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J. Biol. Chem., Vol. 281, Issue 45, 34677-34686, November 10, 2006
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11
2
From the
Laboratory of Human Carcinogenesis and HIV and AIDS Malignancy Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, June 30, 2006 , and in revised form, September 13, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1, and G3BP2a. Understanding the functions of the four splice variants may aid in defining their roles in human carcinogenesis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(7) through a physical interaction with HIF prolyl hydroxylase (HPH)-2C (8), resulting in repressed HIF activation in a chromatin-dependent manner. More recently, it was reported that ING4 acetylates histone H4 lysine 5, 8, and 12 with a histone acetyltransferase, HBO1/MYST2 (MYST2 is MOZ, YBF2/SAS3, SAS2 and TIP60 histone acetyltransferase 2) (9), and interacts with methylated histone H3 (10). Thus, ING4 seems to play several major roles in cells like other ING family proteins (11) that are conserved from yeasts to vertebrates (12).
In this study, we describe three novel splice variants of ING4, ING4_v2 (a 3-bp skip form), ING4_v3 (a 9-bp skip form), and ING4_v4 (a 12-bp skip form), besides ING4_v1 (the originally enrolled ING4, the longest form). These variants are produced from the alternative use of two splice donor sites at the end of exon 4 and two splice acceptor sites at the start of exon 5 of the ING4 gene, although one of the three variants, ING4_v4, was previously attributed to a common deletion mutation (6). The alternative RNA splicing of the ING4 pre-mRNA causes a partial loss of an NLS and affects nuclear localization of the proteins. We show that the small deletion in the ING4 protein leads to functional differences between the ING4 variants. Growth suppressive effects of the variants that have the partially missing NLS were attenuated by weaker activity on p21WAF1 induction. In addition, ING4_v4 showed attenuated suppressive effects on cell spreading and migration compared with the original ING4 (ING4_v1). On the other hand, ING4_v2 only lost the suppressive effect on cell spreading, suggesting an important role for ING4_v2 on the regulation of cell migration. ING4_v4 played dominant-negative roles on these ING4_v1 effects. In addition, we found that ING4_v1 and ING4_v2, but not ING4_v4, have a binding affinity to Liprin 1 that may play a role in the regulation of focal adhesion disassembly (13, 14). In addition, only ING_v1 has a binding affinity to G3BP2a, which is involved in Ras signaling, NF-
B signaling, and the ubiquitin proteasome system (15-17). These differences may affect the function of ING4 splice variants.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Construction of Minigene Plasmids—All sequences for cloning were amplified by PCR using KOD-Plus (Novagen, Madison, WI) and verified by DNA sequencing. A partial ING4 gene from exon 3 to exon 6 and its point mutants were cloned into pcDNA 3.1 (+) (Invitrogen) using the HindIII and XbaI site. Expression plasmids for two major ING4 splice variants (ING4_v2 and ING4_v4) were derived from the original ING4 plasmid (pFLAG-CMV-2 and pcDNA3.1/Hygro) as a template.
Transfection of Plasmids and Isolation of RNA—The plasmids were transfected into cells by Lipofectamine reagent (Invitrogen). After 24 h of transfection, cells were harvested, and total RNAs were isolated from the cells with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Then 5-10 µg of the total RNAs were reverse-transcribed using the SuperScript III first-strand synthesis system (Invitrogen) according to the manufacturer's protocol.
Separation of Each ING4 Variant by PCR-Restriction Fragment Length Polymorphism —The transcribed artificial cDNAs from the series of minigenes were amplified using a forward primer at a vector-specific region (T7 promoter) between the transcriptional starting site and exon 3 of ING4 (5'-TAATACGACTCACTATAGGG-3') and a reverse primer at exon 5 of ING4 (5'-ACTTCTTCTGGGCAGTCTTG-3'). A nested PCR was performed on the first PCR using a forward primer at exon 4 (5'-ACAAACACATTCGGCGGCTG-3'), combined with a reverse primer at exon 5 (5'-ACTTCTTCTGGGCAGTCTTG-3'). One µl of the gel-purified first PCR product was used as a nested PCR template. The nested PCR product was digested by HaeIII (New England Biolabs, Beverly, MA), separated by 9% polyacrylamide gel (AccuGel 19:1, National Diagnostics, Atlanta, GA), and stained with SYBR Gold (Molecular Probes, Eugene, OR).
Real Time PCR Analysis—Total ING4 was detected using variant common primers (5'-CCGGACTCAAAAGGAGAA-3' and 5'-GAGGGGCATCCCATACTCAGGA-3') and a variant common probe (5'-FAM-TCGGATGAAGAAGCCCCCAAGACTG-TAMRA-3'). For specific detection of ING4_v1, a variant-specific forward primer (5'-CCAGCAAAGGCAAAAAGAAAGG-3'), a variant common reverse primer (5'-GAGGGGCATCCCATACTCAGGA-3'), and a variant common probe (5'-FAM-TCGGATGAAGAAGCCCCCAAGACTGTAMRA-3') were designed. For specific detection of ING4_v2,a variant common forward primer an ING4_v2-specific reverse primer (5'-TTCTCCTTTTGAGTCCGGCTCT-3'), and an ING4_v1-and ING4_v2-specific probe (5'-FAM-ATGACAGCTCTTCCAGCAAAGGCAAAA-TAMRA-3') were designed. For specific detection of ING4_v3 and ING4_v4, variant-specific probes (ING4_v3: 5'-FAM-TCCAGCAAAGAAGGCCGGACTCA-TAMRA-3' and ING4_v4:5'-FAM-TCTTCCAGCAAAGGCCGGACTCA-TAMRA-3'), a variant common forward primer (5'-AGGAGAAACAGATTGAGTCAAGTGACTATG-3'), and a variant common reverse primer (5'-TTTGGAACGAGCACGAGCAG-3') were designed. The variant-specific PCR products were continuously measured by means of an ABI PRISM 7700 sequence detection system (Applied Biosystems) during 40 cycles. The annealing temperature was modified for each variant-specific amplification (ING4_v1 and ING4_v3, 64 °C; ING4_v2 and ING4_v4, 66 °C). 
2-Microglobulin RNA was used for normalization using pre-developed TaqMan assay reagent (4310886E; Applied Biosystems). The annealing temperature for the amplification of total ING4 and B2M was 60 °C.
Western Blotting—Plasmid constructs were transfected into cells using Lipofectamine reagent (Invitrogen). After 24 h of transfection, cells were washed with phosphate-buffered saline, harvested in Nonidet P-40 lysis buffer (150 mM NaCl, 1% Nonidet P-40, and 50 mM Tris-HCl, pH 8.0) with Proteinase Mixture Set III (Calbiochem), and sonicated. The extracted proteins were separated by SDS-PAGE. The FLAG-tagged proteins were detected by anti-FLAG M2 antibody (Sigma).
Fractionation of Proteins—Cells were transfected with different plasmids that express FLAG-tagged ING4_v1, ING4_v2, and ING4_v4. 24 h after transfection, the cellular proteins were fractionated by the nuclear/cytosol fractionation kit (BioVision Research Products, Mountain View, CA) and the same amount of proteins from nuclear and cytoplasmic fractions was applied into a gel. Immunoblotting was performed by a standard protocol. FLAG-tagged proteins were detected by anti-FLAG antibody (Sigma). Fractionation was evaluated by a nuclear marker, anti-Lamin A/C antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Indirect Immunofluorescence Assay—Cells were cultured on coverslips in 6-well plates and transfected with different untagged ING4 plasmid constructs using Lipofectamine Reagent (Invitrogen). After 24 h, cells were fixed, permeabilized, and incubated with anti-ING4 antibody (Rockland, Gilbertsville, PA), followed by a secondary antibody (fluorescein isothiocyanate-conjugated donkey anti-goat IgG from Jackson ImmunoResearch, West Grove, PA). Then the slides were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing 1.5 µg/ml 4,6-diamidino-2-phenylindole. The fluorescent images were obtained using a fluorescent microscope (Axioplan 2 imaging; Carl Zeiss, Thornwood, NY).
Colony Formation Assay—Three cancer cell lines, RKO, U-118 MG, and U-2 OS were used for the experiments. Cells were plated on 10-cm dishes (5 x 105 cells/dish), cultured for 12 h at 37 °C, and then transfected with 5 µg of pcDNA3.1/Hygro (5.6 kb) as a control, and 5.7 µg of pcDNA3.1/Hygro-ING4_v1 (6.35 kb), pcDNA3.1/Hygro-ING4_v2 (6.35 kb), and pcDNA3.1/Hygro-ING4_v4 (6.35 kb). Cells were cultured in the selection medium (hygromycin B; Invitrogen) as follows: 100 µg/ml for U-118 MG, 200 µg/ml for U-2 OS, and 300 µg/ml for RKO. After a 2-week selection, cells were fixed on the plates with 4% formaldehyde (Sigma) and stained with 0.1% crystal violet (Sigma). The area of the colonies (pixels) in each dish was calculated by Photoshop CS (Adobe, San Jose, CA). The data are shown as the average and standard deviation of three independent experiments. Statistical analysis was carried out by both Scheffé's F test and Student's t test.
Reporter Gene Assay—Cells were plated into 12-well plates and transfected with 0.1 µg of pEYFP-Nuc that encodes the enhanced yellow-green variant of the Aequorea victoria green fluorescent protein gene (BD Biosciences) and with 0.5 µg of WWW-Luc-p21 using Lipofectamine 2000 (Invitrogen) and with the indicated amount of pcDNA3.1/Hygro empty vector, pcDNA3.1/Hygro-ING4_v1, pcDNA3.1/Hygro-ING4_v2, or pcDNA3.1/Hygro-ING4_v4. The cells were lysed in cell culture lysis reagent (Promega, Madison, WI), and the lysates were collected to measure the p21WAF1 promoter activity 48 h after transfection. The transcriptional efficiency was examined by detecting a signal from enhanced yellow fluorescent protein (the fluorescence excitation maximum is 513 nm and the peak of the emission spectrum is 527 nm), and the promoter activity (the peak of the emission spectrum of luciferase is 562 nm) was examined by the Bright-Glo luciferase assay system (Promega). The activities of enhanced yellow fluorescent protein and luciferase were quantified with an FLX800 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT).
Cell Spreading Assay—RKO cells transiently transfected with ING4 expression plasmids were plated on glass coverslips in medium depleted of serum. Following overnight adherence at 37 °C, the starved cells were stimulated with 5% fetal bovine serum to observe the cellular response in membrane spreading. At designated time points, cells were fixed and stained on F-actin by rhodamine-phalloidin. Quantitative data of cell spreading, the percent of the cells that have filopodia and/or lamellipodia (% S), were derived from the equation % S = (S/T) x 100%, where S is the number of cells containing filo/lamellipodia, and T is the total number of cells counted.
Modified Boyden Chamber Migration Assay—RKO cells transiently transfected with ING4 expression plasmids were subjected to a transmembrane cell migration assay containing 8 µM pore size polystyrene membranes coated with FluoroBlok materials in the migration chamber (BD Biosciences). By following the manufacturer's protocol, the transverse cells were stained with calcein AM (Molecular Probes, Eugene, OR) in the lower chamber, and the magnitude of activated fluorescence was read by fluorescence spectrometry (Victor II; PerkinElmer Life Sciences).
Immunoprecipitation—Cells were transfected with different plasmid vectors that express FLAG-tagged or untagged ING4_v1, ING4_v2, or ING4_v4, and a plasmid that expresses FLAG-tagged G3BP2a that was a generous gift from Dr. Derek Kennedy (University of Queensland, Australia). 24 h after transfection, the cells were lysed in Nonidet P-40-based lysis buffer (0.1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, and protease inhibitor mixture; Calbiochem). Immunoprecipitation was performed using FLAG M2 resin (Sigma). Each precipitate was washed, and proteins were eluted in sample buffer. Immunoblotting was performed by standard protocols. FLAG-tagged ING4_v1, ING4_v2, ING4_v4, and G3BP2a were detected by rabbit anti-FLAG antibody (Sigma). Liprin 1 was detected by chicken anti-liprin 1 antibody (Genway, San Diego, CA). Untagged ING4_v1, ING4_v2, and ING4_v4 were detected by anti-ING4 antibody (Rockland, Gilbertsville, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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10% and less than 10%, respectively. Because ING4_v4 was reported previously as a 12-bp common deletion mutation (6), we examined the genomic DNA sequence of the ING4 gene in H82 that was reported to have a mutation (6), and did not detect any mutations or polymorphisms (data not shown). These variations occurred only at the cDNA level, suggesting they were presumably splicing variants. Determination of Alternative Usage of Splice Donor and Acceptor Sites for the Production of the Splice Variants by Minigene Experiment—To prove our assumption, we analyzed the ING4 gene sequence and identified two potential alternative splice donor sites, D1 and D2, at the end of exon 4 and two potential alternative splice acceptor sites, A1 and A2, at the start of exon 5 (Fig. 1A). The consensus sequences for the splice donor and acceptor sites are MAGGTRAGT and YYYYYYYY-NCAGR, respectively. D1 (AAGGCAAAA) matched 67% with the consensus sequence, whereas D2 (AAG-A-GTGAGG) matched 89% with the consensus sequence but includes an extra "A" base between MAG and GTRAGT. A1 (CCTCTTCCCTAGA) matched 92% with the consensus sequence, whereas A2 (CTTCCCTAGAAGG) matched 85% with the consensus sequence. It was predicted that ING4_v1 might be a spliced product from D1 to A1, and ING4_v2, ING4_v3, and ING4_v4 might be the spliced products from D1 to A2, D2 to A1, and D2 to A2, respectively.
To prove usage of the potential splice sites, a "minigene" approach was utilized (Fig. 1B). The plasmid contains a partial ING4 gene from exon 3 to exon 6, including the introns, a TATA box, a transcriptional starting site, and a T7 promoter just before ING4 exon 3, and a poly(A) signal just after ING4 exon 6. The expected artificial pre-mRNA should undergo alternative RNA splicing with cellular splicing machinery. Along with the wild-type minigene, we also constructed five mutant minigenes with a destroyed D1, D2, or A2 site or a combination of D1A2 and D2A2 site mutations to examine the usage of each splice site.
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Subcellular Localization of the ING4 Variants—So far, several putative motifs, a coiled-coil domain, two NLSs, an endoplasmic reticulum membrane retention signal, and a PHD finger motif were predicted by computer searches (supplemental Fig. S3A, PSORT II Prediction and SMART). The variations of the three novel ING4 variants were located at just one of the predicted NLSs (NLS1). Because two NLSs were predicted by the computer search (PSORT II Prediction), we examined whether the predicted NLSs were functional. One study reported that NLS1 is a functional NLS (4). We hypothesized that NLS2 might also function as an NLS. We constructed FLAG-tagged NLS1, NLS2, and NLS1 + NLS2 deletion mutants (FLAG-
NLS1, FLAG-NLS2, and FLAG-NLS1 + 2; supplemental Fig. S3B). The expression of the mutant proteins was first determined with the anti-FLAG M2 antibody by Western blotting (supplemental Fig. S3C), and their subcellular localization was then examined by indirect immunofluorescence using the same antibody. The deletion of the NLS2 region did not affect nuclear localization of ING4, although NLS2 might alter the distribution of ING4 in the nucleus (supplemental Fig. S3D). These results indicate that NLS1 is the only ING4 NLS and suggest that sequence variations at the NLS1 region because of alternative RNA splicing could affect the localization of ING4 variants.
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) and ING4_v4 (129KKKG132 ). Although the two variants were expressed equally well by Western blotting assays (Fig. 2A), they, especially ING4_v4, were localized primarily in the cytoplasm with a lesser amount in the nucleus (Fig. 2, B-D). This observation is consistent with a reported study that ING4 contained NLS deletion (ING4NLS) exhibited primarily in the cytoplasm (4). These results suggest a different role for the variants in cells through different localization. Attenuated Growth Suppression of ING4 Variants—The overexpression of ING4 variants was conducted using two p53 wild-type cancer cell lines, U-2 OS and RKO, and a p53-mutated cell line, U-118 MG. We confirmed equal expression levels of all of the splice variants by Western blotting (data not shown). Cell growth was suppressed by ING4_v1 effectively in all cell lines we examined, but the variants, ING4_v2 and ING4_v4, that have a partial NLS were less effective (Fig. 3, A and B). We did not observe a significant p53 dependence. The ING4 variant effects on the p21WAF1 promoter were further examined by a reporter assay. Fig. 4A shows that ING4_v1 overexpression could activate the p21WAF1 promoter (1.5-fold when compared with the vector control), but both ING4_v2 and ING4_v4 were weaker inducers (1.2- and 0.8-fold when compared with the vector control, respectively). Equal expression levels of all of the splice variants were confirmed by Western blotting (data not shown). In addition, we found ING4_v4 could block ING4_v1-mediated p21WAF1 induction (Fig. 4B), suggesting a dominant-negative effect of ING4_v4 on ING4_v1.
ING4 Splice Variants Lose the Capacity to Suppress Cell Spreading and Migration—Because we recently found that ING4_v1 interacts with several cytoplasmic proteins that might associate with cell migration and spreading under some conditions,4 we examined the activity of the two major splice variants (ING4_v2 and ING4_v4) in cell spreading and cell migration assays in comparison with ING4_v1. We found that in the cell spreading assay, both ING4 variants (ING4_v2 and ING4_v4) lost the capacity to suppress actin filament polymerization and the consequent membrane spreading (Fig. 5, A-C). In the modified Boyden chamber assay, we found that ING4_v4 completely lost the capacity to suppress cell migration, whereas ING4_v2 retained the capacity (Fig. 6A). These results regarding cell migration were further confirmed by the scratch assay (supplemental Fig. S4). We confirmed equal expression levels of all of the splice variants by Western blotting (data not shown). In light of the loss-of-function characteristic of ING4_v4, which suppressed neither cell spreading nor cell migration, we performed a competition assay between ING4_v1 and ING4_v4. ING4_v4 inhibited the filopodia/lamellipodia formation by ING4_v1 (Fig. 5D) and also completely abrogated the migration suppression activity conducted by ING4_v1 (Fig. 6B), suggesting a dominant-negative regulation of ING4_v4 on ING4_v1.
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1 and G3BP2a—To explore the underlying mechanisms of ING4 variants in cell spreading and cell migration, we focused on two ING4_v1-binding proteins that we have recently determined.4 Liprin 1 may play a role in the regulation of focal adhesion disassembly (13, 14). G3BP2 is a member of Ras-GAP Src homology 3 domain binding protein (G3BP). Evidence to date suggests that G3BPs may modulate cell proliferation through Ras signaling, NF-B signaling, and the ubiquitin proteasome system (15-17). ING4_v1 and ING4_v2 interacted with Liprin 1, but ING4_v4 did not (Fig. 7A). Only ING4_v1 had a binding affinity to G3BP2a (Fig. 7B). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this study, in addition to the original ING4 (ING4_v1) (1), we describe three novel splice variants of ING4, ING4_v2, v3, and v4, that are produced from two alternative splice donor sites at the end of exon 4 and two alternative acceptor sites at the start of exon 5. Although ING4_v4 was reported to be a common deletion-type mutation (6), we proved that the deletion was one of the four splice variants of ING4 by the minigene analysis. Interestingly, although ING5 has a similar sequence with ING4, no ESTs corresponding to ING4 splice variants have been enrolled, probably because small sequence differences between ING4 and ING5 in the region of exon 4 and exon 5 make the ING5 missing the splicing signals seen in the ING4 (supplemental Fig. S5A). These ING4 variants were expressed ubiquitously in all tissues and cell lines examined. All variants were expressed at high levels in the same tissues and cell lines, suggesting that the four splice variants are generated from the same pre-mRNA at the same ratio by cellular splicing machinery. In addition, several stimulations failed to change the composition of the variants. Serum starvation induced expression levels of all splice variants, on the other hand, serum stimulation suppressed expression levels of all splice variants in WI-38 and MRC5 normal fibroblast cells (supplemental Fig. S6A). There was no significant difference between the variants, although this observation might be very important for understanding the mechanism of cell cycle regulation by ING4. Treatment of several cell lines with DNA-damaging reagents, adriamycin and etoposide, also failed to differentially induce type-specific expression of the variants, although both adriamycin and etoposide induced expression levels of all splice variants examined in a particular cell line, U-118 MG cells, at a similar ratio (supplemental Fig. S6B). Considering the presence of putative ESE-like motifs (26) in exons 4 and 5 of the ING4 gene, we overexpressed SF2/ASF, one of the ESE-binding proteins (27-29), in cells to examine whether the protein might induce type-specific expression of the variants, and we did not observe the specific induction of any variants (data not shown). Although the exact mechanism that controls the modulation of each ING4 variant in mammalian cells remains to be understood, we found homologues of ING4_v1,-v2, and -v4 in the EST data base of mouse, rat, and pig and a homologue of ING4_v3 in the EST data base of pig (supplemental Fig. S5B). This conservation across species suggests their significance in biological functions.
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, and Wnt-1, regulate p21WAF1 transcription. ING4 could activate one of these p53-independent pathways as well as the p53-dependent pathway under some conditions. It was reported that ING4_v1 acetylates histone H4 (9), and its PHD finger motif interacts with methylated histone H3 (10). In addition, several binding partners of ING4_v1 in nucleus have been determined (9). Therefore, the nuclear ING4_v1 may have multiple functions.
Recently, we found that ING4_v1 interacts with several cytoplasmic proteins, including Liprin 1 and G3BP2 by protein pulldown and subsequent mass spectrometry analysis,4 suggesting important roles of cytoplasmic ING4_v1 similar to ING2 that have been reported to change subcellular localization dynamically between the cytoplasm and the nucleus under some conditions through its PHD finger motif that has a high identity with that of ING4 (32). Liprin 1 is a cytoplasmic protein necessary for focal adhesion and intracellular vesicle transport (13, 14). G3BP2 has been implicated in Ras signaling, NF-B signaling, and the ubiquitin proteosome pathway (15-17). In addition, G3BP2 is overexpressed in human breast cancer tissue (33). Because we are interested in the function of Liprin 1 in focal adhesion and the association between the Ras signaling pathway and G3BP2, we are investigating the functions of ING4_v1 in cell migration and cell spreading. Because G3BP2 retains IB/NF-B complexes in the cytoplasm, ING4_v1 might also be retained in the cytoplasm by G3BP2 that is overexpressed in cancer (33).
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ING4 has also been shown to inhibit angiogenesis though an interaction with NF-B (2) and to regulate HIF through an interaction with HPH-2/PHD2, a family member of the prolyl hydroxylases (7). NF-B shuttles between the cytoplasm and nucleus dynamically (34), and HPH-2/PHD2 is expressed both in the cytoplasm and the nucleus (7). Our report suggests that there are many chances for spatial interaction between ING4 and their partners both in the cytoplasm and nucleus. The balance among the four splice variants of ING4 and their subcellular localization may modulate the recently reported functions in controlling the cell cycle checkpoint, cell contact inhibition, and angiogenesis.
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FOOTNOTES |
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* This work was supported in part by Japan Society for the Promotion of Science Postdoctoral Fellowships for Research Abroad 2004-2006 and the Intramural Research Program of the Center for Cancer Research, NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S7.
1 Both authors should be considered as first authors.
2 To whom correspondence should be addressed: Laboratory of Human Carcinogenesis, CCR, NCI, National Institutes of Health, 37 Convent Dr., Bldg. 37, Rm. 3068, Bethesda, MD 20892-4258. Tel.: 301-496-2048; Fax: 301-496-0497; E-mail: Curtis_Harris{at}nih.gov.
3 The abbreviations used are: HIF, hypoxia-inducible factor; NLS, nuclear localization signal; Liprin 1, LAR-interacting protein 1; G3BP2, Ras-GTPase activating protein SH3 domain-binding protein 2; PHD, plant homeodomain; NF-B, nuclear factor of B; HPH-2, HIF prolyl hydroxylase 2; ESE, exonic splicing enhancer.
4 J.-C. Shen, M. Unoki, D. Ythier, A. Duperray, K. Kumamoto, L. Varticovski, R. Pedeux, and C. C. Harris, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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