RED CELLS

Repression via the GATA box is essential for tissue-specific erythropoietingene expression*Naoshi Obara,1,2 *Norio Suzuki,1,3 Kibom Kim,1-3 Toshiro Nagasawa,2 Shigehiko Imagawa,2 and Masayuki Yamamoto1-4

1Center for Tsukuba Advanced Research Alliance (TARA), 2Graduate School of Comprehensive Human Sciences (CHS), and 3Exploratory Research forAdvanced Technology (ERATO), Environmental Response Project, Japan Science and Technology Agency (JST), University of Tsukuba, Tsukuba; and4Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan

In response to anemia, erythropoietin(Epo) gene transcription is markedly in-duced in the kidney and liver. To elucidatehow Epo gene expression is regulated invivo, we established transgenic mouselines expressing green fluorescent pro-tein (GFP) under the control of a 180-kbmouse Epo gene locus. GFP expressionwas induced by anemia or hypoxia specifi-cally in peritubular interstitial cells of thekidney and hepatocytes surrounding the

central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphol-ogy and expressed neuronal markergenes. Furthermore, the regulatorymechanisms of Epo gene expression wereexplored using transgenes containing mu-tations in the GATA motif of the promoterregion. A single nucleotide mutation inthis motif resulted in constitutive ectopicexpression of transgenic GFP in renaldistal tubules, collecting ducts, and cer-

tain populations of epithelial cells in othertissues. Since both GATA-2 and GATA-3bind to the GATA box in distal tubularcells, both factors are likely to repressconstitutively ectopic Epo gene expres-sion in these cells. Thus, GATA-basedrepression is essential for the inducibleand cell type–specific expression of theEpo gene. (Blood. 2008;111:5223-5232)

© 2008 by The American Society of Hematology

Introduction

To maintain proper homeostasis during growth and develop-ment, the regulation of gene expression needs to be strictlycontrolled, especially at the transcriptional level. For instance,in response to anemia and hypoxia, erythropoietin (Epo) genetranscription is activated in the kidney and liver (reviewed inEbert and Bunn1). Epo is then secreted from these tissues tostimulate erythropoiesis in hematopoietic tissues. The kidneyproduces approximately 90% of the total Epo during theresponse to anemia,2 indicating that Epo gene expression isunder strict tissue-specific regulation. Thus, the Epo geneprovides an intriguing model for understanding gene expressionin higher eukaryotes. Furthermore, dysfunctional Epo generegulation has been shown to underlie the pathogenesis ofrenal anemia.3,4 As such, recombinant Epo has been used widelyas a powerful drug for treating patients with anemia (reviewedin Jelkmann5).

Epo gene expression in response to anemia and/or hypoxiahas been vigorously analyzed by exploiting the human hepatomacell lines Hep3B and HepG2 that produce Epo in a hypoxia-inducible manner.6 An important finding from these studies wasthat Epo gene expression is regulated by an enhancer located3� to the transcriptional termination site.7 This 3� enhancercontains a hypoxia response element (HRE) that has been shownto bind hypoxia-inducible transcription factors (HIFs).7 Abinding sequence for nuclear receptor also resides in theenhancer.1,8 Thus, these 2 cis-acting elements may controlEpo gene expression in a hypoxia-inducible manner (reviewedin Koury9).

A GATA factor–binding motif (GATA box) has been identifiedin the core promoter region of the Epo gene, where a TATA boxnormally resides.10 We have found that this GATA box activelyparticipates in Epo gene regulation. The GATA box acts as anegative regulatory element in the hepatoma cell lines.10 Duringnormoxic conditions, GATA transcription factors bind to the GATAbox and repress Epo gene transcription, but when exposed tohypoxia, GATA binding markedly decreases, with a markedincrease in Epo gene expression.10,11

Although the mechanisms underlying inducible gene expres-sion of the Epo gene are generally understood, the basis oftissue-specific Epo gene regulation is largely unknown. Constitu-tive expression, as well as ectopic transgene expression, has beenfrequently detected in several transgenic mouse reporter studiesusing constructs containing less than 20 kb of the Epo geneflanking region.12-15 In agreement, our own transgenic reportergene expression analysis demonstrated that an 11-kb mouse Epogene fragment, which incorporates the 2 important regulatoryelements mentioned above (the promoter GATA motif and 3�enhancer), was unable to fully recapitulate Epo gene expression(N.S. and M.Y., unpublished data, March 25, 2002). We thereforerealized that regulatory element(s) critical for Epo gene expressionmust be contained in further upstream or downstream regionssurrounding the Epo locus.

An unsolved but important issue in Epo gene regulation is theidentification of precisely which kidney cells actively produce Epo.This topic is controversial; some reports claim that proximaltubular cells produce Epo,14,16 whereas others present evidence that

Submitted October 1, 2007; accepted January 14, 2008. Prepublished online asBlood First Edition paper, January 17, 2008; DOI 10.1182/blood-2007-10-115857.

*N.O. and N.S. contributed equally to this work.

An Inside Blood analysis of this article appears at the front of this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2008 by The American Society of Hematology

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the Epo-producing cell population might be glomerular,17 mesangi-al,18 or interstitial cells of the renal cortex.13,19-23 We surmise thatthese discrepancies are most likely due to a low sensitivity in thedetection of Epo.

To elucidate in this study how inducible and tissue-specifictranscription of the Epo gene is attained, we established bacterialartificial chromosome (BAC) transgenic mouse lines expressinggreen fluorescent protein (GFP) as a reporter under the control of a180-kb mouse Epo gene locus.24 This 180-kb region can suffi-ciently recapitulate inducible transgene expression in a tissue-specific manner, including expression in the kidney and liver. TheGFP labeling clearly identified the renal Epo-producing (REP)population as interstitial cells. Surprisingly, we discovered thatREP cells have a neuronlike shape and express marker antigensfound in neuronal cells. Furthermore, we also found that a singlenucleotide mutation in the promoter GATA box can cause constitu-tive ectopic expression of GFP in the renal distal tubules, collectingducts, and certain epithelial cells of other tissues. Thus, GATAbox–based repression is essential for inducible and cell type–specific Epo gene expression.

Methods

Bacterial artificial chromosome transgenes

A bacterial artifical chromosome (BAC) clone (Epo-60K/BAC, RP27826)containing the Epo gene and flanking regions was obtained from aC57black/6 mouse genomic library (Incyte Genomics, San Diego, CA). Tomake the wt-Epo-GFP construct, GFP cDNA was integrated into Epo-60K/BAC in Escherichia coli EL250 as described previously.25,26 For construc-tion of the mutant Epo-GFP transgenes (m1-Epo-GFP and m2-Epo-GFP),the polymerase chain reaction (PCR)–based mutant 5� arms of the targetingvectors were used.11,27 Recombinant BAC clones were identified throughsequencing, restriction enzyme analysis, and Southern blotting. BACtransgenic constructs were purified from a bacterial cell suspension culturedin LB/chloramphenicol (25 �g/mL) for 24 hours at 32°C using a Nucleo-bond BAC DNA preparation kit (Macherey-Nagel, Duren, Germany). Thepurified DNA was linearized by digestion with �-terminase (EpicentreTechnologies, Omaha, NE) and purified by pulse-field gel electrophoresis.28

Mice

Purified linear transgenes (5 ng/�L) were microinjected into the pronucleiof fertilized eggs from BDF1 parents. Transgenic mice were screened byPCR genotyping of tail DNA using the following 3 pairs of primers. BAC1F(5�-GTGCGGGCCTCTTCGCTATT-3�) with BAC1R (5�-CAGGTC-GACTCTAGAGGATC-3�) and BAC2F (5�-AGTGTCACCTAAAT-AGCTTG-3�) with BAC2AR (5�-CAGTACTGCGATGAGTGGCA-3�) wereused to amplify the BAC vector sequences at the 3�and 5� inserts,respectively. The primer set GFPs4 (5�-CTGAAGTTCATCTGCACCACC-3�) and GFPas4 (5�-GAAGTTGTACTCCAGCTTGTGC-3�) was used toamplify the GFP cDNA.29 Southern blotting analyses were carried out todetermine the transgene copy numbers. Genomic tail DNA was preparedand digested with ApaI. Southern membranes were hybridized with a32P-labeled probe. The copy numbers of the transgenes were determined bythe intensities of the bands. To induce profound anemia (Hct: � .15[ 15%]), 6- to 8-week-old mice were bled from the retro-orbital plexus(0.3-0.4 mL) at 48, 36, 24, 12, and 6 hours before analysis. To inducemoderate anemia (Hct: .20-.35 [20%-35%]), the amount of bleeding wasreduced to 0.2 mL. The plasma Epo concentrations were measured using aphotometric enzyme-linked immunoabsorbent assay (Epo ELISA kit;Roche, Indianapolis, IN). The Gata2-GFP30 and Gata3-LacZ knock-in31

lines of mice have been described previously. All mice were kept strictly inspecific pathogen-free conditions, and were treated according to theregulations of The Standards for Human Care and Use of LaboratoryAnimals of the University of Tsukuba.32

Immunostaining

Tissues were fixed in 4% paraformaldehyde for 30 minutes and embeddedin OCT compound (Sakura-Finetechnical, Tokyo, Japan). Sections 10 �min thickness were incubated with rabbit anti-GFP polyclonal antibody(diluted 1:1000; Molecular Probes, Eugene, OR) or anti–GATA-4 antibody(diluted 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°Covernight. After treatment with hydrogen peroxide, sections were incubatedwith horseradish peroxidase–conjugated anti–rabbit IgG secondary anti-body. Color detection was performed using diaminobenzidine as a chromo-gen (brown staining). Hematoxylin was used as a counterstain. To detect theexpressions of CD31 and Mac1, frozen sections were incubated withanti-CD31 and Mac1 antibodies (BD Pharmingen, San Diego, CA)conjugated with phycoerythrin (PE) for 2 hours at room temperature. Todetect the expressions of �-fetoprotein (AFP), microtubule-associatedprotein 2 (MAP2), and neurofilament light polypeptide (NFL), sectionswere incubated with primary antibodies (Santa Cruz Biotechnology)against AFP (diluted 1:200), MAP2 (diluted 1:200), and NFL (diluted1:200), respectively, followed by incubation with TRITC-conjugatedanti–goat IgG secondary antibody (ZyMed, South San Francisco, CA).Fluorescent images were observed using the LSM510 confocal imagingsystem (Carl Zeiss, Heidelberg, Germany).

RT-PCR

Liver sections were microdissected with the Leica laser microdissection system(LMD; Leica, Heidelberg, Germany) and RNAs were purified using RNeasy(Qiagen, Hilden, Germany). Total RNAs were purified using ISOGEN (Nippon-Gene, Tokyo, Japan). Reverse transcription was performed with SenscriptRT(Qiagen). Epo mRNAlevels were measured quantitatively using theABI PRISM7700 (Perkin-Elmer, Waltham, MA) and the primer pair 5�-GAGGCAGAAAAT-GTCACGATG-3� and 5�-CTTCCACCTCCATTCTTTTCC-3� with FAM-labeled oligo-DNAprobe (5�-TGCAGAAGGTCCCAGACTGAGTGAAAATA-3�).33 Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as aninternal standard.29 Semiquantitative reverse-transcriptase (RT)–PCR analysisof Epo-GFP transgene expression was performed using the primer pair5�-AAGGATGAAGACTTGCAGCG-3� and 5�-GAAGTTGTACTCCAGCTT-GTGC-3�. Hypoxanthine guanine phosphoribosyl transferase (HPRT) wasused as an internal control using 5�-GCTGGTGAAAAGGACCTCT-3� and5�-CACAGGACTAGAACACCTGC-3� primers.29

Chromatin immunoprecipitation

Normal renal medulla (10 mg) was minced by needling and fixed with 1%formaldehyde for 10 minutes. Sample in a 200-�L aliquot was sonicated for10 seconds on ice 12 times by a 2-mm microtip sonicator (Branson, Danbury,CT). One half of the sonicated sample was used as preimmunoprecipitatedcontrol (input), while the other half was incubated with antibody againstGATA-2, GATA-3, or normal rabbit IgG (Santa Cruz Biotechnology). Antibody-bound chromatin complexes were precipitated with an Immunoprecipitation Kit(Upstate Biotechnology, Lake Placid, NY). To detect the promoter region in theprecipitated chromatin complexes, PCR was performed using the followingprimers: 5�-TCACGCACACACAGCTTCAC-3� and 5�-CACGCTGCAA-GTCTTCATCC-3� for the Epo promoter; 5�-TTATCTGGAGTCCATTAAT-GAGG-3� and 5�-CTGCTCGGCCTTCTGAGCGCTG-3� for the Aquaporin-2(Aqp2) gene promoter; and 5�-TATGGCGGGCAAGAAGTTGA-3� and5�-GTACTAGGCCAGGACTAGTG-3� for the Gata1 gene promoter.34 TheAqp2 and Gata1 promoters were used as positive and negative controls,respectively.

Results

Regulatory region for tissue-specific and inducible Epo geneexpression

To obtain a genomic DNA fragment suitable for our in vivo reporterexpression analysis, we screened several BAC clones containingthe mouse Epo gene and finally selected a 180-kb BAC clone

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(Epo-60K/BAC; Figure 1A). The region spanning exon II to intronIV of the Epo gene in the BAC clone was replaced with a GFPreporter gene through standard homologous recombination.24-26

The GFP gene was inserted in frame in such a manner that GFPexpression would mimic Epo gene expression (Figure 1A). Weconfirmed the authenticity of the recombinant BAC clone bySouthern blotting and DNA sequencing (data not shown).24 TheBAC-derived transgene (wt-Epo-GFP) was microinjected intofertilized eggs and 3 transgenic mouse lines were established.Transgene expression could not be detected under normoxicconditions, yet was unequivocally detected in the kidneys andlivers of the wt-Epo-GFP mice when bleeding-induced anemia wascarried out (Figure 1B panels). These results were quite reproduc-ible; anemia-induced expression of the GFP gene was shown in3 (lines WA, WB, and WC) of 4 lines of mice by semiquantitativereverse transcriptase–PCR (RT-PCR) analysis. This was in excel-lent agreement with the endogenous Epo gene expression detectedby quantitative RT-PCR analysis (Figure 1B bar chart). TheEpo-GFP transgene expression was detected exclusively in both

anemic liver and kidney. Therefore, we concluded that this 180-kbEpo-GFP transgene contained the regulatory regions sufficient fortissue-specific and inducible Epo gene expression in vivo.

While the expression of GFP from the transgenic BAC cloneswas far more stable than that from the plasmid transgenes in micein vivo, we also noticed a variation in the intensity of fluorescencebetween the lines. The green fluorescence intensities (data not shown)were not related to the transgene copy numbers (Figure 1C). These datasuggest that the BAC transgene might be affected to some extent by theintegration position effect. We confirmed integration of the intact BACtransgene for all the lines of transgenic mice by a series of PCR andSouthern blotting analyses (data not shown).

Renal Epo-producing cells are peritubular interstitial cellsexpressing neuronal markers

An important prerequisite for the current study was to elucidate themechanisms regulating Epo gene expression by identifying theprecise cells in the kidney that produce Epo, since earlier conclu-sions were controversial.24 To this end, we exploited normal

Figure 1. Structure and tissue distribution of theEpo-GFP transgene. (A) Strategy for constructing theEpo-GFP transgenes in the BAC recombination sys-tem. Epo-60K/BAC (top), including the 60-kb 5� up-stream and 120-kb 3� downstream regions of themouse Epo gene, was isolated. Exons I to V of the Epogene are depicted by black boxes. The targeting vectorcontained GFP cDNA (white box) and a polyadenyla-tion signal (pA; hatched box) and a Neo cassette(speckled box) between the FRT sequences (whiteovals). The vector was homologously recombined withEpo-60K/BAC within the 5� (1.1 kb) and 3� (0.9 kb)homologous arms. The Neo cassette in the targetedBAC clone was excised using the FLP-FRT system inbacteria. To verify integration of the intact transgeneinto the mouse chromosome, 5� and 3� fragmentsderived from the BAC vector (a and b) and GFP cDNA(c) were amplified by PCR using primers indicated bythe double arrowheads. (B) Expression of the endoge-nous Epo gene (bar chart) and wt-Epo-GFP transgene(panels) in adult mice. Epo mRNA levels under normal(gray bars) and anemic (black bars) conditions weremeasured by quantitative RT-PCR for the organs indi-cated and normalized to the level of GAPDH mRNA(bar chart). Data are the means (� SD) of at least3 independent mice. Semiquantitative RT-PCR analy-sis of transgene expression in the wt-Epo-GFP trans-genic mouse (line WA) under normal and anemicconditions was performed for the organs indicated(panels). HPRT was used as an internal control.(C) Southern blotting analysis of ApaI-digested genomicDNA revealed the copy numbers of the transgenes. TailDNA from each transgenic line was digested with ApaI(Ap; indicated in A) and the endogenous Epo gene(5.2 kb) and transgene (4.4 kb) were hybridized with aradiolabeled probe (indicated in A).

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wt-Epo-GFP (Figure 2A) and wild-type (Figure 2C) mice andwt-Epo-GFP mice bled to induce anemia (Figure 2B,D) and stainedfor GFP expression in the kidneys. The anemic wt-Epo-GFP micedisplayed a GFP expression that was detected reproducibly anduniquely in the peritubular interstitial cells of the border region ofrenal medulla and cortex. We referred to these cells as REP (renalEpo-producing) cells and performed further characterization usingimmunofluorescent techniques.

The REP cells displayed a unique stellar or arboroid shape withprojections extending in various directions (Figure 2D,E; VideoS1A,B, available on the Blood website; see the SupplementalMaterials link at the top of the online article). These cells werelocated between the proximal tubular cells and vascular endothelialcells and were tightly attached to the renal tubular cells (Figure2D). When we stained vascular endothelial cells with anti-CD31,REP cells did not overlap with the red staining representing CD31(Figure 2F). As the dendritelike processes of the REP cellssuggested a neuronal or macrophage origin, we examined whetherREP cells express the macrophage marker Mac1 or neural-specificmarkers such as microtubule-associated protein 2 (MAP2) andneurofilament light polypeptide (NFL). We found that REP cellsare Mac1 negative (Figure 2G), but positive for MAP2 and NFL(Figure 2H and I, respectively). We found that not all interstitialcells positive for MAP2 or NFL express GFP. In our preliminaryanalysis, 5% to 10% of those neural marker–positive cellsexpressed wt-Epo-GFP transgene when Hct was reduced to.15 (15%). Concurring with the previous finding that Epo isproduced in CD73 (ecto-5�-nucleotidase)–positive peritubular cellsin the kidney,22,23 REP cells were found to express CD73 (data notshown). While we used mainly line WA mice in these analyses, weconfirmed that the other 2 lines of wt-Epo-GFP mice showedreproducible results (data not shown). Taking advantage of the GFPlabeling, we executed 3-dimensional imaging of REP cells by

confocal laser microscopy (Video S1A,B). Each REP cell had 4 to6 projections stretching in various directions in the cavity betweenthe tubules, and several REP cells formed a cluster (Video S1B).Taken together, REP cells with long processes seem to form areticular network between renal tubules and capillaries.24

The number of Epo-producing cells correlates with the plasmaEpo concentration

Upon induction of bleeding anemia, the number of GFP-positivecells in the kidney increased exponentially, in excellent agreement

Figure 3. The number of GFP-positive cells in the kidney of wt-Epo-GFPtransgenic mouse is increased as the hematocrit is decreased. Various levels ofanemia were induced in wt-Epo-GFP transgenic mice (line WA) by bleeding over2 days and kidney sections were taken. The plasma Epo concentrations ( ) and thenumbers of GFP-positive cells in the kidney sections (�) increased as the hematocrit(Hct) decreased.

Figure 2. Morphology of renal Epo-producing (REP)cells expressing the wt-Epo-GFP transgene. Immu-nohistochemical staining was carried out with anti-GFPantibody of normal (A) and anemic (B-D) mouse kid-neys from transgenic line WA (A,B,D) and wild-type (C)mice. GFP expression (brown) was detected in theperitubular interstitial cells (arrows in B,D) with longprojections (arrowheads in D) between the proximaltubules (PT) and vessels (V). Fluorescent images ofREP cells from anemic kidneys of transgenic mice (lineWA) taken with the confocal laser scanning microscope(E-I). When costained with cell lineage markers (red),REP cells (green) were negative for CD31 (F) andMac1 (G), but positive for MAP2 (H) and NFL (I). Scalebars represent 50 �m (A-C) and 10 �m (D-I).

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with both increasing plasma Epo concentration and decreasinghematocrit (Hct) score (Figure 3). This analysis revealed that renalEpo production is regulated mainly by the number of REP cells,rather than by the expression levels of the Epo gene in individualREP cells. Indeed, there was no significant difference betweenmildly anemic and severely anemic animals in the fluorescenceintensity of individual GFP-positive cells (data not shown). SinceEpo production was also activated in mice made anemic by the cellcycle inhibitor 5-fluorouracil or �-ray irradiation (data not shown),the increase in GFP-positive cells is likely due to the onset ofEpo-producing ability, but not due to the proliferation of Epo-expressing cells. This result supports previous a hypothesis byKoury et al35 that Epo production is regulated by a small subset ofperitubular interstitial cells with on-off mode.

Epo-GFP expression in hepatocytes

Under severe anemic conditions, Epo production also occurs in the liver,so we examined GFP expression in hepatocytes. Bleeding anemia gaverise to the emergence of GFP-positive hepatocytes, which rapidlyincreased in number as the Hct score fell (Figure 4A-C). EachGFP-positive region in the liver sections was a concentric circular ringsurrounding a central vein (CV; Figure 4B,C). Since oxygen is supplied

from the interlobular artery, the oxygen supply to hepatocytes surround-ing the CV is low compared with other regions. In our microdissectionanalysis, following induction of anemia, the endogenous expression ofEpo mRNA was detected specifically in cells surrounding the CV, butnot in cells surrounding the interlobular triad (IL; Figure 4D). Theseresults thus demonstrate that Epo gene expression is induced inhepatocytes through sensing the hypoxic threshold of the cellularoxygen tensions.

We next analyzed GFP expression in livers from wt-Epo-GFPtransgenic mouse embryos. At embryonic day 13.5 (E13.5), GFPexpression was detected in hepatocytes expressing �-fetoprotein(AFP) (Figure 4E-G). In contrast to REP cells, GFP-positivehepatocytes did not stain positive for neural markers (data notshown). Hematopoietic cells in the fetal liver made direct contactwith the GFP-positive hepatocytes, suggesting that fetal livererythropoiesis is supported by paracrine Epo production from thehepatocytes. Hepatic GFP expression was found in E9.5 embryosand this expression persisted until the neonatal stage (data notshown). GFP expression was slightly enhanced by the induction ofbleeding anemia in the mother (data not shown).

When housed in a 6% oxygen chamber, GFP expression wasinduced in the livers and kidneys of adult wt-Epo-GFP transgenic

Figure 4. Induction of wt-Epo-GFP transgene ex-pression by bleeding anemia in the liver. Anti-GFPimmunohistochemistry of the livers from wt-Epo-GFPtransgenic mice (line WA) under normal (A, Hct:.45 [45%]) and anemic (B, Hct: .30 [30%]; C, Hct:.15 [15%]) conditions is shown. At the lowest Hct(C), GFP-positive cell regions (brown) had expandedaround the central vein (�), but hepatocytes surround-ing the interlobular triad (sharp) did not express GFP.(D) Relative Epo mRNA levels in the kidneys and liversfrom line WA mice under normal (n, ) and anemic(a, ) conditions were measured by quantitativeRT-PCR and normalized to the levels of GAPDHmRNA. Hepatocytes surrounding the interlobular triad(IL) and central vein (CV) under anemic conditionswere collected using laser microdissection and theexpression of Epo mRNA was examined by quantita-tive RT-PCR. Data are the means (� SD) of 3 indepen-dent mice. (E) GFP expression was also detected inE13.5 fetal liver from a wt-Epo-GFP transgenic embryo(line WA). The GFP-positive cells were fetal hepato-cytes that were also positive for �-fetoprotein (AFP; redimmunofluorescence in panel F). Panels E and F weremerged in panel G. Scale bars are 300 �m (A-C) and20 �m (E-G).

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mice, as was the case for bleeding anemia (data not shown).Throughout this analysis, we did not find GFP-expressing cellsin any tissues other than the kidney and liver, irrespective of thenormoxic or hypoxic/anemic condition. Based on these observa-tions, we concluded with the following 3 points. First, inanimals, Epo is produced in REP cells and hepatocytes throughsensing the hypoxic threshold. Second, Epo production iscontrolled by the increase in the number of Epo-expressingcells, but not through the enhancement of Epo in individualcells. Third, the wt-Epo-GFP transgene contains the regulatoryregions necessary for sufficient expression of Epo in physiolog-ically important Epo-producing tissues.

A mutation in the GATA box results in constitutive GFPexpression in epithelial cells

Our previous analyses showed that in Hep3B and HepG2 cells, GATA-2represses Epo gene expression by binding to its GATA box located30-bp 5� to the transcription start site.10,11 To ascertain whether thisrepression occurs in vivo, we examined GATA box activity using the

Epo-GFP BAC transgenic reporter system. For this purpose, wemutated the GATA box in the wt-Epo-GFP construct to TATA (Figure5A m1-Epo-GFP), to which the GATA factors cannot bind.11 As therewas the possibility that TATA-binding factor (TBP) may recognize the� 30 TATA box in the m1-Epo-GFP construct and activate Epo geneexpression in place of the GATA factors,10,36 we prepared anothermutant that is not recognizable by either GATA or TBP (TTTA mutant,m2-Epo-GFP transgene; Figure 5A).

We established 3 transgenic mouse lines each for the m1-Epo-GFP (lines 1A, 1B, and 1C) and m2-Epo-GFP (lines 2A, 2B, and2C) constructs (Figure 1D) and evaluated GFP expression underanemic conditions. To our surprise, GFP was highly expressed inthe distal tubules and collecting ducts of the m1-Epo-GFP mutanttransgenic mice, even in nonanemic conditions (Figure 5B,C,E,F).This ectopic expression was reproducible in all 3 lines of m1-Epo-GFP mutant transgenic mice (data not shown). GFP was alsoexpressed in both normal and anemic conditions in the bile ductepithelia, bronchial epithelia, and thymus epithelia of the m1-Epo-GFP mutant transgenic mice (Figure 5H,I,K,L; Table 1), however

Figure 5. Expression profiles of the Epo-GFP trans-genes with mutations in the promoter GATA se-quence. (A) Sequences near the GATA factor–bindingsite in the promoter region of the mouse Epo gene. Thewild-type GATA-box in the wt-Epo-GFP transgene wasmutated to create the transgenes m1-Epo-GFP andm2-Epo-GFP. Capital T indicates the point 30-bp up-stream from the transcription initiation site. Sections ofkidneys (B-G), livers (H-J), and lungs (K-M) fromthe m1-Epo-GFP transgenic mouse line 1A (B,C,E,F,H,I,K,L) and wt-Epo-GFP transgenic mouse line WA(D,G,J,M) under normal (B,E,H,K) or anemic (C,D,F,G,I,J,L,M) conditions were stained with anti-GFP antibody(brown). The distal tubules (d) constitutively expressedthe mutant transgene (E,F), but not wt-Epo-GFP(G). Arrows indicate the kidney interstitial cells, whichexpressed GFP only after the induction of bleedinganemia in both m1-Epo-GFP (F) and wt-Epo-GFP(G) transgenic mice. Arrowheads indicate the bileducts, which were constitutively positive for GFP in themutant Epo-GFP transgenic mice (H-I), but negative forGFP staining in the wt-Epo-GFP transgenic mice(J). Hepatocytes surrounding the central vein (�) ex-pressed GFP only in anemic conditions in both m1-Epo-GFP (I) and wt-Epo-GFP (J) transgenic mice. Thebronchial epithelium (�) was also positive for GFPantibody staining only in the mutant Epo-GFP trans-genic mice (K,L). # indicates, interlobular triad. Scalebars are 300 �m (B-D), 20 �m (E-G), and 100 �m(H-M).

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the intensity of expression varied among the lines. This ectopicexpression in nonanemic conditions was also observed in m2-Epo-GFP mutant transgenic mice and the expression did not changesubstantially among the lines (data not shown).

Upon induction of bleeding anemia in the both mutant Epo-GFPtransgenic mice, GFP expression was induced in REP cells and inhepatocytes surrounding the central vein (Figure 5F,I; Table 1), as wasthe case for wt-Epo-GFP transgenic mice (Figure 5G,J). These resultsdemonstrate that the GATA box is essential for repression of the Epogene in epithelial cells in both nonanemic and anemic/hypoxic condi-tions. It should be noted that the GATA box–based repression appears tobe abrogated for expression of the Epo gene in the authentic Epo-producing cells (ie, REP cells and hepatocytes).

GATA-2 and GATA-3 in epithelial cells bind to the GATA box

Using Gata2-GFP knock-in and Gata3-LacZ knock-in mouselines,30,31 we examined which GATA family members are ex-pressed in the epithelial cells that constitutively expressed themutant Epo-GFP transgenes. As described in the previous para-graph, constitutive ectopic expression of GFP from the mutantEpo-GFP transgenes was detected in epithelial cells of the kidneydistal tubules and collecting ducts (Figure 5E,F; Table 1). Inkidney, both Gata2 and Gata3 were expressed in the distal tubulesand collecting ducts, as seen by GFP immunostaining of Gata2-GFP knock-in mouse kidney (Figure 6A,B) and LacZ staining ofGata3-LacZ knock-in mouse kidney (Figure 6C,D). Immunohisto-chemical staining with anti–GATA-4 antibody of wild-type mousekidney showed that the distal tubules and collecting ducts werenegative for GATA-4 expression. Both Gata2-GFP and GATA-4were detected in REP cells (Figure 6B,F arrows; Table 1).

The specific binding of these GATA factors to the GATAsequence in vivo was investigated by a chromatin immunoprecipi-tation (ChIP) assay, exploiting the samples from the normal renalmedulla where most of the cells expressed the mutant Epo-GFPtransgenes and Gata2 and Gata3 genes. We found that the promoterregion of the Epo gene did indeed immunoprecipitate withanti–GATA-2 and anti–GATA-3 antibodies (Figure 6G top panel).The promoter region of the Aquaporin-2 (Aqp2) gene was alsoimmunoprecipitated. The Aqp2 promoter served as a positivecontrol, as it is known to be a target of the GATA factors in renaltubules (Figure 6G middle panel).37 Binding of GATA-2 andGATA-3 to the Epo promoter seemed to be specific, since we didnot detect the Gata1 gene promoter containing GATA sequence inthe ChIP assay (Figure 6G bottom panel).34 These results support

our contention that GATA-2 and GATA-3 constitutively repressEpo gene expression in renal tubular cells through binding to theGATA element. As for the GATA factors in the other epithelial celllineages that were positive for the mutant transgene expression, wesuggest concomitant GATA factor expression (Table 1). WhileGATA-6 expression is not examined in this study, it has beenreported that GATA-6 is expressed in the bile duct and bronchialepithelium.38-40 Although GATA-3 expression was not detected inthe thymic epithelium in this study (Table 1), expression andessential contribution of GATA-3 to the thymic epithelium has beenfound (T. Moriguchi and J.D. Engel, personal oral communication).Based on these observations, we conclude that the renal tubularcells and certain other epithelial cells have the potential to expressEpo in the absence of anemic or hypoxic stresses, and that GATAfactors constitutively inhibit Epo gene expression in these cells.

Discussion

To address the mechanisms underlying the regulation of Epo geneexpression in a tissue-specific manner and in response to anemia/hypoxia, we used a BAC-based GFP reporter transgenic mousestrategy, and the coding region of the Epo gene was replaced withGFP. GFP expression in wt-Epo-GFP BAC transgenic micerecapitulated the inducible and tissue-specific expression of theEpo gene. Importantly, our analysis identified REP cells in thekidney as the Epo-producing cells, which showed specific peritubu-lar localization, a unique arboroid shape and neuronal markerexpression. Furthermore, a novel regulatory mechanism of Epogene regulation was discovered that used the GATA box.

RT-PCR analysis demonstrated that the wt-Epo-GFP transgenefaithfully recapitulated endogenous Epo gene expression in mousetissues under both normal and anemic/hypoxic conditions. Previ-ous Epo transgenic mouse studies revealed that a 33-kb genomicfragment of the human Epo transgene gave rise to hypoxia-inducible expression of human Epo in the kidney and liver.12,13,15 Inmost of the mouse lines, the transgene expression was accompa-nied by ectopic expression and also resulted in polycythemia,indicating that the transgene used did not contain the full regulatoryregion to stably control Epo gene expression with cell type–specificmanner in vivo. On the contrary, the BAC-based wt-Epo-GFPtransgene used in our study not only reiterated the endogenous Epogene expression, but avoided the ectopic expression of Epoobserved in other tissues and cell types. We therefore concluded

Table 1. Expression of Epo-GFP transgenes and GATA factors

Tissue

Epo-GFP transgene GATA factors

Wild type GATA mutant Gata2 Gata3 GATA-4

N A N A N A N A N A

Kidney

Peritubular interstitial � � � �

Distal/collecting tubules � � � �

Liver

Hepatocyte � � � � � �

Bile duct epithelium � � � � � � � � � �

Lung: bronchial epithelium � � � � � � � � � �

Thymus: thymic medullary epithelium � � � � � � � � � �

The GFP expression of wild-type (wt-Epo-GFP) and GATA mutant (m1-Epo-GFP and m2-Epo-GFP) transgenic mice was examined under normal (N) and anemic (A)conditions. Gata2 and Gata3 expression was examined using GFP and LacZ knock-in mouse lines, respectively. Anti–GATA-4 antibody was used for detecting endogenousGATA-4–expressing cells.

� indicates negative; ��, positive with a high expression level; �, positive in all lines examined; and �, positive in lines 1A, 1B, and 2A, but negative in lines 1C, 2B, and2C.

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that the 180-kb genomic region in the BAC contained virtually allthe necessary regulatory elements needed for Epo expression.

We identified and characterized REP cells in the kidney usingwt-Epo-GFP transgenic mice. REP cells are found in the interstitialspace between tubules and capillaries, showing good agreementwith previous reports.13,19-23 The stellar or arboroid shape of theREP cell expresses the neural cell marker genes, and forms clustersin the anemic kidney. These observations suggest that the reticularnetwork of REP cells may contribute to the regulation of Epoproduction by sensing a low oxygen tension.

A widely believed idea in need of validation is that Epo must beproduced by specifically sensing the cellular oxygen tension.Epo-producing cells are localized in the most hypoxic regions ofthe kidney and liver, supporting their ability to rapidly sensehypoxia.24 We found that the numbers of GFP-positive cells in thekidney and liver increased exponentially with escalating anemia,showing a counter-parallel relationship with the Hct. These datafurther implied that Epo production is controlled through theincrease in the number of Epo-expressing cells rather than by thechanges in the expression level of each Epo-producing cell.35 Incontrast, the expression level of Epo appeared to be much higher inREP cells than in hepatocytes on the basis of GFP fluorescenceintensity. This suggested a differential expression level of Epodependent on the cell type. Indeed, most Epo in the adult body isproduced by the kidney,2 even though the population of REP cells isminor compared with that of hepatocytes.

In this regard, it is interesting to note that Epo is also minimallyexpressed in several tissues besides kidney and liver. TheseEpo-producing tissues include the brain, heart, and testis (reviewedin Suzuki et al24 and Brines and Cerami41). However, we could notdetect GFP expression from the wt-Epo-GFP transgene in any ofthese tissues. One plausible explanation for this discrepancy is thatthe expression level of Epo in these tissues was below the level ofdetection. That the 180-kb genomic fragment lacks specific enhanc-ers important for Epo gene expression in these tissues seemsunlikely, as Epo expression other than in the kidney and liver wasnot detected by our sensitive quantitative RT-PCR analysis. In fact,we previously demonstrated that restricted Epo receptor expressionin hematopoietic lineages was sufficient to sustain mousedevelopment.42

We previously reported that GATA factors repressed the produc-tion of Epo in cultured hepatoma cells under normoxic conditionsthrough binding to the Epo gene promoter, and this repressiondecreased in response to hypoxic stress.3,10,11 To examine whetherthis GATA regulation is actually operating in vivo in response tohypoxia, a transgenic mouse study was conducted. Although animportant feature of GATA-based repression of the Epo geneexpression was also observed in the present study, this study furtherrevealed the GATA box in the Epo gene promoter is required for theconstitutive repression of gene expression in a set of epithelialcells, but it does not affect the hypoxia-inducible production of Epoin REP cells and hepatocytes. These differences in the results are

Figure 6. Expression of GATA factors in renaltubular cells and binding of the Epo gene to theGATA box promoter. (A,B) Immunohistochemical stain-ing of GFP in the kidney of a Gata2-GFP knock-inmouse. (C,D) X-gal staining in the kidney of a Gata3-LacZ knock-in mouse. (E,F) Immunohistochemicalstaining with anti–GATA-4 antibody in the kidney ofwild-type mouse. Note that the distal tubules (d) andcollecting ducts are positive for both Gata2-GFP (brown)and Gata3-LacZ (blue), but negative for GATA-4. Onthe other hand, the expression of Gata2-GFP andGATA-4 was detected in REP cells (arrows in B,F).(G) Chromatin immunoprecipitation (ChIP) assays ofGATA-2 and GATA-3 and the Epo promoter. Chromatincomplexes from the normal renal medulla were immu-noprecipitated with anti–GATA-2 or anti–GATA-3 anti-bodies and the presence of Epo, Aqp2, and Gata1 genepromoter fragments was examined by PCR. Preprecipi-tated samples (input) were used as the internal positivecontrols for PCR. Normal rabbit immunoglobulinG (IgG) was used as a negative control. Scale bars are300 �m (A,C,E) and 20 �m (B,D,F).

5230 OBARA et al BLOOD, 15 MAY 2008 � VOLUME 111, NUMBER 10

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most likely caused by the experimental systems exploited, and thisargues that transgenic mouse analyses are important to clarifytranscription regulation mechanisms operating in vivo.

As for the molecular mechanism underlying the function ofGATA factors, we suggest one possibility, as depicted in Figure 7.In this scenario, we assume that there may be an enhancer in thevicinity of the Epo gene that is important for the expression of othergenes in the epithelial cell lineage. GATA factors may act toinsulate the regulatory influence of this presumptive epithelialenhancer by binding to the GATA box. In contrast, while wedetected the expression of GATA-2 and GATA-4 in REP cells, theseGATA factors did not appear to affect Epo gene expression throughits promoter GATA box and GFP expression from the GATA mutanttransgene was induced normally upon bleeding anemia. Thisobservation indicates that the hypoxia-responsive enhancer candrive Epo gene expression regardless of GATA-based repression.In addition to the authentic Epo-producing cells (REP cells and

hepatocytes; Figure 7A) and epithelial cells (Figure 7B), there is athird cell lineage in terms of Epo gene regulation. In these cells,Epo expression would be permanently silenced by epigeneticmechanisms that are independent of the GATA box and hypoxicthreshold (Figure 7C).

GATA factors have been found to activate transcription,43 butrecently repressive gene regulation by GATA factors has also beendiscovered, including the observation that the PPAR� gene inpreadipocytes is repressed by GATA-2 and GATA-3 in the imma-ture stage, but that this repression is abrogated after cellulardifferentiation.44 In contrast, this study demonstrated that Epo geneexpression in epithelial cells is constitutively repressed by GATAfactors. This is a novel regulatory system exploiting the GATA box,and this element shows extremely high conservation amongmammalian Epo gene sequences.45 We surmise that this novelmechanism may rely on the insulator-like functions of GATAfactors.46

Finally, it should be noted that tumors can derive from renaltubular epithelia and are occasionally accompanied by polycythe-mia. Many of these tumors constitutively produce Epo.47,48 Since asingle nucleotide mutation in the GATA box of the Epo-GFPtransgene caused constitutive transgene expression in renal tubularcells, this study suggests that there may be some defects in theGATA signaling pathway in the epithelial tumor cells that leads toectopic Epo gene expression. This observation led us to speculatethat we may be able to develop an alternative treatment forEpo-dependent anemia by controlling GATA factor function inepithelial cells.

Acknowledgments

We thank Naomi Kaneko, Mitsuru Okano, Yuko Kikuchi, andMasako Yamagishi for their assistance on mouse maintenance, andDrs Doug Engel, Tania O’Connor, and Jon Maher for advice.

This work was supported by grants from the Ministry ofEducation, Science, Sports and Culture; the Ministry of Health,Labor and Welfare; and the ERATO project of JST Agency.

Authorship

Contribution: N.O. and N.S. designed the study, performed theresearch, generated transgenic mice, analyzed the data, and wrotethe paper; K.K. assisted with RT-PCR analyses; T.N. analyzed data;S.I. analyzed the data and supervised the study; and M.Y. super-vised the study, analyzed data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Masayuki Yamamoto, Department of MedicalBiochemistry, Tohoku University Graduate School of Medicine,2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan; e-mail:masi@mail.tains.tohoku.ac.jp.

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online January 17, 2008 originally publisheddoi:10.1182/blood-2007-10-115857

2008 111: 5223-5232  

YamamotoNaoshi Obara, Norio Suzuki, Kibom Kim, Toshiro Nagasawa, Shigehiko Imagawa and Masayuki 

gene expressionerythropoietinRepression via the GATA box is essential for tissue-specific  

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