected cell extracts using the anti␣-Flag antibody. to evaluate whether ARD1 modulates the hypoxia-response element (HRE)-containing VEGF gene.2 ␮g) and 2 ␮g of pCMV-ARD1 or pCMV alone as indicated. or pEF-BOS alone (0. RTPCR analysis (top two) was performed to detect gene expression using specific primer for VEGF and ␤-actin. thereby confirming the effect of ARD1 on the downregulation of HIF-1␣ stability (Figure 3C). In the presence of MG132. a specific inhibitor of the 26S proteasome. ARD1 Inhibits Transcriptional Activation Mediated by HIF-1 To investigate whether ARD1 protein is involved in HIF-1 transcriptional activity. This suggests that the stability of the HIF-1␣ protein is reduced by ARD1 protein. and EpoHRE-Luc vectors. HT1080 cells were stably transfected with the Flag-tagged ARD1 expression vector. Upon transient transfection of HIF-1␣. the GFP-HIF-1␣ protein was coimmunoprecipitated (Figure 1C). luciferase activity was not changed by either hypoxia or the ARD1 protein. pSV40promoter-EpoHRE-Luc. the half-life of the HIF-1␣ protein was decreased in HT1080-ARD1 cells compared to HT1080 cells. we used a luciferase reporter system. In cells transfected with the mutated EpoHRE reporter. These results suggest that the ARD1 protein negatively regulates the hypoxia-inducible transcriptional activity of HIF-1. pBOS-hARNT (0. in pulse-chase experiments. the overexpression of ARD1 was shown to decrease the transcriptional activity of HIF-1 in a dose-dependent manner.1 ␮g). pCMV-ARD1. To confirm this notion. ARD1 Decreases the HIF-1␣-Mediated Transactivation Function and VEGF Expression (A) HEK293 cells were cotransfected with pSV40pro-EpoHRE-Luc (1 ␮g) or mutated EpoHRE-Luc (1 ␮g).

however. respectively. To obtain in vivo evidence showing the acetylation of HIF-1␣ protein. To determine if HIF-1␣ can be acetylated by the acetyltransferase activity of ARD1. HIF1␣ protein levels by Western blot were examined (top two) and diminished ARD1 mRNA by the antisense ARD1 expression vector was checked using RT-PCR (bottom two). A histone deacetylase inhibitor. cause HIF-1␣ is not present in this condition. Next. we first assessed whether stimulated acetylation affects the protein level of HIF-1␣. Cell lysates were immunoprecipitated with anti-HIF-1␣ antibody.Cell 712 Figure 3. (C) HT1080 (open circles) and HT1080-ARD1 (closed circles) cells were pulse labeled with [35S]methionine and chased in unlabeled medium for the indicated times under hypoxia. which negatively regulates HIF-1␣ stability. because the rate of HIF-1␣ acetylation may be not equal under these conditions. To do that. As shown in Figure 4A. These results suggest that the degradation of HIF-1␣ is stimulated by protein acetylation. (B) HIF-1␣ protein in HT1080 and HT1080ARD1 cells exposed to 1% O2 for 2 hr followed by the addition of 5 ␮M MG132 for 1 hr as indicated. These data strongly suggest that endogenous ARD1 induces the degradation of HIF1␣ under normoxia. the acetylation level of . The results shown in Figure 4B indicate that lysine residue(s) in ectopic HIF-1␣ was acetylated in vivo and that this acetylation was stimulated by the overexpression of the ARD1 protein. TSA. The HIF-1␣ protein level under hypoxia was set to 100% (right). sodium butyrate dramatically decreased the hypoxiainduced HIF-1␣ protein level. These results suggest that ARD1 could function as a negative regulator of HIF-1␣ stability by accelerating the recruitment of pVHL to HIF-1␣. (E) Endogenous pVHL was immunoprecipitated from 5 ␮M MG132 treated HT1080 and HT1080-ARD1 cell extracts with anti-pVHL antibody. HIF-1␣ stability under normoxia was significantly increased by transfection of the antisense ARD1 expression vector (Figure 3D). HIF-1␣ protein levels and mRNA levels were examined by Western blot analysis (top two) and RTPCR analysis (bottom two). we examined the relative acetylated HIF-1␣ under normoxic and hypoxic conditions. The relative HIF-1␣ protein level from six independent assays was quantified by densitometry. we transfected HIF-1␣ expression vector into HEK 293 cells and performed an in vivo acetylation assay using an anti-Ac-Lys antibody. ARD1 Decreases HIF-1␣ Stability and Regulates Association between HIF-1␣ and pVHL (A) HT1080 and HT1080-ARD1 cells were exposed to 1% or 21% O2 for 2 hr. The interaction of HIF-1␣ with pVHL in HT1080 cells was set to 100% (bottom). an inhibitor of protein deacetylases. also decreased the expression of HIF-1␣ protein (Figure 4A). such as S. Immunoprecipitated pVHL was visualized by Western blot analysis with anti-pVHL antibody for loading control (bottom). However. the function and the substrates of ARD1 in mammalian cells have not been identified. In addition. Cell lysates were harvested and subjected to immunoblot analysis of HIF-1␣. ARD1 Acetylates the ODD Domain of HIF-1␣ The function of the ARD1 protein as an acetyltransferase is known in lower eukaryotes. we treated HT1080 cells with sodium butyrate. cerevisiae. Although tests revealed that the protein level of HIF-1␣ was almost the same under these two conditions when treated with MG132. We found that under normoxic conditions. The intensities of HIF1␣ signals from three independent experiments were quantified and plotted. (D) HEK293 cells were transfected with the antisense ARD1 expression vector and left untreated or exposed to 1% O2 for 2 hr. we investigated whether ARD1 participates in the association of HIF-1␣ and pVHL. and coprecipitated HIF-1␣ was detected by Western blot analysis. Similarly. ARD1 overexpression increased interaction of HIF-1␣ with pVHL (Figure 3E). HIF-1␣ stability under hypoxia was increased by the antisense ARD1 expression vector. The relative interaction from three independent experiments was quantified by densitometry.

whereas acetylated ODD was not detected in the absence of either ARD1 or Ac-CoA. Interestingly. The masses of these peptides were . and then the resulting peptides were analyzed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The HIF-1␣ protein level under hypoxia was set to 100% (right). (C) HT1080 cells were exposed to 5 ␮M MG132 or 1% O2 for 2 hr as indicated and cell lysates were immunoprecipitated with anti-Ac-Lys antibody. namely K443. ODD-K532R was not acetylated by ARD1. and K547. -K477R. K477. Our next question to answer was whether HIF-1␣ could serve as a direct substrate for ARD1 and whether ARD1 protein can function as a HIF-1␣ acetyltransferase by direct interaction with the ODD domain. As shown in Figure 4D. we performed site-directed mutagenesis using pET-ODD expression vector to change the six single lysines to arginine residues. the position-specific acetylation of a target protein is mediated by distinct acetyltransferases (Gu and Roeder. the in vitro acetylated ODD protein by ARD1 protein was purified by immunoprecipitation with Ac-Lys antibody and digested with V8 protease. To test these possibilities. K460. These results strongly suggest that the ARD1 protein acetylates the ODD domain of HIF-1␣ by transferring acetyl group from Ac-CoA to lysine residues in the ODD domain through direct interaction. HIF-1␣ in normoxia was much higher than that of HIF-1␣ in hypoxia or under a MG132-treated hypoxic condition (Figure 4C). The relative acetylated HIF-1␣. K532. -K460R. and -K547R) were acetylated to a similar level as the wild-type ODD protein. The input ODD domain proteins were shown using Coomassie brilliant blue staining (bottom). (B) For ectopic expression of HIF-1␣. and immunoprecipitates were subjected to Western blot analysis for HIF-1␣.ARD1 Acetylates and Destabilizes HIF-1␣ 713 Figure 4. Extracts were immunoprecipitated with anti-Ac-Lys antibody. the reaction products were separated by SDSPAGE and visualized by autoradiography (top) and Western blot analysis with anti-AcLys antibody (middle). as shown in Figure 5A. the other mutated ODD proteins (ODDK443R. -K538R. However. (D) Following incubation of purified ODD domain (400 ng) with 50 nCi [14C]acetyl-CoA (top) or 10 mM acetyl-CoA (middle) and 100 ng of purified ARD1 protein for 2 hr at 30ЊC. 1997). To identify the acetylated lysine(s) of the ODD domain by ARD1. The six mutated ODD proteins were purified and an in vitro acetyltransferase assay was executed. There are six lysine residues in the ODD domain of HIF-1␣. lysine residues of the ODD domain were acetylated in the presence of ARD1 protein and Ac-CoA. we performed an in vitro acetyltransferase assay using purified recombinant ODD and ARD1 proteins. the acetylated HIF-1␣/expressed HIF-1␣ ratios. HEK293 cells were transfected with pBOS-hHIF-1␣ expression vector. we suggest that ARD1 protein specifically acetylates Lys532 in the ODD domain of HIF-1␣. the acetylated HIF-1␣/ expressed HIF-1␣ ratios. ARD1 Acetylated the ODD Domain of HIF-1␣ (A) HT1080 cells were exposed to 1% O2 for 2 hr with addition of 10 mM NaBu or 300 ng/ml TSA as indicated. K538. was normalized to the results of immunoprecipitation and Western blot analysis using densitometer from three independent assays (right). To confirm the acetylation site of the ODD domain. was normalized to the results of immunoprecipitation and Western blot analysis from five independent assays (right). Cell lysates were harvested and subjected to immunoblot analysis of HIF1␣. ARD1 Acetylates the Lys532 in HIF-1␣ Protein In general. Therefore. Cell lysates and immunoprecipitates were subjected to Western blot analysis with anti-HIF-1␣ antibody. The relative acetylated HIF-1␣. The relative HIF-1␣ protein level from four independent assays was quantified by densitometry.

New peptide at m/z 1580. 2001). 10% of the materials used in this assay was shown by staining with Coomassie brilliant blue (bottom). ARD1-treated (acetylated) wild-type ODD protein increased the interaction with pVHL. we first performed a pull-down assay to test for the interaction between the ODD domain and pVHL. which are oxygen sensitive. To determine the half-life of the HIF-1␣-K532R protein. whereas HIF-3␣. Based on the finding that the K532R mutant protein was not acetylated by ARD1. The result in Figure 6D indicates that acetylation by ARD1 increased ubiquitination of the ODD domain. the transfected HIF-1␣-K532R protein was stable in normoxia.0106 Da increase) 525-SDMVNVFKLELVE-537 peptide containing oxidation of Met (15. 2000). Moreover. we transfected the Flag-tagged HIF-1␣-K532R expression vector into HEK 293 cells and checked the protein level under normoxic conditions. To prove this point. This result confirmed the target site of ARD1 as Lys532 in HIF-1␣. To examine this possibility. a Lys532-mutated N-terminal transactivation domain (N-TAD) of HIF-1␣ was not degraded under normoxic conditions (Tanimoto et al. we performed an in vitro ubiquitination assay using purified ODD protein. we suggest that the acetylation of Lys532 by ARD1 is actively involved in the degradation of HIF-1␣ under normoxic conditions. and ARD1 decreased the stability of HIF-1␣. the ODDK532R mutant protein was ubiquitinated at the same level as the wild-type. Lys532 is the target site for acetylation by ARD1 protein. the interaction of pVHL with HIF1␣-K532R protein was much weaker than that with wildtype protein (Figure 6B). whereas wild-type HIF1␣ was degraded under the same condition. The asterisk denotes the lysine residue that acts as a target for ARD1-induced acetylation.9950 Da increase) (Figure 5B). These results suggested that HIF-1␣-K532R protein is stable under normoxic conditions when pVHL interaction is reduced. and then was subjected to MALDI-TOF MS. implying that Lys532 is not a ubiquitination site (Figure 6E). As shown in Figure 6C. In vitro acetylated proteins were subjected to Western blot analysis with antiAc-Lys antibody. (C) Sequence alignments of human and mouse HIF-␣ proteins. The authors suggested that Lys532 is critical to the degradation of HIF-1␣ protein.Cell 714 Figure 5. compared with masses of expected peptides calculated from the known sequence (Figure 5B). Since Lys532 is the acetylation site. we observed . we expected that the acetylation of Lys532 in HIF-1␣ stimulates the degradation of this protein through increasing pVHL-mediated ubiquitination. In a previous report. Moreover. we performed pulse-chase experiments using transiently transfected HEK293 cells under normoxia. It is worth noting that this lysine residue is conserved in HIF-1␣ and -2␣. To test whether acetylation by ARD1 can stimulate HIF-1␣ ubiquitination. This is the expected mass of acetylated (42. However.. In addition. stable even in normoxia (Hara et al. The Acetylation Site of HIF-1␣ by ARD1 Is Lys532 in ODD Domain (A) Purified recombinant ODD and mutated ODD domain proteins were incubated with acetyl-CoA and ARD1 protein for acetylation reaction. does not contain this residue (Figure 5C). As shown in Figure 6A. The arrow indicated the acetylated peptide peak. Two dots denote the conserved prolines (Pro402 and Pro564) for hydroxylation. (B) In vitro acetylated ODD protein with acetyl-CoA and ARD1 was purified using antiAc-Lys antibody and cleaved with V8 protease. The results confirmed that HIF-1␣-K532R protein is more stable than wild-type HIF-1␣ protein under normoxia (Figure 6A). two proline residues for hydroxylation are conserved in the three HIF-␣ proteins.. whereas ARD1treated ODD-K532R mutant protein did not. ARD1 as a HIF-1␣ Acetyltransferase Stimulates HIF-1␣ Degradation As described above.7529 Da was detected in the ODD domain acetylated by ARD1 protein.

skeletal mus- cle. we performed reverse transcriptase PCR. ARD1 Expression Is Decreased under Hypoxic Conditions The EST data from UniGene Cluster Mm. cobalt chloride and 2. whereas the protein level of HIF-1␣ was increased. The intensities of Flag-HIF-1␣ signals from three independent experiments were quantified and plotted. The interaction of wild-type HIF-1␣ with pVHL was set to 100% (bottom). Levels of total and ubiquitinated ODD in the second and third lanes from left were quantified by densitometery. Cell extracts were prepared and subjected to immunoblot analysis with anti-Flag antibody (top two). (E) 35S-labeled ODD and ODD-K532R were preincubated with HeLa cell extract. Pulldown reaction was performed with 35Slabeled pVHL for 1 hr and precipitants were subjected to SDS-PAGE and autoradiography. Cell lysates were immunoprecipitated with anti-Flag antibody. ARD1 mRNA level decreased upon exposure to hypoxia for 2 and 4 hr. Wild-type (open circles) and K532R mutant (closed circles) HIF-1␣-transfected HEK293 cells were pulse labeled with [35S]methionine and chased in an unlabeled medium for the indicated times under normoxia. Endogenous pVHL was immunoprecipitated and coprecipitated HIF-1␣ was detected by Western blot analysis. The ratio of ubiquitinated/total ODD was determined and normalized to the result obtained in the second lane to yield the relative HIF-1␣ ubiquitination (right). including brain. The in vitro ubiquitination assay was performed at 30ЊC for 2 hr and the reaction products were subjected to SDS-PAGE and autoradiography. (B) After transfection of HEK293 cells with the indicated vectors. heart. the hydroxylation of Pro564 in the ODD domain under ARD1-mediated acetylation by the MALDI-TOF MS analysis (data not shown). The ARD1 mRNA level was decreased in cells exposed to 1% O2 and was also decreased when cells were treated with hypoxia-mimicking agents. mediated by the pVHL ubiquitination complex under proline hydroxylated conditions.2Ј-dipyridyl (Figure 7B). (D) 35S-labeled ODD was acetylated by ARD1 as indicated and the products were preincubated with HeLa cell extract. and liver. 5 ␮M MG132 was added for 2 hr and total cell extracts were isolated. To examine the expression level of ARD1 during hypoxia. 5934 indicated that mouse ARD1 is expressed in almost every cell type.ARD1 Acetylates and Destabilizes HIF-1␣ 715 Figure 6. suggesting that ARD1 is broadly expressed. Our next question was the relation between ARD1 mRNA reduction and HIF-1␣ stability. The interaction signals from three independent experiments were quantified (bottom). To confirm the expression of ARD1 mRNA. ARD1 was detected in all tissues tested. As shown in Figure 7A. These results indicate that the acetylation of Lys532 by ARD1 protein is a key regulatory event for the maximal degradation of HIF-1␣. The acetylation of HIF-1␣ was also decreased in proportion to the increase of hypoxic exposure. The relative interaction from three independent experiments was quantified by densitometry. As shown in Figure 7C. The HIF-1␣-K532R Mutant Is Not Degraded under Normoxic Conditions (A) Flag-tagged wild-type or mutant HIF-1␣ (substitution of Lys532 to Arg) was transiently expressed in HEK293 cells and incubated under normoxic or hypoxic conditions for 6 hr. (C) Purified His-ODD or His-ODD-K532R protein was acetylated by ARD1 as indicated and products were preincubated in the presence of HeLa cell extract at 30ЊC for 2 hr. The in vitro ubiquitination assay was performed at 30ЊC for 2 hr and the reaction products were subjected to SDS-PAGE and autoradiography. we performed Northern blot analysis of RNAs from various human tissues. We therefore checked the mRNA level of ARD1 and the acetylation and protein levels of HIF-1␣ after hypoxic exposure for 2 and 4 hr. These data support the .

respectively. and Western blot analysis for HIF-1␣ and ␣-tubulin (bottom two) were performed. These results strongly suggest that ARD1 functions as a negative regulator of the HIF-1␣ protein.. leading to rapid proteasomal degradation (Ivan et al. 2001.2Ј-dipyridyl.. Extracts were subjected to Western blot analysis with anti-Flag antibody. Arrowheads at left indicate nonspecific bands (top). The modulation of HIF-1␣ protein by interaction with other proteins is known to play an important role in its stabilization and activation. Immunoblotting was used to confirm purity of nuclear and cytosolic extracts using antisera against histone H1 and ␣-tubulin. In this report. acetylation assay for HIF-1␣ (middle). To obtain information on the subcellular localization of ARD1 protein. then Northern blot analysis using specific probe for ARD1 and ␤-actin (top two).Cell 716 Figure 7. (D) The nuclear and cytosolic extracts were prepared from HT1080 or HT1080-ARD1 cells exposed to 1% or 21% O2 for 2 hr. We found that binding of ARD1 to HIF-1␣ was stronger under normoxic conditions than under hypoxic conditions. (B) HT1080 cells were incubated for 2 hr under hypoxic conditions or treated with 100 ␮M cobalt chloride and 100 ␮M 2. A human ␤-actin probe was used as a loading control. Moreover. Discussion The regulation of HIF-1␣ stability and its activity occur at multiple levels. whereas inhibition of ARD1 expression by transfection with an antisense ARD1 expression vector significantly increased protein stability. hypothesis that decreased expression of ARD1 causes increased HIF-1␣ stability by reduction of acetylation under hypoxia. Jaakkola et al. Recent work with mammalian cells demonstrated that posttransla- tional modification by prolyl hydroxylation targets HIF1␣ to the pVHL ubiquitination complex. not in nucleus (Figure 7D). indicating that ARD1 may act on HIF-1␣ in the cytosol. we separately prepared nuclear extracts and cytosolic extracts of ARD1 transfected cells. ARD1 protein was detected only in the cytosolic fraction. Moreover. . ARD1 Expression Is Decreased under Hypoxic Conditions (A) The expression of hARD1 mRNA was detected by Northern blot analysis using human multiple-tissue blot. 2001). forced expression of ARD1 diminished HIF-1␣ protein stability. we characterized ARD1 as a protein acetyltransferase and HIF-1␣ protein as a substrate of ARD1. we show the interaction of ARD1 with HIF-1␣ using the yeast two-hybrid system and characterize ARD1 as an important regulator of HIF-1␣ stability. RT-PCR analysis was performed using specific primers for ARD1 and ␤-actin. (C) mRNA and cell lysates were isolated from HT1080 cells exposed to 1% O2 for 2 and 4 hr.

whereas the level of HIF-3␣ was not affected by O2 concentration (Hara et al. our data show that ARD1. we suggest that Lys532 of HIF-1␣ is the target site for acetylation by ARD1. 1998). human ARD1 homolog was found in all tissues examined. So far. and protein levels increased in response to hypoxia. TCF (Waltzer and Bienz. 2001). For luciferase assay. Recently. 2001). 1994). the acetylation of lysine residues in transcription factors by PCAF or p300/CBP has been described for p53 (Gu and Roeder.. we report that the acetylation of Lys532 by ARD1 in the ODD domain is critical to the proteasomal degradation of HIF-1␣. Here. In a previous report. We also found that the acetylation of Lys532 in HIF-1␣ strongly increased the interaction with pVHL and induced pVHL-mediated ubiquitination. and GFP antibodies were purchased from Pharmigen. TbDn-1 alone shows acetyltransferase activity without ARD1 or other acetyltransferases (Gendron et al. Moreover. Experimental Procedures Materials Sodium butyrate (NaBu) and trichostatin A (TSA) were purchased from Sigma and MG132 from Calbiochem. however. functions for EKLF and TCF acetylation have not been established (Bannister and Miska. Binding of pVHL is dependent on hydroxylation of Pro402 and Pro564 in the ODD domain of HIF-1␣ through an enzymatic process that requires O2 as well as iron and is inhibited by cobalt chloride (Ivan et al. Moreover. we found that HIF-1␣-K532R was ubiquitinated at the same level as wild-type HIF-1␣ and that its binding to pVHL was not changed by ARD1. Our results suggest that the HIF-1␣ protein is acetylated and that this modification is essential to the regulation of HIF-1␣ stability.. To construct antisense ARD1 vector.. in mammalian cells. several nonhistone proteins. (2000) examined three lysines (K532. the acetylation of p53. 1999). cerevisiae as a component of the NAT complex and was found to have the ability to form a homodimer or heterodimer with NAT1 protein in S. pVHL. not a ubiquitination site. The level of acetylation of HIF1␣ was shown to gradually decrease as the length of hypoxic exposure time increases. we found that the ARD1 protein itself acts as an acetyltransferase without a partner acetyltransferase and regulates HIF1␣ stability. and accordingly suggested that Lys532 is critical for the degradation of HIF-1␣. Multiple Tissue Northern (MTN) blot was purchased from CLONTECH. 2000). 2000). including PCAF/GCN5. functions mainly under normoxic conditions due to the decreased ARD1 mRNA and a decreased affinity to HIF-1␣ under hypoxia. Park (College of Medicine. In fact. 2000) that targets HIF-1␣ for proteasomal degradation (Salceda and Caro. E2F1 (MartinezBalbas et al. Huang et al. and -3␣ have two conserved prolines that are target sites for hydroxylation and pVHL binding in the ODD domain. and CLONTECH. which we found is partly due to the reduced expression of ARD1. These enzymes function as histone acetyltransferases and mainly exist in the nucleus... 1997). which is the E3 ubiquitin-protein ligase (Cockman et al. Plasmids and Recombinant Proteins The full-length ARD1 expression vector was constructed by PCR and subcloned into pCMV-Tag (Stratagene).. MyoD (Sartorelli et al. SRC1. not HIF-3␣. 2001. 1998).. Kamura et al. and MyoD occurs close to the DNA binding domain and results in stimulation of DNA binding (Martinez-Balbas et al.. 1999. Similarly. Several families of acetyltransferases are known. HIF-1␣ antibody was kindly provided by Dr. Moreover.. K538. p53 and Ac-Lys antibodies were purchased from Upstate Biotechnology. have been identified as substrates for PCAF and/or p300/CBP (Kouzarides. 1997). 2000). and K547) of N-TAD for degradation of HIF-1␣ during normoxia. acetylation of the ODD domain by ARD1 increased the interaction with pVHL and stimulated ubiquitination of HIF-1␣ protein.ARD1 Acetylates and Destabilizes HIF-1␣ 717 ARD1 protein was discovered originally in S. 2000. and that the mutant protein was stabilized and showed decreased interaction with pVHL during normoxia. TAF250. Korea). We found that a K532R mutant of HIF-1␣ was not acetylated by ARD1 protein. the results reported here establish a key mechanism for the modulation of HIF-1␣ protein stability through acetylation. Seoul National University.1 (Invitrogen) in reverse orientation.. although its functions and substrates have not been characterized (Tribioli et al. 2001). p300/CBP. Therefore. Seoul. 2000). The functional consequences of posttranslational modification by acetylation of these transcription factors appear to be quite varied (Kouzarides. 2000).. 1999). pBOS- .. 2000). Recently. Evaluating the collective evidence. a subunit of the N-acetyltransferase complex that functions with the ARD1 protein in S. From these data. E2F1. and MOZ (Kouzarides. we suggest that ARD1 is a new mammalian acetyltransferase.. HIF-1␣ and -2␣ were shown to be degraded under normoxic conditions. cerevisiae (Park and Szostal. as a negative regulator of HIF-1␣ stability. Furthermore. Masson et al. cerevisiae (Gendron et al. From these data. such as general transcription factors and DNA binding proteins. whereas the lysine residue acetylated by ARD1 is conserved only in HIF-1␣ and -2␣. -2␣. HIF-1␣. Therefore. Tanimoto et al. 1999). For instance.. Sigma. we showed ARD1 acetylates Lys532 in the ODD domain of HIF-1␣ by transferring acetyl group from Ac-CoA. EKLF (Zhang and Bieker. 2001. To construct pETARD1 and GST-ARD1 for in vitro translation and bacterial induction. we found that neither TbDn-1 nor mouse NAT-1 homolog affected the stability of HIF-1␣ protein in human cells (data not shown).. 1998. 2000). Previously. In mammalian cells.W. TbDn-1 was isolated in mouse and found to be homologous to the yeast NAT1 protein. Further. These investigators showed that the K532R mutation stabilized HIF-1␣ during normoxia. we conclude that ARD1 acetylates Lys532 in the ODD domain of HIF-1␣ by direct interaction and plays a central role in HIF-1␣ stability by accelerating HIF-1␣ interaction with pVHL. and Sp3 (Braun et al. 1992). these data further support our finding that the acetylation of lysine residue plays a critical role in the degradation of HIF-␣ proteins in response to O2 concentration... 2000) through the interaction with pVHL. 2000. respectively. Flag. The ODD domain encompasses sequences that mediate O2dependent ubiquitination of HIF-1␣ protein (Sutter et al.. Sartorelli et al. GATA-1 (Boyes et al. J. the ARD1 cDNA was inserted into pET-28 (Novagene) and pGEX4T (Amersham Pharmacia Biotech). However. Hung et al. Jaakkola et al. ARD1 cDNA fragment (1–308) was inserted into pCDNA3.

594–598. Y. A. Received: June 3. and mutated EpoHRE-Luc vector was provided by Dr.A.Cell 718 hHIF-1␣.. The luciferase and ␤-galactosidase enzyme assays were performed as described (Bae et al..E. Nucleic Acids Res. R. Bae. 1184– 1192.. with proper recombinations of effector plasmids using calcium phosphate-mediated methods (Sambrook et al. we used the MS-Digest program available on the World Wide Web site of the University of California at San Francisco (http://prospector... Regulation of activity of the transcription factor GATA-1 by acetylation.K. T. 2002).J.. Mansfield. 2001) using 100 ng purified bacterially expressed ODD protein and 40 ng ARD1 protein. 29.. Inc. Applied Biosystems. Eric Huang for the mutated EpoHRE-Luc vector. P. Cockman. 277. Preparation of protein extracts. Ratcliffe..W.. cells were washed and chased with complete DMEM for the indicated amount of time in hypoxic chamber. Japan) (Ema et al. Liu. and McKnight. Bae...H. Financial support was provided by the National Research Laboratory Fund (2001-N-NL-01-C-015). Hypoxia inducible factor-␣ binding and ubiquitination . Science 294... 2.. Cell. 1997).. M. 4994–5000.. and Dr. 615–620. P.. immunoprecipitation. Masson.. 1337–1340.J. Fujii-Kuriyama for his gifts of the pBOS-hHIF-1␣. A. 1996). Subcellular Fractionation of Cell Lysates HT1080 and HT1080-ARD1 cells were incubated in 21% or 1% O2 for 2 hr and separated into cytoplasmic and nuclear fractions as described (Kim et al.. M.K. Bethesda.R. the Ministry of Science and Technology. Eric Huang (NCI.)..G. respectively. were used to assay protein-protein interactions and for library screening. (2002). G... For the generation of ARD1-overexpressing cells. (2000). Ahn. and Berger. N. Chang. Protein Acetyltransferase Assay Protein acetyltransferase assays were performed as described (Ito et al. EMBO Rep. pBOS-hARNT.R. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. K.H..K. GST Pull-Down Assay The 35S-labeled deletion mutants of HIF-1␣ were prepared by TNT system (Promega). Mole. P.. Two-Hybrid Library Screening and Evaluation of Protein-Protein Interactions Yeast strains for two-hybrid experiments were obtained from CLONTECH as components of the MATCHMAKER Two-hybrid System (CLONTECH). K. GST-fusion proteins were purified as described (Shuman et al.L. 35S-labeled ODD (5 ␮l) was incubated in the presence of HeLa cell extracts (200 ␮g). S. 57. purified His-ODD protein (1 ␮g) was preincubated with HeLa cell extracts for 2 hr at 30ЊC and then binding assay was performed as described (Ohh et al. (2001). Bruick.. ucsf. Regulation of gene expression by transcription factor acetylation. and Miska. E.. J. 2000). Pugh.. was added to growing HT1080 cells to a final concentration of 1 mM for 16 hr. pET-ARD1 and pET-ODD vectors were transformed and His-ARD1 and His-ODD proteins were purified on a Talon metal affinity column (CLONTECH) according to the instructions of the manufacturer. All proteins were dialyzed using Sephadex G-25 column (Amersham Pharmacia Biotech). C. D.Y.. and Maxwell. Two-hybrid assays using the GAL4 system were performed according to the instructions of the manufacturer (CLONTECH). VHL-HIF-1␣ Binding Assay and In Vitro Ubiquitination Assay For the binding assay.. Roux. Dr. Band intensities were measured using an autoradiopraph image analyzer (Fuji). (2001). Cell 8. Harris. V. A conserved family of prolyl4-hydroxylases that modify HIF. Nacht. Chem. Koop. Maher. an in vitro acetyltransferase assay was preperformed for 2 hr at 30ЊC. cells were incubated at a CO2 level of 5% with 1% O2 balanced with N2 using a hypoxic chamber (Forma). Dr. Total cell extracts were isolated from sodium butyrate-treated cells and immunoprecipitated with 1 ␮g Ac-Lys antibody. N. 2002 Revised: September 17. L. MD.).. Acknowledgments We thank Dr.W.. pBOS-hARNT. For production of His fusion proteins. 2000).M. Hypoxia-inducible factor-1␣ (HIF-1␣) escapes O2-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm. L.R. and Suske.. H. an inhibitor of protein deacetylase. 1989). In Vivo Acetylation Assay Sodium butyrate. For the acetylation-combined experiments. 2000) with [35S]pVHL. Park. For the hypoxic condition.. Korea (to K. 1997) and the GST pull-down assay was performed as described (Bae et al.W. 1243–1254. Kim. D. Ertmer.. Cell Culture and Hypoxic Condition HEK293 and HT1080 cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotics in a humidified 5% CO2 incubator. Richard. 2000). and pSV40pro-EpoHRE-Luc vectors were kindly provided by Dr.. For the ubiquitination assay. Fujii-Kuriyama (Tohoku University.. 2002) using GST or GST-ARD1.. 2002). M. Yeast strains. National Institutes of Health. (2001).. After labeling. Coimmunoprecipitation Transiently transfected HEK 293 cells or stably transfected HT1080 cells were treated for 6 hr or 2 hr under hypoxic conditions. P. Jaakkola. USA). S. and Ogryzko. K. Boyes. K. Clifford. before harvesting the cells. G.. L.. Y. 9–12. For interpretation of the mass spectra. Barlev. Jong-Wan Park for the anti-HIF-1␣ antibody. Braun. E.. respectively. 2002 References Bae. In-Gel Digestion and Mass Spectrometric Analysis The acetylated ODD protein was excised from Coomassie-stained gel and purified as described (Song et al.edu). Life Sci. and the ubiquitination assay was performed as described (Ohh et al. Transient Transfection and Luciferase Assays Five micrograms of plasmids was transfected to HEK293 cells.H. pGBT9-ODD. Mol. M..K. Byung Pal Yu for the critical reading of the manuscript. J. Site-Directed Mutagenesis Site-directed mutagenesies of six lysine residues to arginine in both pET-ODD expression vector and Flag-tagged HIF-1␣ expression vector were performed using the Quick Change site-directed mutagenesis kit (Stratagene). S. Berra.. J. Jeong. S. Mol. (2001).. and Kim.W. The immunoprecipitates were subjected to SDS-PAGE and Western blot analysis with HIF-1␣-specific antibody. Halazonetis. S.. Transcription factor Sp3 is regulated by acetylation. Lee. Nature 396. J. and Western blot were performed as described previously (Tanimoto et al. R.A.C. E. Pulse-Chase Experiments HT1080/HT1080-ARD1 cells and HEK 293 cells were labeled with 200 ␮Ci of [35S]methionine per plate in methionine-free DMEM (Invitrogen) under hypoxia for 4 hr.E. and GFP-HIF1␣ vectors were prepared as previously described (Bae et al.-W.L.W. D. Peptide mixtures were analyzed with MALDI-TOF MS using a delayed ion extraction and ion mirror reflector mass spectrometer (Voyager-DE STR. Cells were subsequently subjected to immunoprecipitation as described above. Bannister. (1998). Byfield. Nakatani. J. HT1080 cells were transfected with pCMV-ARD1 and selected using G418 (Invitrogen). SFY526 and HF7c. and Pouyssegur. Biol. N. Jab1 interacts directly with HIF-1␣ and regulates its stability. (2000)..D. deletion mutants of HIF-1␣.. Chehab. and pSV40pro-EpoHRE-Luc vectors.

T. 1474–1480. Ito.. R. Artemov. 1331–1340.. Chae. Conaway. N. C. (2000). 16. A. 20. B. 88. J... S.M.W.. Hamamori. Mukherji.A. J. (2000).L. Activation of HIF-1␣ ubiqitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Kim. M.. 287. C.. Pereira. E. (1999). 4748–4753.. Acetylation: a regulatory modification to rival phosphorylation? EMBO J. G. D. 9. Ogryzko. 808–813. Lando.L. Sci. K.J. C. 43–54...W. G.. Parasitol. (2002). Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha. S. J.P. 19. Biol.H. U. H. B.S.. 4273–4278... 14. and Kaelin. Chung. J. (2001). (2000). D. (1999).J. M.J.V. Gorman. et al. Howard. V. et al. (1996). L.H. P. EMBO J. (1989).. M. Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch... Asara..H.E.. Kim. Regulation of E2F1 activity by acetylation.. Curr.C. Proc. N. Wenger.Y. J. T. A.K. Tian. Hewitson.. Appl. M.H... (1999).S.A. Y. Czyzyk-Krzeska. (1998). (Cold Spring Harbor. Gu. Berra.. Yang. 309–317. Gu.. Proc. Gleadle. Kotani.W. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1 ␣ (HIF-1 ␣) and enhance the transcriptional activity of HIF-1.J. Ema. M... D.. p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. Sambrook. 271.. Chem. Salceda.J.W. and Blobel. 725–734.... Wiesener..F. (1997). Wilson. D. and Imura.. A.M. M...R. 34–44. Bhujwalla. N. D. 271–275. Commun.. T.. 468–472. Yang.R. (1997). 272. J. 15. Kouzarides.. Cell 90. Peet. Bullock.S... E. EMBO J. S. L.. A. 19. A. Clifford. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. and Poellinger.S.. Park. Cell. W. M.C. Y. Gothie. Leung.. J..L.J.A.S.. M. 275. and Bruick. and Yao. Mechanism of regulation of the hypoxia-inducible factor-1 ␣ by the von Hippel-Lindau tumor suppressor protein.. Chung. N. J.. Pugh. 19. Chang. and Roeder.G.... Nakatani. (2001). 32631–32637. C. Mole.Y. Cell 94. HIF-1␣ protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutation. et al. Gaskell. Regulation of hypoxi-inducible factor 1 ␣ is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Natl.. Ubiquitination of hypoxia-inducible factor requires direct binding to the b-domain of the von-Hippel-Lindau protein.. Kouzarides.K. Tubedown-1. M. J. (2002a). Mukherji. Nat. The role of carboxylterminal basic amino acids in Gq ␣-dependent activation. J. NY: Cold Spring Harbor Laboratory). Biochemistry 39. Kotch. Cell Biol. Kondo. Biol. Brehm.. Y. Fritsch. E. (1997).. J... Madan. USA 97. Nature 399. Acad. P. (2000). L.. Wang. X. E. D..R. 300–315. R. R. Res.. M.. USA 94. J. Gendron. Park. A. 551–578. D. M. N. 1466–1471. J.. Riordan.Y. and Paradis. EMBO J. D. Oldham.K. Lawler. P/CAFmediated acetylation regulates the function of the basic helix-loophelix transcription factor TAL1/SCL. Huang. (1992). K. Sci. 2. ARD1 and NAT1 proteins form a complex that has N-terminal acetyltransferase activity.. E. 5197–5206.. 858–861.J.M.. 595–606.. Dev. Kobayashi. E. Cell Dev. V. Biophys. E. K.. USA 95. C. J. a novel acetyltransferase associated with blood vessel development. S.. Natl..F. Science 292. Xu. F.N..A. A.. X. Zhao. S. S. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. 3496– 3505. Acad. Laughner. D. 22642–22647. Dhanda. Sci.E. 19. Gorman. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. 272. Shuman. G. D. Chau. 662–671. Salic. and Ratcliffe.L. Cell 107. R. W.E... (2001). 12.. Biol. Opin.J. Shi. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Y. Martinez-Balbas. X. Genes Dev. A. Zeng. S. Schiltz. Science 295.. Chem.. Acad. D. Pugh.L. Masson.. Molecular Cloning: A Laboratory Manual. Biol... Sutter.H. Whelan. G.Y. Z. Genes Dev.. Qiu.I. (2001)..M. T. K.. and Poussegur.G. 277. O’Rourke. Ogryzko. J. (1999).D. R... Chem. H. Histone acetylases and deacetylases in cell proliferation. Kamura. C. T. McNeill. S..J. S. Hamada. and Lee. 21187–21192. (1998). Metzen. USA 97. HIF-1: mediator of physiological and pathophysiological responses to hypoxia.L.. Maxwell.C. McNeill.. Y. Hamilton. G. (2000).H. Sutter.. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1␣.H.. and Rhee. and Maniatis. Semenza. C. Gleadle. Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. Kim.. Mol.... 10090–10097. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. 6792–6803. 149–162. Cell 4. Tian.J. Zhang. A.. Jaakkola. (2001). and Kedes.. Valiando. Roux. Natl. and Ratcliffe.. P. J.. L..E. H.. Mol. EMBO J.. Adams.. Biol.. 10430–10435. Biol. (2000).. G.V.. Y. (2002b). 12793–12800.. 218. and Coligan. Tanimoto.J.. Chem.. 4298–4309.. R.M.C.A.M.. Y. Biol. Elkins. 18. P. 11. Wilson. A. Lane. Science 292.. L. N.. F. Hewitson. Hypoxia-inducible Factor (HIF) asparagine hydroxylase is identical to Factor Inhibiting HIF (FIH) and is related to the cupin structural family. 2087–2093.D. S.. A. M..C. (2001). Mol. Bauer. (2000).. EMBO J. Qin. Hebestreit. HIF ␣ targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.. M. Histone-like TAFs within the PCAF histone acetylase complex. R. A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. J. Ivan. and Nakatani... J. K. Puri.. Appella.. Mole. H. Acad. Epstein. D. Wykoff. M. CREB-binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites.. and Caro. M. L. Proc..C.J.G. ATF-2 and C/ EBPalpha can form a heterodimeric DNA binding complex in vitro. Independent function of two destruction domains in hypoxia-inducible factor-␣ chains activated by prolyl hydroxylation. Sato. H. Pavletich. 423–427. K..J... Natl. Gielbert.. 19.. Semenza.F. T. Cockman. Whelan. and Bunn. Biol.E.. V. Gearhart. Iyer. Functional implications for transcriptional regulation. Dyn. and Brandt. Semenza. M. 2nd Ed. T. and Kouzarides. Dillehavy.J. Biochem. Bhattacharya. 11. M..G. Howard.. particulate association. 274.L. J.. and Yu. Makino. W... J. Chem. L.L..A.. Proc. Gassmann. Nielsen. P. Cheong. T. Kriegsheim. Annu.. and nuclear localization of phospholipase C-␤1. E. E...Y. (1999). Huang. M. Cross.. Y.J..G. C. J. Song. (1999). and FujiiKuriyama. S. Its stabilization by hypoxia depends on redox-induced changes.E. T.. Lau. Z. Targeting of HIF-1 ␣ to the von Hippel-Lindau ubiquitination complex by O2-regulated prolyl hydroxylation. Laughner. Sogawa.. Kim. and Semenza. S. C.. Chem. Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program.. Lando.M.M. Expression and characterization of hypoxia-inducible factor (HIF)3␣ in human kidney: suppression of HIF-mediated gene expression by HIF-3␣. Y. J. Hara. C... (2000). Iwai. W. P. Maxwell. J. J.. and Kaelin. (1998). A.A. (2000). Kim. Willam. Schau. 35–44. C.. Ohh. Huang.. Y. (1997). D. Genes Dev.. Mookerjee.W. Matsuda. J.A. . 26351– 26355.. Q. Ivan.E. Physiol. Biochem. G. E. Sartorelli. Sci. 25733–25741. S. Dev.. Vaux. (2000). A. and Horn. L. Kim. Welford. Y.. Yokotani.L.ARD1 Acetylates and Destabilizes HIF-1␣ 719 by the von Hippel-Lindau tumor suppressor protein. EMBO J.L. M. Hoffman. Rev.R.M. and Szostal. E. J. V. 464–468. G. Taya.. Ingram. and Bedi. J.I. J..H. J. and Conaway. S. (2000). Park.W.C. Genet.. Lai.. Hung.W. 40–48. L. Kondo. (2000). 1176–1179. M. R. Ohh.. W. Maher. Genetic manipulation indicates that ARD1 is an essential N␣-acetyltransferase in Tripanosoma brucei. Hypoxia-inducible factor 1 ␣ (HIF-1 ␣) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Agani. Saito. Whitelaw... J. and Whitelaw. Ravi... M. Peet.V. 7987–7992.M. Y. T. Schofield.. J.W.. Richard... Weiss. K. C.

. Natl. (1995).Cell 720 Tribioli. C.. S. Mancini. Torri.J.. Jiang. and Toniolo. Waltzer. G.. Isolation of new genes in distal Xq28: transcriptional map and identification of a human homologue of the ARD1 N-acetyltransferase of Saccharomyces cerevisiae. Acad. Wang.. USA 92. Mol. M. (1998). Natl. Hum. S.L. Bione. E. C. Nature 395. (1998). and Semenza. G... Sci. 5510–5514. W. Sci. Plassart. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Sala. J.H. Acad. USA 95.. 521–525. Genetics 3. Proc. G. Drosophila CBP repressed the transcription factor TCF to antagonize Wingless signaling. . and Bienz. Proc. 9855–9860.. Acetylation and modulation of erythroid Kruppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Rue. (1994).. M. Rivella. E. and Bieker. D.A.. B. Zhang..L. 1061–1067. L.

Sign up to vote on this title
UsefulNot useful