hypoxia regulates alternative splicing of hif and non-hif ......chromatin, gene, and rna regulation...

Chromatin, Gene, and RNA Regulation

Hypoxia Regulates Alternative Splicing of HIF and non-HIFTarget Genes

Johnny A. Sena1, Liyi Wang2, Lynn E. Heasley2, and Cheng-Jun Hu1,2

AbstractHypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-

inducible transcription factor 1a (HIF1a) and 2a (HIF2a/EPAS1) to activate gene transcription, which promotestumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code forfunctionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression aswell as proper RNA splicing of theseHIF-dependent transcripts. However, it is unclear if and how hypoxia regulatesRNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF andnon-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, butreduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found tobe necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenasekinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increaseexon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by othertranscription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing.

Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes.Mol Cancer Res; 12(9); 1233–43. �2014 AACR.

IntroductionHypoxia is a common characteristic of many solid tumors.

The hypoxicmicroenvironment stabilizes hypoxia-inducibletranscription factor 1a (HIF1a) and 2a (HIF2a) that arenormally degraded under normoxia. The stabilized HIF1aand HIF2a proteins translocate to the nucleus, where theydimerize with aryl hydrocarbon receptor nuclear translocator(ARNT) to form HIF1a–ARNT (HIF1) and HIF2a–ARNT (HIF2) complexes. HIF1 and HIF2 activate genetranscription to promote tumor progression and metastasis(1–4). Thus, HIF-mediated transcriptional response is veryimportant in cancer biology.Recently, it was found that 95% of human genes are

alternatively spliced and RNA isoforms produced from asingle gene code proteins with distinct or opposing functions(5). Thus, it becomes increasingly clear that assessing iso-

forms expression will be more informative than quantifyingtotal transcripts to appreciate the functional consequence ofgene regulation.Exon arrays contain probe sets interrogating every exon

of the transcriptome, thereby permitting the distinguish-ing of different isoforms of a gene as well as measuringgene expression levels. So far, two studies have examinedthe effect of hypoxia in regulating RNA splicing usingexon arrays (6, 7). However, both studies used endotheliacells, a normal cell type. So far, hypoxia-mediated RNAsplicing changes have not been measured in cancer cells. Inaddition, these previous studies in endothelial cellsfocused on non-HIF target genes and reported that hyp-oxia, like other stresses, inhibits RNA splicing of non-HIFtarget genes by promoting exon skipping and/or introninclusion (6, 7). Thus, role of hypoxia in regulating RNAsplicing of HIF-induced genes, the most important genesin hypoxia response is still unknown. Furthermore, noneof these studies have addressed the molecular mechanismsabout how hypoxia regulates alternative splicing (AS) ofHIF target genes. In this study, we used exon arrays toassess global effects of hypoxia on RNA splicing of HIFand non-HIF target genes in Hep3B cells, a cancer cellline. In addition, we studied if HIF or hypoxia per se isimportant for RNA splicing of several HIF target genes.These findings have important implications for our under-standing of how HIFs promote tumorigenesis in responseto hypoxia.

Authors' Affiliations: 1Molecular Biology Graduate Program and 2Depart-ment of Craniofacial Biology, School of Dental Medicine, University ofColorado Anschutz Medical Campus, Aurora, Colorado

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Cheng-Jun Hu, Department of Craniofacial Biol-ogy, School of Dental Medicine, University of Colorado Anschutz MedicalCampus, Aurora, CO 80045. Phone: 303-724-4574; Fax: 303-724-4580;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0149

�2014 American Association for Cancer Research.

MolecularCancer

Research

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on August 18, 2021. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst May 21, 2014; DOI: 10.1158/1541-7786.MCR-14-0149

http://mcr.aacrjournals.org/

Materials and MethodsCell cultureHep3B cells were cultured in Minimum Essential Medi-

um with Earle's Balanced Salt Solution (Hyclone) contain-ing 10% FBS, 2 mmol/L L-glutamine, 1 mmol/L sodiumpyruvate, 100,000 U/L penicillin/streptomycin, 1.5 g/Lsodium bicarbonate, and 1� nonessential amino acids(NEAA). HEK293T, RCC4, RCC4T, and UM-SCC-22B cells were grown in high-glucose Dulbecco's ModifiedEagle Medium (DMEM; Hyclone) with 10% FBS, 2mmol/L L-glutamine, 100,000U/L penicillin/streptomycin,and 1�NEAA. SK-N-MC cells were grown in RPMI-1640(Hyclone) with 10% FBS, 2 mmol/L L-glutamine, 100,000U/L penicillin/streptomycin, and 1� NEAA. Before hyp-oxia treatment, 25 mmol/L HEPES was added to growthmedia and cells were incubated under normoxia (21%O2) orhypoxia (1.5%O2) for 12 to 16 hours. All parental cell lineswere purchased from ATCC. After completing the experi-ments, the parental (Hep3B,HEK293T, RCC4, UM-SCC-22B, and SKNMC) and modified cell lines (RCC4T) wereauthenticated by DNA profiling or "fingerprinting" by theUniversity of Colorado DNA Sequencing & Analysis Core.

Exon array analysis of ASThe genome-wide effect of hypoxia on ASwas determined

in Hep3B cells using the Affymetrix GeneChip HumanExon 1.0 ST array. Hep3B cells were cultured, as describedabove, under normoxic (21%) or hypoxic (1.2%) conditionsfor 12 hours. RNAwas prepared using aQiagen RNeasy PlusRNA extraction kit. RNA was reverse transcribed andassessed for hypoxic induction of HIF target genes usingqPCR. RNAs that passed quality control were submitted tothe Gene Expression Core at the University of ColoradoComprehensive Cancer Center for exon array analysis.Three independent replicate samples were analyzed for eachtreatment. The Core performed target labeling, hybridiza-tion, and chip scanning. To assess AS events, raw CEL fileswere analyzed using the freely available AltAnalyze software(http://www.altanalyze.org; ref. 8) with the followingsettings: species, human; gene and exon set, both core; andgene and exon level normalization FIRMA. The array datacan be accessed at GEO (GSE57613). Raw CEL files werealternatively analyzed using easyExon (http://microarray.ym.edu.tw:8080/tools/module/easyexon/index.jsp?mod-e¼home), with default settings and using RMA filtering (9).In addition, AltAnalyze software was used for gene expres-sion analysis. Genes predicted to undergo AS by AltAnalyzeand easyExon were then compared with genes that wereinduced by hypoxia (AltAnalyze) using Venny (http://bioinfogp.cnb.csic.es/tools/venny/) to identify genes thatwere predicted to undergo AS and hypoxia induction.

Functional clustering of hypoxia-inducible genes andalternatively spliced genes using DAVID bioinformaticsresourcesDAVID Bioinformatics (10) was used to create function-

ally annotated clusters of (i) genes that were hypoxia

induced, (ii) genes that were predicted to undergo hypox-ia-induced AS, and (iii) genes that were hypoxia induced andexhibiting AS.

Knockdown of endogenous mRNA using smallinterfering RNAsControl (Qiagen; 1027281) or siRNAs specific for human

ARNT (Qiagen; equal mix of SI00304220, SI00304234,and SI03020913), HIF1a (Qiagen; SI02664053), andHIF2a (Qiagen; SI00380212) mRNAs were transfectedinto Hep3B cells at 40% confluency using HiPerFectTransfection Reagent (Qiagen) according to the manufac-turer's protocol. Thirty-two hours after transfection, cellswere cultured at 21% or 1.5% O2 for 12 to 16 hours andthen collected for mRNA or protein purification.

Viral transduction of Hep3B cellsThe GFP-Flag, HIF1aTM-Flag (triple mutation to make

HIFa protein active under normoxia, with Flag tag), andHIF2aTM-Flag lentiviral constructs were described (11).The GFP-Flag, HIF1aTM-Flag, or HIF2aTM-Flag plas-mid was cotransfected with psPAX2 packaging vector(Addgene), and pMD2.G envelope vector (Addgene) intoHEK293T cells. Twenty-four hours after transfection,transfection media were replaced with complete Hep3Bmedia. The following day, viral media were filtered toremove possible 293T cells and viral particles were concen-trated using high-speed ultracentrifugation at 26,000�g for3 hours. The viral pellet was resuspended in 1mL of PBS andadded to 6 cm Hep3B cells (30% confluency) for 6 hours.The following day, a second round of concentrated virus wasadded to the same Hep3B cells. Twenty-four hours after thesecond viral transduction, Hep3B cells were harvested forRNA and protein analysis.

Plasmid constructsThe CA9P/ADM, PAI1P/ADM, and PAI1PmHRE/

ADM constructs were described previously (11). TheCA9P/PDK1, PAI1P/PDK1, and PAI1PmHRE/PDK1constructs were generated by replacing the ADM gene withPDK1 minigene that was PCR amplified from humangenomic DNA and contained exon 3 to exon 5, includingintrons 3 and 4. The HIF1aTM-Flag, HIF2aTM-Flag,HIF1aDBD-Flag, HIF1aDBD/VP16-Flag, HIF1aDBD/E2F-Flag, and upstream stimulatory factor 2 (USF2) expres-sion plasmids were described previously (11–13). The G5/PDK1 minigene was generated by replacing the ADM genewith PDK1 minigene in the G5/ADM construct that wasdescribed previously (11). The Gal4 DNA binding domain(Gal4), Gal4/HIF1aTAD, Gal4/VP16, and Gal4/E2F1have been described in ref. (11).

PDK1 splicing reporter assayTypically, HEK293T cells were grown to approximately

30% confluency in 6-well plates and cotransfected with200 ng of splicing reporters (CA9P/PDK1, PAI1P/PDK1,PAI1PmHRE/PDK1, or G5/PDK1) and 1.8 mg of His(empty vector control), HIF1aTM, HIF2aTM, USF2,

Sena et al.

Mol Cancer Res; 12(9) September 2014 Molecular Cancer Research1234




HIF1aDBD, HIF1aDBD/E2F1, or HIF1DBD/VP16expression constructs or Gal4 expression construct forG5/PDK1. Forty-eight hours after transfection, cells werecollected for RNA or protein preparation. Results were theaverage of at least three experiments.

Protein analysisWhole-cell lysates were prepared and quantified for pro-

tein concentration. Western blot analysis was performedusing standard protocols with the following primary anti-bodies: anti-CA9 (H-120) pAb (SC-25599; Santa CruzBiotechnology), anti-ANGPTL4 (H-200) pAb (SC-66806; Santa Cruz Biotechnology), anti-Flag mAb(F3165; Sigma), anti-Gal4DBD pAb (SC-577; Santa CruzBiotechnology), or anti-beta Actin pAb (SC-1616; SantaCruz Biotechnology).

RNA preparation and reverse transcription PCR orquantitative PCRRNA was isolated from cells using the RNeasy Plus mini

kit (Qiagen), which removes DNA, then was reverse tran-scribed using the iSCRIPT Advanced cDNA synthesis kit(Bio-Rad) containing both oligo-dT and randomhexamer asprimers because using both random hexamers and oligo-dTallows efficient reverse transcription of the whole mRNAtranscript as well as 18 rRNA. Alternative splicing analysiswas then validated by RT-PCR using forward and reverseprimers flanking AS events predicted by both AltAnalyze andeasyExon software. Genes that expressedmultiple transcriptswere TopoTA cloned and submitted to theGene ExpressionCore at the University of Colorado Cancer Center forsequencing. qRT-PCR using iQ SYBR Green Supermix(Bio-Rad) in triplicate on the CFX384 Real-Time System(Bio-Rad) was used to quantify the levels of RNA isoforms.All primer sets for qRT-PCR designed to measure mRNAlevels were validated for their specificity and amplificationefficiency (85%–110%) using melt curve analysis, qRT-PCR product sequencing, and standard dilution analysis.qRT-PCR results were normalized using the PfafflDDCTmethod using18s rRNA and b-actin as reference genesbecause their expression are not affected by hypoxia anduntreated normoxia samples, GFP lentivirus, or emptyvectors (His) as controls. The RNA ratio of splice variantswas calculated by dividing the expression level of the full-length (FL) isoform by the expression level of the DEisoform. The expression level for each isoform was deter-mined by the following equation: Expression level ¼efficiencytarget

DCt target (control � sample)/efficiencyreferenceDCt

reference (control � sample), where the efficiency ¼ 10�1/slope

(slope generated by standard curve analysis). At least threeindependent experiments were performed to generatethe results presented in the Figures. All the primers usedfor RT-PCR and qRT-PCR were included in Supplemen-tary File S3.

Statistical analysisOne-way analysis of variance was performed unless oth-

erwise stated. Error bars in figures indicate standard devi-

ation. Asterisks indicate statistical significance as follows:�, P < 0.05; ��, P < 0.01. Controls for statistical analysisare specified in each figure. All experiments were per-formed at least three separate times.

ResultsGenome-wide exon array analysis determines thathypoxia alters RNA splicing of HIF and non-HIF targetgenes in Hep3B cellsTo assess the effects of hypoxia on RNA splicing, Affy-

metrix GeneChip Human Exon 1.0 S T arrays were used toprobe RNAs isolated from normoxic or hypoxic Hep3Bcells. Hep3B cells were chosen because of their high expres-sion levels of HIFa proteins and high hypoxic induction ofHIF target genes (12–14). AltAnalyze (FIRMA) and easy-Exon software predicted that hypoxia regulated AS of about2,005 (Fig. 1A, yellow and gray) and 3,919 genes, respec-tively (Supplementary File S1; AS by Firma or AS by easy-Exon). Venn diagrams using Venny indicated that 1,012genes were predicated to undergo AS by both programs,whereas an additional 957 and 2,634 genes were predicatedto undergo AS by FIRMA and easyExon, respectively.FIRMA was used to further characterize the hypoxia-medi-ated splicing events, as FIRMA predictions of AS are morestringent than easyExon. FIRMA analysis indicated thathypoxia produced 3,059 AS events in 2,005 genes, averaging1.53 splicing events per gene (Fig. 1A, all genes). Asexpected, alternative cassette-exons were the most abundanttype of splicing events, making up 51% of all splicing events.Other AS events included alternative 50 splice-sites (16%),alternative 30 splice-sites (14%), intron retention (11%),mutually exclusive exon (4%), and others (4%).In addition, AltAnalyze determined that hypoxia induced

2,439 genes, for at least 1.4 fold, when compared withnormoxic Hep3B controls (Fig. 1A, blue and gray; also seeSupplementary File S1, gene expression). Venny analysis ofhypoxia-inducible genes and AS genes indicated that 134genes were hypoxia induced and alternatively spliced andthese 134 genes were called Hx AS genes (Fig. 1A, in gray).Interestingly, these 134 Hx AS genes had 238 splicingevents, averaging 1.78 AS events per gene (Fig. 1A, Hx-induced genes), suggesting that AS is enriched in hypoxia-induced genes, compared with the total population of genesthat underwent AS (1.53 AS events per gene). Similar to thetotal population of AS genes, alternative cassette-exons werethe most abundant type of splicing events (62%) in Hx ASgenes. Other AS events for these Hx AS genes includedalternative 50 splice sites (16%), alternative 30 splice sites(13%), intron retention (9%), and mutually exclusive exons(0%). However, Hx AS genes exhibited 1.2-fold enrichmentin alternative cassette-exon inclusion and a 1.22-fold reduc-tion in intron retention in comparison with the total pop-ulation of AS genes.Because cassette-exon AS is the most abundant splicing

event, hypoxia-mediated cassette-exon regulation was fur-ther analyzed. In the total population of cassette-exon genes(all cassette, Fig. 1B), 44% of cassette-exon genes exhibitedexon inclusion (454 out of 1,022 genes), whereas 56% of the

Hypoxia Regulates Alternative Splicing

www.aacrjournals.org Mol Cancer Res; 12(9) September 2014 1235




genes exhibited exon skipping, suggesting that hypoxiagenerally promotes exon skipping in Hep3B cells. However,76% of cassette-exon Hx AS genes underwent exon inclu-sion (63 out of 83 genes), whereas 24% of genes exhibitedexon skipping, suggesting that hypoxia promotes exoninclusion of hypoxia-inducible genes (Fig. 1B, Hx-inducedAS). In contrast, hypoxia promoted exon skipping of 75% ofhypoxia-reduced cassette-exon genes (Hx reduced, Fig. 1B),whereas the number of exon inclusion to exon skippingevents was equal (50%) for genes whose expression was notchanged by hypoxia (no change, Fig. 1B).

Functional clustering of hypoxia-mediated AS genesusingDAVID reveals newpathways regulated byhypoxiaTo determine whether hypoxia-induced AS was enriched

in specific cellular pathways, DAVID (10) was used to createfunctional annotation clusters of genes that were hypoxia

induced (Fig. 1A, left column), the entire population of ASgenes (Fig. 1A, right column), and theHx AS genes (Fig. 1A,top; see Supplementary File S2 for the gene lists). Asexpected, hypoxia induced the expression of genes in path-ways allowing cancer cells to adapt to a hypoxic microen-vironment such as vascular development, glycolysis, oxido-reductase activity, and cell adhesion/cadherins (Fig. 1A, leftcolumn). On the other hand, when analyzing all AS genes,pathways involved in oxidoreductase activity, glycolysis, andpositive regulation of glucose transport that were enriched ingene expression were also enriched in AS (Fig. 1A, rightcolumn and Supplementary File S2). However, for the samepathway, the genes involved in AS could be different fromthe genes enriched in expression analysis, demonstrating theimportance of these pathways in the hypoxia response.Importantly, several novel cellular pathways, includingATP-binding/protein kinase activity, pleckstrin homology

Hexose metabolic process Glycolysis

Oxidoreductase activity

L-ascorbic acid binding

Membrane- bounded vesicle

Bloodvessel morpho-genesis Gluconeo

-genesis

Leucine-ZipperTranscriptionfactor Cell death

Basement membrane

ATP-binding/protein kinase activity


Rho protein signal transduction

Zinc finger

Glycolysis

Positive regulation of glucose transport

Cell death

Nuclear lumen

ES: 7.1P = 2.35x10−8

ES: 5.6P = 1.45x10−8

ES: 2.5P = 0.001

ES: 2.2P = 4.99x10−4

ES: 1.5P = 0.021

ES: 1.5P = 0.023

ES: 1.4P = 0.015

ES: 1.3P = 0.036

ES: 1.2P = 0.047

ES: 1.2P = 0.01

ES: 6.1P = 1.53x10−8

Pleckstrin homologyES: 4.7P = 5.8x10−6

ES: 3.4P = 3.81x10−4

ES: 3.8P = 3.41x10−4

ES: 3.4P = 9.78x10−5

Cytoskeleton organization

ES: 3.3P = 2.36x10−4

ES: 3.2P = 1.31x10−4

ES: 3.1P = 5.36x10−4

ES: 2.8P = 4.55x10−4

ES: 2.7P = 5.92x10−4

Vascular development

Glycolysis

L-ascorbic acid binding

Gluclose metabolism

Leucine-zippertranscription factor

G-protein coupled receptor

Gluconeogenesis

Transmembrane


Celladhesion/cadherins

ES: 6.1P = 5.08x10 −7

ES: 5.7P = 4.08x10 −8

ES: 5.4P = 7.82x10 −7

ES: 3.7P = 4.21x10 −5

ES: 3.2P = 2.22x10 −4

ES: 3.2P = 1.22x10 −4

ES: 3.1P = 1.83x10 −4

ES: 3.0P = 1.88x10 −4

ES: 2.9P = 0.0011

ES: 2.8P=0.003

44% 52%

76%

25%

56%48%

24%

75%

0%10%20%30%40%50%60%70%80%90%

100%

All cassetteAS

No changeAS

Hx-inducedAS

Hx-reducedAS

Exon skippingExon inclusion

568

454

63

20

275

92299

273

All Genes Hx Induced GenesTotal Events 3,059 238Total Genes 2,005 134Events/Gene 1.53 1.78

A

B

2,305 134 1,871

Figure 1. Hypoxia regulates gene expression and RNA splicing in Hep3B cells. A, Venn diagrams of genes that are hypoxia induced (blue and gray), genes thatare alternatively spliced under hypoxia (yellow and gray), and genes whose expression are induced by hypoxia and alternatively spliced under hypoxia (gray).Left column, top 10 enrichment pathways whose genes are induced at least 1.4 fold by hypoxia. Top row, top 10 enrichment pathways whose genes that arehypoxia induced and undergo hypoxia-induced AS. Right column, top 10 enrichment pathwayswhose genes undergo hypoxia-induced AS. Numbers next toeach box represent the DAVID enrichment score (ES), higher score representing more enriched, followed by the Fisher exact P value, smaller scorerepresenting more enriched. P values equal to or less than 0.05 are considered strongly enriched. Boxes of the same color, excluding white, representoverlapping or functionally related gene pathways. White boxes are unique pathways for each group. Additional statistics and gene list associated with eachgroup can be found in Supplementary File S2. Below the Venn diagram in (Fig. 1A are the splicing events per genes for all alternatively spliced genes (all genes,left) and hypoxia-inducedgenes (Hx-induced genes, right). B, summary of exon skipping or inclusion events in all cassette ASgenes, geneswhose expressionare not changed during hypoxia (no change AS), genes induced by hypoxia (Hx-induced AS), and genes reduced by hypoxia (Hx-reduced AS).

Sena et al.





(related to rho protein signal transduction and cytoskeletonorganization), rho protein signal transduction, cytoskeletonorganization, zinc fingers, cell death, and nuclear lumengenes, were also enriched (Fig. 1A, right column andSupplementary File S2). Correspondingly, Hx AS geneswere largely overlapped with pathways identified in geneexpression and/or AS (Fig. 1A, top).

RT-PCR and qRT-PCR validation of AS for select HIFtarget genes identified in exon array analysisNext, we sought to validate the exon array data using RT-

PCR and qRT-PCR. We focused on Hx AS genes as thehypoxia-inducible genes are the most important genes in thehypoxia response. First, we selected Hx AS genes as deter-mined by both AltAnalyze FIRMA and easyExon. Next, weused easyExon to identify regions of potential AS. Then weused RT-PCR to identify the RNA isoforms and qRT-PCRto quantify the levels of these isoforms in normoxic andhypoxic Hep3B cells or other cancer cells. For example,carbonic anhydrase 9 (CA9), a well-known HIF target gene(15, 16), was predicted to undergo AS in exon 2 and in aregion spanning exons 6 to 10 as determined by the splicingindex (Fig. 2A). AS was considered significant if the splicingindex was greater than þ1.5 or less than �1.5 (negative SIindicates exon inclusion by hypoxia) using easyExon. RT-PCR was not able to detect an AS event in exon 2. RT-PCR,using primers that amplify exons 4 to 10 (Fig. 2B), followedby Topo TA cloning and sequencing confirmed that Hep3Bcells expressed two CA9 isoforms, a full-length isoform (FL),and an isoform in which exons 8 and 9 were skipped(DE89; Fig. 2C, left). qRT-PCR determined that hypoxiainduced the expression of both isoforms but significantlyfavored FL in Hep3B cells; thus, hypoxia increased the FL/DE89 ratio by 9.3 fold (Fig. 2D). Similar CA9 isoformexpression patterns and increased CA9 FL/DE89 ratio byhypoxia were observed in a neuroblastoma cell line,SKNMC, suggesting that this splicing event is not cell-typespecific (Fig. 2E and F). In addition,Western blot analysis ofCA9 in Hep3B cells confirmed that both FL and DE89protein isoforms were expressed and FL CA9 protein waspreferentially induced by hypoxia (Fig. 2C, right). The largerCA9 FL band in hypoxic Hep3B cells was likely the result ofposttranslational modifications and was also observed pre-viously (17).Angiopoietin-like 4 (ANGPTL4) was predicted to under-

go AS in exon 4 (Supplementary Fig. S1A). RT-PCR, TopoTA cloning, and sequencing confirmed that ANGPTL4expressed a FL isoform, and an exon 4 skipping isoform(DE4) in Hep3B cells (Supplementary Fig. S1C, left). qRT-PCRdetermined that hypoxia induced the expression of bothisoforms but FL was favored; thus, hypoxia increased the FL/DE4 ratio by 4.9 fold (Supplementary Fig. S1D). In addition,hypoxia increased the ANGPTL4FL/DE4 ratio by 3.5 fold inUM-SCC-22B, a head and neck squamous cell carcinomacell line (Supplementary Fig. S1E and S1F). The expressionof ANGPTL4 FL and DE4 protein isoforms and preferentialinduction of ANGPTL4 FL protein by hypoxia were alsoobserved in Hep3B cells (Supplementary Fig. S1C, right).

Expression of multiple isoforms and hypoxia-regulatingRNA splicing of additional HIF target genes, pyruvatedehydrogenase kinase 1 (PDK1, FL, and DE4), WNK lysinedeficient protein kinase 1 (WNK1, FL, and DE11–12), andprolyl 4-hydroxylase alpha polypeptide II (P4HA2, FL, andDE2), were also confirmed by RT-PCR and qRT-PCR(Supplementary Fig. S2A–C).CA9, ANGPTL4, PDK1, WNK1, and P4HA2 exhibited

preferential induction of the FL isoform. However, AltA-nalyze (FIRMA) predicts that hypoxia promotes exon skip-ping of someHIF target genes, including procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2, FL, and DE14)and enolase 2 (ENO2, FL, and DE8), which are alsoconfirmed (Supplementary Fig. S2D and S2E). These datademonstrated that hypoxia promotes exon inclusion formost HIF target genes; however, hypoxia promotes exonskipping for some HIF target genes such as PLOD2 andENO2.

Hypoxia changes isoform ratios ofHIF target genes at thetranscriptional levelAfter confirming AS of several HIF target genes, we

addressed the molecular mechanism on how hypoxia reg-ulates AS of HIF target genes. Although AS was proposed toexplain the observed FL to skipping isoform ratio changes inthe above sections, the hypoxia-induced isoform ratio varia-tions could be caused by RNA splicing or othermechanisms.For instance, preferential degradation of exon skipping iso-forms under hypoxia might explain the hypoxia-inducedisoform ratio changes as exon skipping RNA isoforms mayincur premature termination codons and such transcripts areoften targeted for nonsense-mediated decay (NMD; ref. 18).In the seven genes analyzed, only RNA of CA9DE89 (E8–9,172 bp), but not ANGPTL4 (E4, 114 bp), PDK1 (E4,138 bp), WNK1 (E11–12, 609 bp), P4HA2 (E2, 136 bp in50-UTR), PLOD2 (E13, 114 bp), and ENO2 (E8, 198 bp)had premature stop codon. To test the possibility thatpreferential instability of CA9 DE89 contributes to theincreased CA9 FL/DE89 ratio under hypoxia, Hep3B cellswere placed under hypoxia for 16 hours to increase the levelsof bothCA9FL andDE89 transcripts, followed by treatmentwith actinomycin D to inhibit de novo transcription. Cellswere then placed back under normoxia or hypoxia for 0, 2, 4,or 8 hours to allow RNA decay. Using qRT-PCR, both CA9FL and DE89 transcripts were found to be very stable, as90% of the FL andDE89 transcripts were detected even after8 hours. Moreover, CA9 FL and DE89 transcripts exhibitedsimilar stability under normoxia and hypoxia at every timepoint (data not shown). ANGPTL4 FL and DE4 transcriptswere far less stable than CA9 transcripts as only 40%, 28%,and 20% of the transcripts remained after 2, 4, and 8 hours.However, ANGPTL4 FL and DE4 transcripts also exhibitedsimilar stability under normoxia and hypoxia (data notshown). Furthermore, actinomycin D treatment in Hep3Bcells blocked hypoxic induction of HIF target genes andblocked splicing change of HIF target genes, indicating thatthe hypoxia-mediated isoform shift required active transcrip-tion. These data supported the idea that transcription






regulation, not posttranscriptional regulation is responsiblefor the hypoxia-induced increased CA9 and ANGPTL4 FL/exon-skipping ratio.

HIF activity, not hypoxia per se, is necessary to change ASof HIF target genesTo test whether hypoxic stress or HIF activity is respon-

sible for the splicing changes of HIF target genes, ARNT,HIF1a, or HIF2amRNA levels were reduced by 80%usingsiRNAs in normoxic or hypoxic Hep3B cells (data notshown). ARNT and HIF1a, but not HIF2a knockdowndramatically reduced the hypoxic induction of CA9,ANGPTL4, and PDK1, consistent with the idea that CA9,ANGPTL4, and PDK1 are primarily regulated by HIF1 inHep3B cells (Fig. 3A). qRT-PCR confirmed that ARNT andHIF1a knockdown significantly reduced the levels of both

FL and exon skipping isoforms of CA9, ANGPTL4, andPDK1 and prevented the splicing changes of these genes(Fig. 3B–D). In contrast, HIF2a knockdown only mildlyreduced hypoxic induction of CA9FL (1.44 fold) andPDK1DE4 (1.6 fold), similarly reduced hypoxic inductionof ANGPTL4FL and DE4. Thus, HIF2a knockdown onlyreduced the CA9FL/DE89 ratio by 1.33 fold (Fig. 3B),maintaining the ANGPTL4FL/DE4 ratio (Fig. 3C), butenhanced the PDK1FL/DE4 ratio 1.5 fold (Fig. 3D). Knock-down of ARNT and HIF1a also inhibited hypoxic induc-tion of the FL and exon skipping isoforms of WNK1,PLOD2, ENO2, and P4HA2 in Hep3B cells and preventedsplicing ratio changes for these genes (data not shown). Thesedata suggested that HIF activity, but not hypoxia per se isnecessary for increased gene expression as well as hypoxia-mediated splicing changes of these HIF target genes.

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Figure 2. Hypoxia regulates AS of the HIF target gene, CA9. A, easyExon analysis of differentially expressed probes for the CA9 gene in Hep3B cells. Lines,probe intensities for normoxia or hypoxic samples. Differentially expressed probes were considered positive for AS if the splicing index was greaterthan 1.5 or less than�1.5 (negative SI indicates exon inclusion is increased by hypoxia). Vertical numbers below the splicing index indicate Affymetrix probeIDs. B, diagram of the confirmed CA9 gene isoforms. Gray boxes, constitutive exons; black boxes, alternatively spliced exons. Solid arrows, primerused for RT-PCR (E4//E10) and qRT-PCR (E7/E10/7 for CA9DE89, andE7/E8/7 for CA9 FL). C, RT-PCR andWestern blot analysis of CA9mRNAandprotein innormoxic and hypoxic Hep3B cells. CA9 FL proteins exhibit two bands with different sizes. D, qRT-PCR analysis of CA9FL and DE89 transcripts innormoxic and hypoxic Hep3B cells. E, RT-PCR analysis of CA9 mRNAs in normoxic and hypoxic SKNMC cells. F, qRT-PCR analysis of CA9FL andDE89 transcripts in normoxic and hypoxic SKNMC cells. Numbers in D and F here and other qRT-PCR panels represent the ratio of the FL isoformversus the exon-skipping isoform � the standard deviation. Fold of ratio change is also indicated. One-way analysis of variance was performed for this andother studies in this article unless otherwise stated. �, P ¼ 0.05; ��, P ¼ 0.01. Controls for statistical analysis are specified in each Figure.

Sena et al.





HIF activity is sufficient to regulate AS of HIF targetgenesNext, we wanted to determine whether HIF activity is

sufficient for hypoxia-regulated AS of HIF target genes. Totest this, normoxic Hep3B cells were transduced withlentiviruses expressing normoxia active, flag-tagged, HIF1a,or HIF2a, or GFP as a negative control (Fig. 4A). HIF1aand HIF2a transduction induced the expression of CA9,ANGPTL4, and PDK1 as determined by RT-PCR (Fig. 4B).More importantly, qRT-PCR determined that HIF1a andHIF2a increased both FL and exon skipping isoforms ofCA9 (Fig. 4C), ANGPTL4 (Fig. 4D), and PDK1 (Fig. 4E).However, FL transcripts of CA9, ANGPTL4, and PDK1were preferentially induced (Fig. 4C–E). Thus, HIF1 orHIF2 increased the FL/exon skipping ratio for CA9 by 14.5or 6.0 fold (Fig. 4C), ANGPTL4 by 3.41 or 5.7 fold(Fig. 4D), and PDK1 by 1.43 or �1.2 fold (Fig. 4E).To further validate these results, the RNA splicing of CA9,

ANGPTL4, and PDK1 were examined in RCC4 cells, arenal cell carcinoma cell line that expresses constitutivelyactive HIF1a and HIF2a proteins due to loss of functionalpVHL (15, 19). In addition, the RNA splicing of the abovethree genes were also assessed in RCC4T cells in whichfunctional pVHL is reintroduced into RCC4 cells and,therefore, HIF proteins are only active under hypoxia(15, 19). RT-PCR and qRT-PCR analysis determined thathypoxia increased the levels of CA9FL and DE89 by 41.8and 7 fold, respectively, in RCC4T cells, increasing the FL/DE89 ratio by 6.2 fold (Supplementary Fig. S3B). Incontrast, hypoxia did not induce the levels of CA9FL andDE89 isoforms nor did hypoxia enhance the FL/DE89 ratioinRCC4 cells (Supplementary Fig. S3B).However, theCA9

FL/DE89 ratio in RCC4 cells was already high even undernormoxia (Supplementary Fig. S3B). Similar findings wereobserved for AS of ANGPTL4; however, hypoxia was stillable to enhance the ANGPTL4 FL/DE4 ratio in RCC4(Supplementary Fig. S3C). Although hypoxia was able toincrease the expression of the PDK1FL and DE4 isoformsand to enhance the PDK1FL/DE4 ratio in RCC4T cells,PDK1 FL/DE4 ratio was not elevated in normoxic orhypoxic RCC4 cells (Supplementary Fig. S3D).Taken together, these data suggested that HIF activity is

necessary and sufficient to regulate AS of a subset of HIFtarget genes independent of hypoxia.

PDK1 splicing reporters recapitulate splicing changesobserved for the endogenous PDK1 gene when activatedby HIF under normoxiaNext, we wanted to see if similar splicing changes could be

observed in splicing reporter gene. We selected the PDK1gene for splicing reporter model as both the FL and DE4isoforms were highly expressed in 293T cells (data notshown). Exons 3 to 5, including introns 3 and 4 of thePDK1 gene, were PCR amplified from human genomicDNA and placed downstream of the CA9 promoter, a HIF1target gene promoter, or the PAI1 promoter, a HIF2 targetgene promoter (Fig. 5A). RT-PCR using minigene-specificprimers determined that the PDK1minigene expressed bothFL and DE4 isoforms in 293T andHep3B cells (Fig. 5B). Inaddition, the PDK1DE4 isoform was the dominantlyexpressed isoform in both 293T and Hep3B cells (Fig.5B). Furthermore, PDK FL isoforms were also highlyexpressed in 293T cells (Fig. 5B). These data demonstratedthat the PDK1 splicing reporter recapitulated the expression

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Figure 3. HIF activity, but nothypoxia per se is necessary topromote AS of HIF target genes.A, RT-PCR analysis of CA9,ANGPTL4, and PDK1 RNAtranscripts in normoxic or hypoxicHep3B cells targeted with control(Ctrl), ARNT, HIF1a, or HIF2asiRNAs. B to D, qRT-PCR analysisof FL and exon-skipping isoformsof CA9 (B), ANGPTL4 (C), andPDK1 (D) in the normoxic orhypoxic Hep3B cells targeted withcontrol or siRNA to ARNT, HIF1a,or HIF2a.






patterns of the endogenous PDK1 gene in 293T andHep3Bcells (Supplementary Fig. S2A and data not shown).To assess AS of the PDK splicing reporter in response to

HIF activation, 293T cells were transfected with the PDK1minigene and normoxia active HIF1a expression plasmids.HIF1a induced the levels of total PDK1 minigene tran-scripts, FL, and DE4 isoform by 3.65, 24.9, and 10.6 fold,respectively, and enhanced the FL/DE4 ratio by 2.34 fold(Fig. 5D). Next, similar experiments were performed usingthe PAI1P/PDK1 splicing reporter (Fig. 5A). Normoxiaactive HIF2a induced the expression of PDK1FL and DE4isoforms by 4.22 and 2.36 fold, respectively, thus enhancingthe FL/DE4 ratio 2.08 fold (Fig. 5E). These results indicatedthat the PDK1 splicing reporters recapitulate splicingchanges observed for the endogenous PDK1 gene whenactivated by HIF under normoxia.Some transcription factors have dual roles in RNA splicing

and gene transcription by recruiting splicing factors andtranscription cofactors using their transactivation domains(20–22). To determine whether the activation domain ofHIF1a protein is important for PDK1 pre-mRNA splicing,fusion constructs containing the HIF1a DNA bindingdomain fused to the transactivation domains from the VP16or E2F1 transcription factors were used to activate CA9P/PDK1 splicing reporter (Fig. 5C). HIF1aDBD/VP16induced the expression of PDK1FL (12.2 fold) andPDK1DE4 (6.4 fold), and enhanced the FL/DE4 ratio by1.9 fold (Fig. 5D). Similarly, HIF1aDBD/E2F1 inducedPDK1FL (21.7 fold) and PDK1DE4 (9.4 fold), andincreased FL/DE4 expression ratio by 2.3 fold (Fig. 5D).

These data indicated that HIF transactivation domain is notrequired for AS of the PDK1 minigene.In summary, these data indicated that HIF1 or HIF2-

mediated activation of the PDK1 minigene is sufficient toincrease PDK1 FL/DE4 ratio.

Activation of endogenousHIF target genes contributes toincreased FL/DE4 ratio of PDK1 splicing reporterAs stated above, transcription activation of PDK1 by HIF

or HIFDBD hybrid constructs is sufficient to increase thePDK1 minigene FL/DE4 ratio. However, HIF and thefusion constructs can activate endogenous HIF target genes;therefore, it is possible that activation of endogenous HIFtarget genes may increase FL/DE4 ratio. To rule out orconfirm this possibility, we used USF2 and the PAI1P/PDK1 minigene as USF2 can activate the PAI1 promoter(13), but not endogenous HIF target genes. USF2 inducedthe expression of the PDK1FL and DE4 isoforms by 2.95and 2.96 fold, respectively, but was not able to enhance theFL/DE4 ratio even though USF2 was able to activate theminigene to a similar extent as HIF2a (USF2 ¼ 1.97 andHIF2a ¼ 1.52-fold induction of total transcripts; Fig. 5E).To test if activation of endogenous HIF target gene couldregulate AS of the PDK1minigene, HIF-binding site on thePAI1 promoter was mutated to produce the PAI1PmHRE/PDK1 minigene (Fig. 5F). Interestingly, although HIF2awas not able to activate the expression of PAI1PmHRE/PDK1 as assessed by total as well as FL transcripts, HIF2aincreased the FL/DE4 ratio by reducing the levels of thePDK1 DE4 transcript (Fig. 5F).

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Figure 4. HIF activity is sufficient topromote AS of HIF target genes. A,Western blot analysis of Flag-tagged proteins in normoxicHep3B cells transduced withlentiviruses expressing GFP, orFlag-tagged, normoxia-activeHIF1a or HIF2a proteins. B, RT-PCR analysis of CA9, ANGPTL4,and PDK1 transcripts in normoxicHep3B cells transduced with GFP,or normoxia-active HIF1a, orHIF2a proteins. C to E, qRT-PCRanalysis of FL and exon-skippingRNA isoforms of CA9 (C),ANGPTL4 (D), and PDK1 (E) in theabove described cells.

Sena et al.





To further validate that transcription activation of endog-enous HIF target genes increases the PDK1 FL/DE4 ratio,the PDK1 splicing reporter was placed under the control of apromoter containing five copies of the Gal4 DNA bindingelement (Supplementary Fig. S4A, 5xUAS). Fusion con-structs containing the Gal4 DNA binding domain fused tothe transactivation domains of normoxia active HIF1a,VP16, or E2F1 were used to activate the G5P/PDK1minigene in the presence or absence of normoxia-activeHIF1a and HIF2a that activate endogenous HIF targetgenes (Supplementary Fig. S4B). Interestingly, althoughGal4/HIF1aTAD, Gal4/VP16TAD, and Gal4/E2F1TADincreased the PDK1 FL/DE4 ratio by 1.27, 2.44, and 1.47fold, activation of endogenous HIF target genes furtherincreased the PDK1 FL/DE4 ratio to 2.04, 3.28, and

3.29 fold. Importantly, cotransfected HIF1a and HIF2aincreased the expression of endogenous HIF target genes,including CA9 and LOX (data not shown), but not theexpression of G5/PDK1 (Supplementary Fig. S4C, sameamount of PDK1 total transcripts with or without HIF).These data demonstrated that activation of endogenous HIFtarget genes contributes to AS of the PDK1 minigene.

DiscussionMost analyses of hypoxia-mediated gene expression

changes are conducted at the gene level using traditionalmicroarrays (15, 23–26). However, due to significant func-tional difference of the proteins encoded by RNA isoformsand the fact that a majority of genes express multiple RNAisoforms (5), gene expression analysis by exon array or RNA

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Figure 5. Transcription activation is not sufficient to regulate splicing of a PDK1 minigene. A, schematic of a PDK1 splicing reporter driven by CA9 or PAI1promoter. Arrows, primers used for RT-PCR (E3/vector R) and qRT-PCR (E3/5, E4/5, or total with Vector R for DE4, FL, or total transcript respectively). B, RT-PCR analysis of PDK1 transcripts expressed from CA9P/PDK1 in 293T and Hep3B cells. C, Western blot analysis of Flag-tagged HIF1a, HIF1aDBD/VP16TAD, and HIF1aDBD/E2F1TAD proteins in 293 T cells for experiments whose results are presented in Fig. 5D. HIF1aDBD/E2F1TAD constructproduces two proteins. D, qRT-PCR analysis of minigene-specific PDK1 isoforms in 293 T cells cotransfected with CA9P/PDK1 minigene and HIF1a orHIF1aDBD expression constructs. E, qRT-PCR analysis of minigene-specific PDK1 isoforms in 293 T cells cotransfected with PAI1P/PDK1 minigene andHIF2a or USF2 expression constructs. F, qRT-PCR analysis of minigene-specific PDK1 isoforms in 293 T cells cotransfected with PAI1PmHRE/PDK1minigene and HIF2a or USF2 expression constructs.






deep sequencing is necessary to fully understand geneexpression programs. Our exon array analysis in normoxicand hypoxic Hep3B cells not only confirms hypoxic induc-tion of previously identified hypoxia-inducible genes, butalso reveals significant roles of hypoxia in regulating RNAsplicing of genes whose transcription are induced by hypoxiaor genes whose transcription are not changed or reduced byhypoxia.Here, we first reported novel hypoxia-regulated genes/

pathways that cannot be identified using traditional micro-arrays as expression levels of these genes are typically notchanged by hypoxia or reduced by hypoxia. In addition, wefound that hypoxia primarily promotes exon skipping of thesenon-HIF target genes in Hep3B cells, a result consistent withwhat is reported in endothelial cells (6, 7). We proposed thatincreased exon skipping of these non-HIF target genes alsohave functional consequences. For example, genes involved inATP binding and protein kinase activity are not hypoxiainduced, but exhibit AS. Increased exon skipping of thesegenes in hypoxicHep3B cells, presumably acts to reduce ATPusage tomaintain cellular ATP levels. AS of some of these nonhypoxia-induced genes are not associated with enriched path-ways identified by DAVID Bioinformatics; however, theirsplicing are also altered and expected to be functionallyimportant. For example, the KRAS 4B isoform (exon 4a isskipped inKRAS 4B, but included inKRAS 4A) is induced inhypoxic Hep3B, thus reducing the KRAS 4A/4B ratio.Interestingly, a decreased KRAS 4A/4B ratio is often observedin human colorectal cancer, but not in normal colon (27, 28).Furthermore, decreased KRAS 4A/4B ratios through reduc-tion ofKARS4A expression or increasedKRAS4B expressionpromotes DMH-induced colonic tumorigenesis in in vivomouse models independent of KRAS mutations (29), dem-onstrating the functional consequence of reduced KRAS 4A/4B ratio in tumor biology.More importantly, we reported here, for the first time the

role of hypoxia in regulating RNA splicing of hypoxia-inducible genes. We found that hypoxia promotes exoninclusion for 75% of Hx AS genes. Although no functionaldifferences have been examined for the isoforms ofmostHIFtarget genes, FL CA9 protein was reported to be a mem-brane-associated, tumor-promoting protein by regulatingintercellular pH via transporting protons out of the cellsduring hypoxia. In contrast, the CA9DE89 protein is asoluble protein and is not able to transport protons out ofthe cells (17). Thus, it makes sense that CA9 FL is prefer-entially induced by hypoxia. In addition, preferential induc-tion of nerve growth factor receptor TrkA DE679 isoform,but not FL leads to nerve growth factor–independent recep-tor activity and neuroblastoma tumor-promoting activity(30). Again, specific promotion of Cysteine-Rich protein 61FL, but not intron-3 retaining isoform (producing nofunctional protein) promotes tumor angiogenesis (31).While isoform ratio changes of several HIF target genes

have been reported under hypoxia (17, 30–34), none ofthese studies have addressed the molecular mechanismsabout how hypoxia regulates isoform ratio changes of HIFtarget genes. We demonstrated in this study that the

increased FL/exon-skipping ratio of severalHIF target genes,including CA9 and ANGPTL4, is the result of AS, notpreferential degradation of exon-skipping isoforms. More-over, we determined thatHIF activity, but not hypoxic stressper se is necessary and sufficient to regulate AS of CA9,PDK1, and ANGPTL4 pre-mRNAs. Furthermore, wefound that activation of endogenous HIF target genescontributes to AS of PDK1 minigenes.Our studies also indicated that there is an opposing effect

on HIF target genes versus non-HIF target genes for RNAsplicing in which hypoxia primarily promotes exon inclusionof HIF target genes, but inhibits exon inclusion of genesrepressed under hypoxia. This opposing effect is similarlyobserved for gene transcription and protein translation inwhich, transcription and protein translation of non-HIFtarget genes are generally reduced, whereas HIF target genesare transcriptionally induced and protein translation of genesinvolved in hypoxia response such as HIF1A, HIF2A, andVEGF are maintained due to internal ribosome entry sites(IRES). Our data indicated that exon inclusion ofHIF targetgenes is due toHIF activity, but not hypoxic stress.However,we do not know if exon skipping of hypoxia-repressed genesis dependent on HIF activity or hypoxic stress. This is a veryinteresting question that should be assessed in future studies.In summary, this study suggests that hypoxia regulates AS

of hypoxia-induced genes, hypoxia-reduced genes, and geneswhose transcription is not changed by hypoxia. In addition,HIF activity, not hypoxic stress is found to be necessary andsufficient to regulate AS of a subset of hypoxia-induciblegenes. Moreover, activation of endogenous HIF target genescontributes to AS of some HIF target genes. These findingssignificantly increase our understanding of how cells regulategene transcription as well as RNA splicing to adapt to ahypoxic microenvironment. In the future, it will be inter-esting to examine the role of hypoxia in regulating RNAsplicing in primary tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.A. Sena, L. Wang, C.-J. HuDevelopment of methodology: J.A. Sena, L. Wang, C.-J. HuAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): J.A. Sena, L. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): J.A. Sena, L. Wang, C.-J. HuWriting, review, and or revision of the manuscript: J.A. Sena, L.E. Heasley,C.-J. HuAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J.A. Sena, C.-J. HuStudy supervision: C.-J. Hu

Grant SupportThis work was supported by the National Cancer Institute (RO1CA134687,

to C.-J. Hu) and Cancer League of Colorado (to C.-J. Hu). J. A. Sena was supportedby "Research Supplemental to Promote Diversity" (RO1CA134687-S3 andRO1CA134687-S4).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received March 19, 2014; revised April 21, 2014; accepted May 6, 2014;published OnlineFirst May 21, 2014.

Sena et al.





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2014;12:1233-1243. Published OnlineFirst May 21, 2014.Mol Cancer Res Johnny A. Sena, Liyi Wang, Lynn E. Heasley, et al. GenesHypoxia Regulates Alternative Splicing of HIF and non-HIF Target

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hypoxia regulates alternative splicing of hif and non-hif ......chromatin, gene, and rna regulation...

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