DNA AND CELL BIOLOGYVolume 21, Number 8, 2002© Mary Ann Liebert, Inc.Pp. 561–569

The Mouse Alpha-Fetoprotein Promoter is Repressed in HepG2 Hepatoma Cells by Hepatocyte Nuclear

Factor-3 (FOXA)

MEI-CHUAN HUANG,1 KELLY KE LI,1 and BRETT T. SPEAR1,2

ABSTRACT

The mouse alpha-fetoprotein gene is expressed at high levels in the fetal liver and is transcriptionally silencedat birth. The repression is governed, at least in part, by the 250 base pair (bp) AFP promoter. We show herethat the AFP promoter is dramatically repressed by HNF3 in HepG2 hepatoma cells. This repression is gov-erned by the region between 2205 and 2150. Furthermore, this fragment can confer HNF3-mediated re-pression on a heterologous promoter. The repression is abolished by a mutation that is centered at 2165.EMSA analyses using in vivo and in vitro synthesized proteins indicate that HNF3 proteins do not bind DNAfrom the 2205 to 2150 region. We propose that HNF3 represses AFP promoter activity through indirectmechanisms that modulate the binding or activity of a liver-enriched factor that interacts with the 2165 re-gion of the AFP promoter.

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INTRODUCTION

ALPHA-FETOPROTEIN (AFP), A MAJOR FETAL SERUM PROTEIN

in mammals, is involved in ligand transport and maintain-ing physiologic osmolarity (reviewed in Deutsch, 1991). TheAFP gene is transcribed at high levels in the yolk sac and fe-tal liver, and to a much lesser extent in the fetal gut and kid-ney (Tilghman, 1985). AFP transcription in the liver declinesdramatically after birth, resulting in barely detectable AFPmRNA levels in the liver by 4 weeks of age (Tilghman and Be-layew, 1982). Five cis-acting elements that regulate AFP ex-pression, the promoter, repressor, and three upstream enhancers,have been identified and characterized using transgenic mouseand in vitro studies (Chen et al., 1997; Spear, 1999). The AFPpromoter, localized within the first 250 bp upstream of the tran-scriptional start site, is active only in tissues where AFP is nor-mally transcribed (Godbout et al., 1986). The repressor region,between 2250 and 2800, is required for complete postnatal si-lencing of AFP transcription (Vacher and Tilghman, 1990).Three distinct upstream enhancer regions, each roughly 300 bpin length, have also been identified (Godbout et al., 1988);transgenic studies indicated that all three are active in AFP-ex-

pressing tissues in both fetal and adult mice (Hammer et al.,1987; Ramesh et al., 1995).

Extensive analysis of the AFP promoter from mice, rats, andhumans have shown that binding sites for several transcriptionfactors exist within this region (Fig. 1A; reviewed in Chen etal., 1997). Overlapping binding sites for the liver-enriched tran-scription factors Hepatocyte Nuclear Factor 1 (HNF-1) and theubiquitous factor Nuclear Factor 1 (NFI) exist in the 2130 to2110 region (Feuerman et al., 1989; Zhang et al., 1991; Bernieret al., 1993; Bois-Joyeux and Danan, 1994). Constructs inwhich this HNF1/NFI binding site was mutated exhibited sub-stantially less activity than wild-type promoter constructs whentransiently transfected into AFP-expressing HepG2 human he-patoma cells (Feuerman et al., 1989; Bernier et al., 1993). In-terestingly, a naturally occurring mutation in this region in thehuman AFP gene leads to incomplete postnatal AFP repression,resulting in continued AFP transcription in the adult liver(McVey et al., 1993). This single base substitution results inincreased HNF1 binding, suggesting that the competition be-tween HNF1 and NFI binding could contribute to the develop-mental control of promoter activity. The region centered around2160 is also important for AFP promoter activity. Wen et al.

Departments of 1Microbiology, Immunology, & Molecular Genetics and, 2Pathology & Laboratory Medicine, University of Kentucky, Lex-ington, Kentucky.

(1993) identified a 12-bp inverted repeat located from 2166 to2155 termed the promoter-linked coupling element (PCE),which was required for the stimulation of the rat AFP promoterby the upstream enhancers. Additional studies identified a fac-tor, nkx-2.8, that bound this region (Apergis et al., 1998). Thisfactor belongs to the nk-2 family of homeodomain-containingtranscription factors. Separate studies showed that this 12 bpelement from 2166 to 2155 also binds the factor FetoproteinTranscription Factor (FTF; Bernier et al., 1993), an orphan nu-

clear receptor of the Drosophila fushi tarazu F1 (FTZ-F1) classof receptors (Galarneau et al., 1996). In addition to these well-defined elements, sequence analysis indicates the presence ofa CAAT/enhancer binding protein (C/EBP) binding site at 275of the AFP promoter (Zhang et al., 1991; Thomassin et al.,1992; Bois-Joyeux and Danan, 1994; Galarneau et al., 1996).

HNF3 (FOXA) was originally identified as a liver-enrichedfactor that regulated the rat transthyretin gene (Costa et al.,1989). Additional analysis revealed three HNF3 isoforms,HNF3a, HNF3b, and HNF3g, that are encoded by distinctgenes (Lai et al., 1990, 1991). The HNF3 family of factors reg-ulate numerous liver genes, and are among the earliest factorsto be expression during hepatogenesis (Kaestner, 2000). Al-though HNF3 proteins are most often associated with activa-tion, there are several examples whereby HNF3 acts to represstranscription (Gregori et al., 1994; Rouet et al., 1995). TheHNF3 proteins are related to the Drosophila forkhead protein,which is important for foregut and hindgut development(Weigel and Jackle, 1990). Members of the HNF3/forkheadfamily of proteins are defined by a novel “winged-helix” DNAbinding motif (Clark et al., 1993). Studies with the albumin en-hancer suggest that HNF3 proteins may regulate transcription,at least in part, by positioning nucleosomes (Shim et al., 1998).

AFP is among the earliest genes that are induced during he-patogenesis and, like many liver genes, is regulated by HNF3.An HNF3 site is found in the 59 end of enhancer III, and mu-tational studies have demonstrated that this site is important forfull enhancer activity (Groupp et al., 1994; Thomassin et al.,1996). AFP enhancers I and II do not contain consensus HNF3binding sites and do not appear to be regulated by HNF3 (K.Li and B. Spear, unpublished observation). The mutually ex-clusive binding of HNF3 and p53 at the 2850 region appearsto govern AFP repressor activity (Lee et al., 1999). We previ-ously showed by transient transfection in Hela cells and in vitrotranscription assays that a 1-kb fragment containing the AFPpromoter and repressor is regulated by HNF3 (Crowe et al.,1999). To further define this regulation, we tested whether the250-bp promoter could be the target of this control. We showhere that the 250-bp promoter is activated by HNF3 in Helacells. However, we found that HNF3 represses AFP promoteractivity in HepG2 cells. This repression acts through the 2205to 2150 region, and mutation of the nkx2.8/FTF site at 2165alleviates this repression. EMSA analysis indicates that HNF3does not bind this region. Based on these data, we propose thatHNF3 downregulates AFP promoter activity by modulating thebinding or transactivating potential of other liver-enriched tran-scription factors.

METHODS AND MATERIALS

Plasmids and DNA fragments

The D44–lacZ and (HNF3)12–D44–lacZ expression vectorswere described previously (Spear et al., 1995). The 250–lacZand 150–lacZ expression vectors were generated by replacingthe promoter of D44–lacZ with fragments of the AFP promoterfrom 2250 to 123 or 2150 to 123, respectively. Two mu-tated versions of the 250–lacZ vector, in which the region cen-tered at 2165 was changed from CTTTGTCC to ACTGCAGA

HUANG ET AL.562

FIG. 1. (A) Region of the AFP promoter used in this study.The AFP promoter is defined as a 250-bp fragment upstreamof the AFP transcription start site. Binding sites for nkx2.8/FTF,NF-1, HNF1, and C/EPB and the endpoints of the “A” and “B”fragments are shown. (B) AFP promoter mutations used in thisstudy. Sequence of the AFP promoter regions centered at 2165and 275 and the corresponding mutations, designated by lowercase letters, that were used to alter these regions. (C) HNF3 re-presses AFP promoter activity in HepG2 cells. Cells were trans-fected with vectors containing lacZ fused to the entire 250-bpAFP promoter, 150-bp promoter, or 150-bp promoter fused tothe AFP “A” or “B” fragments, pCAT, and either empty vetor(e.v) or HNF3b expression vectors as designated. The b-galac-tosidase activity normalized to CAT is shown. Error bars rep-resent standard deviation.

or the region centered at 275 was changed from TGTTTGCTto ACTGCAGA, were generated by PCR-based mutagenesis asdescribed (Fig. 1B; Zaret et al., 1990) to produce 250m165–lacZand 250m75–lacZ, respectively. The AFP promoter “A” frag-ment, extending from 2205 to 2137, was generated by PCRamplification. A mutated version of the “A” fragment, with theregion centered at 2165 mutated as described above, was gen-erated by PCR amplification as described (Zaret et al., 1990).The AFP promoter “B” fragment, extending from 2250 to2192, was generated by PCR amplification. The A-150–lacZexpression vector was generated by PCR amplifying the AFPpromoter from 2205 to 223; this amplified product was usedto replace the promoter of D44–lacZ. The “B” fragment waslinked to 150–lacZ in its normal orientation to generate B-150–lacZ. Wild-type and mutated versions of the AFP promoterA and B fragments were fused in their normal orientations 59 of the promoter of D44–lacZ to produce A-D44–lacZ,Am165–D44–lacZ, and B-D44–lacZ. All fragments generated byPCR were confirmed by DNA sequencing. The pCAT plasmidwas obtained from Promega (Madison, WI). The CMV-basedrat HNF3a and HNF3b eukaryotic expression vectors were pro-vided to us by Dr. Robert Costa (University of Illinois atChicago; Qian and Costa, 1995; Qian et al., 1995; Rausa et al.,1997). The entire cDNA insert containing HNF3a was removedas an EcoRI fragment from the CMV–HNF3a expression vec-tor to generate the CMV empty vector (E.V.). A truncated ver-sion of the rat HNF3b cDNA that encodes the DNA-bindingdomain (DBD; amino acids 121–272) was produced by PCRamplification and inserted into the CMV expression vector; thistruncated insert was confirmed by DNA sequencing. Full-lengthcDNAs for rat HNF3b and mouse HNF6 in pBluescript wereprovided by Dr. Robert Costa and used for in vitro protein syn-thesis.

Transfections

The human Hela cell line was maintained in Dulbecco’smodified Eagle medium (DMEM) supplemented with 10% fe-tal bovine serum (FBS), Penicillin/Streptomycin (Pen/Strep),and glutamine. The human hepatoma HepG2 cell line was main-tained in DMEM/F12 (1:1) medium with 10% FBS, PenStrep,glutamine, and 10 mg/ml insulin. Insulin was obtained fromSigma Chemical Corporation (St. Louis, MO), all other reagentswere from Life Sciences (Gaithersburg, MD). Transient trans-fections were carried out by the calcium phosphate method asdescribed for HepG2 cells (Spear and Tilghman, 1990) or us-ing Lipofectamine (Life Technologies) according to manufac-turer’s directions for Hela cells. For Hela cotransfections, cellswere plated at 1 3 106 cells/10 cm dish. The following day,cells were transfected with a total of 20 mg of DNA consistingof 12 mg of the lacZ reporter construct, 3 mg of the HNF3 ex-pression vector, and 5 mg of pCAT. For HepG2 cotransfections,cells were plated at 0.5 3 106 cells/6 cm dish. The followingday, cells were transfected with 6 mg of the lacZ reporter con-struct, 1.5 mg of the HNF3 expression vector, and 1 mg ofpCAT. For Hela synthesized HNF3 proteins, cells were platedat 1 3 106 cells/10 cm dish and transfected with 15 mg of HNF3vectors. Forty-eight hours after the addition of DNA, cells werewashed three times in 13 PBS and scraped from plates into 1.5ml of PBS and transferred to 1.5 ml microcentrifuge tubes. Cells

were pelleted by centrifugation and used for bgal/CAT assaysor the preparation of nuclear extracts as described below.

bgal/CAT assays

Cell pellets from transient transfections were resuspended in100 ml of 0.25 M Tris, pH 7.4, and thoroughly vortexed. Cellswere lysed by three cycles of freeze thawing using dry ice, fol-lowed by centrifugation at 14,000 3 g for 5 min. Protein con-centrations were determined by the BCA assay kit (Pierce Bio-chemicals, Rockford, IL). The b-galactosidase activity wasmeasured using the colorimetric substrate chlorophenolred-b-D-galactopyranoside (CPRG; Boehringer Mannheim, Indi-anapolis, IN) as described (Spear et al., 1995). Cell extracts(10–50 ml) were added to microfuge tubes containing 200 mlof buffer A (100 mM NaPO4, pH 7.2, 10 mM KCl, 10 mMMgCl2, and 10 mM 2-mercaptoethanol [2-ME]). Thirty micro-liters of CPRG solution (15 mg CPRG/ml Buffer A lacking 2-ME) was added to the tubes and reactions were incubated at37°C. Reactions mixtures were stopped by the addition of 23

sample volumes of Buffer A lacking 2-ME. Absorbance wasmeasured at 570 nm using a control reaction without cell ex-tracts as a blank. The b-gal activity was normalized to CAT ac-tivity to control for variations in transfection efficiency. Chlo-ramphenicol acetyltransferase (CAT) assays were performed asdescribed (Sambrook et al., 1989). Briefly, cell extracts wereincubated at 65°C for 10 min to inactivate deacetylases. Par-ticulate material was removed by centrifugation at 14,000 3 gfor 2 min at 4°C. Cell extracts (10–50 ml) were mixed with 10ml of the CA solution (10 ml of 3H-chloramphenicol [1 mCi/ml,NEN, Boston, MA], 10 ml of nonradioactive chloramphenicol[20 mM, Sigma, St. Louis, MO], and 380 ml of 0.25 M Tris-HCl, pH 8.0), 10 ml of n-butyryl Coenzyme A (5 mg/ml,Sigma), and 0.25 mM Tris-HCl (pH 8.0) buffer to bring the to-tal volume to 125 ml. The n-butyryl Coenzyme A was addedlast as it initiated the reaction. Reaction mixtures were incu-bated at 37°C and 300 ml of xylene (Baxter, Deerfield, IL) wereadded to stop the reactions. Reactions were mixed and the xy-lene was removed. The xylene was back-extracted once with100 ml of 0.25 M Tris-HCl buffer. The resulting xylene layerwas transferred to a scintillation vial containing 3 ml of scin-tillation fluid (Bio-Safe NA, RPI, Mount Prospect, IL) and thecpms were determined using a scintillation counter.

Nuclear extracts/in vitro synthesized proteins

Nuclear extracts were prepared from Hela or HepG2 cells(Li et al., 2000). Monolayers of untransfected or transfectedcells were harvested by scraping as described above and re-suspended in buffer containing 10 mM HEPES, pH 7.9, 1.5 mMMgCl2, 10 mM KCl, 0.5 mM dithiolthreitol (DTT), and 0.2 mMphenylmethylsulfonyl fluoride (PMSF). After a 10-min incu-bation on ice, nuclei were collected by centrifugation at14,000 3 g for 2 min. The nuclear pellet was resuspended inbuffer containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420mM NaCl, 0.2 mM EDTA, 25% (v:v) glycerol, 0.5 mM DTT,and 0.2 mM PMSF. After a 20-min incubation on ice, sampleswere centrifuged at 14,000 3 g for 2 min. The supernatant wasremoved and stored in aliquots at 280°C. Protein concentra-tions were determined using the BCA assay kit (Pierce Bio-chemicals). The TNT-coupled reticulocyte lysate system

HNF3 AND REPRESSION OF AFP PROMOTER ACTIVITY 563

(Promega Biotech) was used for the production of in vitro syn-thesized HNF3 and HNF6 proteins according to the manufac-turer’s instructions. Proteins were aliquoted and stored at280°C until use.

EMSAs

Dephosphorylated, gel-purified DNA fragments were end-labeled with 32P-ATP using T4 polynucleotide kinase as de-scribed (Li et al., 1996). Oligonucleotides corresponding to2111 to 285 of the rat transthyretin promoter were chemicallysynthesized, annealed, and used as the HNF-3 site probe (Costaet al., 1989). The AFP promoter “A” fragments from 2205 to2137 (wild-type and 2165 mutation) were amplified by PCR,dephosphorylated, and gel purified prior to labeling. EMSAswere carried out using 10 mg of extract and 2 mg of polydI:dCas described (Li et al., 1996). Reaction mixtures were resolvedon nondenaturing 8% polyacrylamide gels in 13 TBE (2.2 mMTris, 2.2 mM boric acid, 0.5 mM EDTA) running buffer. Gelswere dried and subjected to autoradiography using Kodak X-OMAT AR film.

RESULTS

HNF3a and HNF3b repress AFP promoter activity inHepG2 hepatoma cells

We showed previously that HNF3 could transactivate anAFP(1.0)–lacZ reporter gene in Hela cells (Crowe et al., 1999).Additional studies found that the 250-bp AFP promoter wasalso activated by HNF3 in Hela cells (M.-C. Huang and B.T.Spear, unpublished observations). AFP is not normally ex-

pressed in Hela cells, but is synthesized in several hepatomalines including HepG2 human hepatoma cells, which have beenused extensively to study AFP regulation. To investigate fur-ther HNF3 control of the AFP promoter, transient cotransfec-tions were performed in HepG2 cells. A construct with the 250-bp AFP promoter fused to lacZ (250–lacZ) was transientlytransfected into HepG2 cells along with a CMV expression vec-tor that contained no insert (empty vector) or cDNA insert en-coding HNF3b (Fig. 1C). In addition, pCAT was included tocontrol for variations in transfection efficiency. Forty-eighthours after the addition of DNA, cell lysates were prepared andlacZ and CAT activities were determined. In contrast to the ac-tivation that was seen in Hela cells, the 250 bp AFP promoterwas repressed nearly fivefold by HNF3b in HepG2 cells (Fig.1C). This repression was specific to the AFP promoter becausethe (HNF3)12–D44–lacZ vector was transactivated nearly 26-fold by HNF3a in HepG2 cells (see Fig. 3). To further local-ize the region that is required for this repression, additionalAFP–lacZ constructs were analyzed in HepG2 cells (Fig. 1C).A promoter extending from 2150 was linked to lacZ to obtain150–lacZ; this was, in turn, fused to regions of the AFP pro-moter from 2205 to 2150 (A-150–lacZ) or from 2250 to 2192(B-150–lacZ). When cotransfections were performed with theempty vector, 150–lacZ exhibited roughly 10% of the activityof 250–lacZ, whereas the b-gal levels in cells transfected withA-150–lacZ were nearly identical to cells transfected with250–lacZ. This indicates that the 2205 to 2150 region con-tributes substantially to promoter activity. The b-gal levels incells transfected with B-150–lacZ were roughly the same ascells transfected with 150–lacZ, suggesting that the region be-tween 2250 and 2192 does not contribute to promoter activ-ity in HepG2 cells. Similar results were observed with these

HUANG ET AL.564

FIG. 2. (A) HNF3a, HNF3b, and 3b-DBD are equally capable of repressing the AFP promoter in HepG2 cells. Cells weretransfected with 250–lacZ, RSV-CAT, and HNF3 expression vectors at the designated concentrations. The b-galactosidase ac-tivity normalized to CAT is shown. (B) Mutations at 2165 and 275 reduce AFP promoter activity and the 2165 mutation re-lieves HNF3-mediated repression in HepG2 cells. Cells were transfected with lacZ fused to the wild-type 250-bp AFP promoteror 250-bp promoter with mutations centered at 2165 or 275 (250-lacZ, 250m165-lacZ, and 250m75-lacZ, respectively), pCAT,and HNF3 expression vectors as shown. The b-galactosidase activity normalized to CAT is shown. Error bars represent standarddeviation.

AFP–lacZ constructs were transfected in the absence of theempty vector (data not shown). When cotransfections were per-formed with the HNF3b expression vector, A-150–lacZ was re-pressed similarly to the 250–lacZ (4.3-fold versus 4.2-fold re-duction, respectively). In contrast, the AFP(150)–lacZ andB-150–lacZ constructs were not repressed, but were instead ac-tivated slightly by HNF3b. This indicates that HNF3 repressesAFP promoter activity through the 2200 to 2150 region.

DNA binding by HNF3 proteins is mediated by the internalwinged-helix domain. Previous studies had shown that this do-main could act in a dominant negative manner to repress numer-ous liver genes when stably transfected in hepatoma cells (Valletet al., 1995). A 152-amino acid form of HNF3b that contains theinternal DNA binding domain (HNF3b-DBD) was generated byPCR. The ability of this truncated HNF3b-DBD to repress250–lacZ was tested in HepG2 cells (Fig. 2A). HNF3b-DBD wasalmost as effective as HNF3a and full-length HNF3b at repress-ing AFP promoter activity in HepG2 cells. We also tested whetherlower amounts of the HNF3 expression vectors could still repressAFP promoter activity. HNF3a, HNF3b, and HNF3b-DBD wereall still potent repressors when titrated to 0.1 mg, which is 30-foldlower than the levels used in Figure 1C.

HNF3-mediated repression of the AFP promoter isabolished by a mutation at 2165

HNF3-mediated repression of AFP promoter activity is gov-erned by the 55-bp region between 2205 and 2150, but se-quence analysis failed to identify potential HNF3 sites in thisregion. However, this region contains overlapping binding sitesfor two factors, nkx-2.8 and FTF, that are important regulatorsof AFP promoter activity (Fig. 1A). To explore the potentialrole of this motif in HNF3-mediated repression, this site cen-tered at 2165 was mutated (Fig. 1B). The mutation was con-firmed by DNA sequencing and the mutated AFP promoter wasfused to lacZ to generate 250m165–lacZ. When cotransfectedwith the empty vector, 250m165–lacZ had roughly 10% of theactivity of 250–lacZ (Fig. 2B), consistent with a role for thenkx-2.8/FTF site in promoter function. Whereas the wild-typepromoter was repressed when HNF3a or HNF3b was cotrans-fected, the mutated promoter was resistant to repression andwas, in fact, slightly activated. This modest activation is simi-lar to what was seen previously with 150–lacZ and B-150–lacZ.This demonstrates the importance of the 2165 site for normalpromoter activity and HNF3-mediated repression.

The AFP promoter constructs that were subjected to repres-sion by HNF3 were much more active in HepG2 cells than thosethat were not repressed. This raised the possibility that the lessactive promoters were simply too weak to be repressed byHNF3. To test this, a putative C/EBP binding site at 275 wasmutated to generate 250m75–lacZ (Fig. 1B). This mutationshould diminish promoter activity, but should not influenceHNF3-mediated repression through the AFP promoter A frag-ment. Both 250m165–lacZ and 250m75–lacZ had equally low ac-tivity when transfected into HepG2 cells alone (data not shown)or with the empty vector (Fig. 2B). Despite its low activity,250m75–lacZ could still be repressed by HNF3b and HNF3a

(Fig. 2B).If the AFP promoter region between 2205 and 2150 is re-

sponsible for HNF3-mediated repression, this fragment should

confer repression when fused to a heterologous promoter. Wepreviously used a minimal promoter (D44) from the humanliver/bone/kidney alkaline phosphatase gene (Spear et al., 1995)because this promoter is highly responsive to linked elements(Kiledjian and Kadesch, 1990). The AFP A fragment, the Afragment with the 2165 mutation, and the AFP B fragmentwere fused to D44–lacZ. The (HNF3)12–D44–lacZ vector wasused as a positive control. Cotransfections were performed inHepG2 cells, and changes in lacZ levels with the HNF3a ex-pression vector versus empty vector was determined (Fig. 3).Most of the D44–lacZ-based reporter constructs had little ac-tivity in the presence of the empty vector, consistent with stud-ies showing low basal activity of the D44 promoter. The oneexception was A-D44–lacZ, which was nearly as active as250–lacZ. This confirms the robust activity of the A fragmentin HepG2 cells. Activities of D44–lacZ and B-D44–lacZ re-porter constructs were increased roughly three- to fourfold bythe HNF3a. This demonstrated that D44–lacZ was somewhatresponsive to HNF3a, and that the B fragment did not influ-ence this response. The positive control (HNF3)12–D44–lacZvector was activated 26-fold when HNF3a was cotransfected,consistent with previous studies by us and others (Pani et al., 1992; Spear et al., 1995). In contrast, A-D44–lacZ was repressed fourfold by HNF3a; this repression is similar to the 3.7-fold HNF3-mediated inhibition of 250–lacZ. TheAm165–D44–lacZ vector was activated roughly fourfold, simi-larly to D44–lacZ, further demonstrating that this mutation abol-ished HNF3-mediated repression.

HNF3 AND REPRESSION OF AFP PROMOTER ACTIVITY 565

FIG. 3. The AFP promoter “A” fragment confers HNF3a-mediated repression to a heterologous promoter. A minimal pro-moter of the human liver/bone/kidney alkaline phosphatasegene (D44), was fused to lacZ to generate D44–lacZ. Fragmentsfrom the AFP promoter or tandem copies of the rat transthyretinpromoter HNF3 binding site were inserted upstream ofD44–lacZ. These lacZ reporter constructs were transfected withpCAT and the HNF3a expression vector or empty vector asshown. The b-galactosidase activity normalized to CAT isshown. Error bars represent standard deviation.

HNF3a or HNF3b proteins do not bind the 2205 to2150 region of the AFP promoter in vitro

Computer analysis did not reveal any obvious HNF3 bind-ing sites in the region between 2205 and 2150. However, therepressive effects of HNF3 on this fragment prompted us to useelectrophoretic mobility shift assays (EMSAs) to investigate thepossibility that HNF3 binding sites might exist within this re-gion. Nuclear extracts were prepared from Hela cells that weretransfected with the CMV expression vector that contained noinsert, HNF3a, or HNF3b or from untransfected HepG2 cells.These extracts were incubated with radiolabeled DNA frag-ments containing the rat transthyretin promoter HNF3 site (286to 2111), the wild-type AFP “A” fragment, or the “A” frag-ment with the 2165 mutation (A*; Fig. 4). The HNF3 probedid not bind any proteins in extracts from Hela cells transfectedwith the empty vector (Fig. 4, lane 4). A predominant band wasdetected when this probe was incubated with extracts from Helacells transfected with HNF3a or HNF3b (Fig. 4, lanes 7 and10). These two bands had roughly the same mobility, althoughthe HNF3a band had a slightly slower mobility when gels wererun further (data not shown), consistent with previous studies(Lai et al., 1990). A faint band corresponding to HNF3a/b wasalso present in HepG2 cell extracts (Fig. 4, lane 13). The Helaextracts contained three faint complexes that bound to the AFP

“A” fragment (Fig. 4, lanes 5, 8, and 11). These complexes werealso present in extracts from untransfected Hela cells (data notshown). Interestingly, the intensity of the complex with inter-mediate mobility (asterisk) increased when cells were trans-fected with HNF3a or HNF3b. These three complexes werealso present in HepG2 extracts, as was another complex ofhigher mobility that was not present in Hela extracts (Fig. 4,lane 14). Although extracts from transfected Hela cells andHepG2 cells contained HNF3 proteins, HNF3 binding to theAFP “A” fragment could not be detected with these extracts.The mutated “A*” fragment did not bind any proteins in Helaor HepG2 cell extracts, indicating that the site at 2165 is crit-ical for all the protein binding to this fragment (Fig. 4, lanes 6,9, 12, and 15).

To explore further the possibility of HNF3 binding to theAFP “A” fragment, EMSAs were performed with in vitro syn-thesized proteins. When the HNF3, wild-type “A” and mutated“A” fragments were incubated with lysates that were pro-grammed with BlueScript alone (B/S), no complexes were seen(Fig. 5, lanes 4–6). Extracts programmed with the HNF3b vec-tor contained a complex that bound the HNF3 probe (Fig. 5,lane 7). However, the HNF3b extract did not bind the wild-type or mutant “A” fragments (Fig. 5, lanes 8 and 9). Previousstudies have shown that some HNF3 sites, including that fromthe transthyretin promoter, can also be recognized by the CUT-

HUANG ET AL.566

FIG. 4. In vivo synthesized HNF3a or HNF3b do not bindthe AFP promoter “A” fragment. EMSAs were performed withradiolabeled probes corresponding to the rat transthyretin pro-moter HNF3 binding site (HNF3), the AFP promoter “A” frag-ment extending from 2205 to 2137 (A), or the AFP promoter“A” fragment containing the mutation centered at 2165 asshown in Figure 1B (A*). Probes were incubated with no ex-tract (free probe, F.P.; lanes 1–3), or extracts from Hela cellsthat were transfected with the empty vector (E.V.; lanes 4–6),HNF3a expression vector (lanes 7–9), HNF3b expression vec-tor (lanes 10–12), or extracts from untransfected HepG2 cells(lanes 13–15). The asterisk to the right of the figure designatesa band that is found in Hela cells and HepG2 cells; the inten-sity of this band is increased with extracts from cells transfectedwith HNF3a or HNF3b compared to cells transfected with theempty vector.

FIG. 5. In vitro synthesized HNF3a or HNF6 fail to bind theAFP promoter “A” fragment. EMSAs were performed with ra-diolabeled probes corresponding to the rat transthyretin pro-moter HNF3 binding site (HNF3) or the wild-type or mutatedAFP promoter “A” fragment (A and A*, respectively). EMSAswere performed with no proteins (free probe, F.P.; lanes 1–3)or with proteins generated from TnT reticulocyte lysates thathad been programmed with BlueScript (B/S; lanes 4–6), or vec-tors for HNF3b (lanes 7–9) or HNF6 (lanes 10–12).

Homeodomain protein HNF6 (Lemaigre et al., 1996; Samadaniand Costa, 1996). Therefore, we tested whether this factor mightbind the AFP “A” fragment. Extracts programmed with theHNF6 vector did bind the transthyretin HNF3 site, as expected,but showed no binding to the AFP promoter fragments (Fig. 5,lanes 10–12).

Because a truncated form of HNF3b containing only theDNA binding domain was a potent inhibitor of AFP promoteractivity in HepG2 cells, it was somewhat surprising that theEMSA data indicated that HNF3 did not bind to the AFP pro-moter “A” fragment. The truncated form is substantially smallerthan the full-length HNF3b protein, so we tested whether wecould detect HNF3b-DBD binding to the AFP “A” fragment inEMSAs. Nuclear extracts were prepared from Hela cells trans-fected with the empty vector, HNF3a, HNF3b, or HNF3b-DBD, and used in EMSAs with the HNF3 probe and AFP “A”fragment (Fig. 6). Consistent with Figure 4, Hela-synthesizedHNF3a and HNF3b could bind the HNF3 probe (Fig. 6, lanes5 and 7). HNF3b-DBD could also bind effectively to this probe;the HNF3 probe/HNF3b-DBD complex had a high mobilitythat could be readily distinguished from other bands in thisEMSA gel (Fig. 6, lane 9). However, none of these HNF3 mol-ecules bound to the AFP “A” fragment (Fig. 6, lanes 6, 8, and10). As was seen in Figure 4, the “A” probe bound three com-

plexes in Hela cells and the intensity of the middle band wasenhanced by the presence of HNF3 proteins including HNF3b-DBD (asterisk in Fig. 6).

DISCUSSION

We previously showed that HNF3a and HNF3b can activateAFP-linked reporter genes in Hela cells through the 1-kb pro-moter/repressor region. We extended these studies using transienttransfections to show that this induction in Hela cells is medi-ated, at least in part, by the 250-bp AFP promoter. In contrast tothe transactivation that was seen in Hela cells, we have shownhere that HNF3 acted as a potent repressor of AFP promoter ac-tivity when transient transfections were performed in AFP-ex-pressing HepG2 hepatoma cells. A truncated form of HNF3b thatcontained the winged-helix DNA binding domain, but lacked theN-terminal and C-terminal activation domains, could still repressAFP promoter activity. Repression was governed by the regionbetween 2205 and 2137 of the AFP promoter, and was abol-ished by a mutation centered at 2165. EMSA analysis indicatedthat HNF3 could not bind to a fragment spanning the 2205 to2137 region. This argues that repression does not require the di-rect binding of HNF3 to this region.

The ability of HNF3 to transactivate or repress the AFP pro-moter in Hela or HepG2 cells, respectively, argues that HNF3-mediated control is modulated by factors that are differentially ex-pressed in these two cell lines. In Hela cells, the AFP promoterhas little, if any, activity. Forced expression of HNF3 in thesecells is able to induce AFP promoter activity. This data is con-sistent with previous in vitro chromatin-based transcription assays(Crowe et al., 1999). In contrast to Hela cells, the AFP promoterhas robust activity in HepG2 cells. This activity is due to the pres-ence of multiple liver-enriched factors, including FTF and nkx-2.8, both of which bind to a site centered at 2165 of the AFPpromoter. This site is required for full promoter activity (this pa-per and Wen and Locker, 1994; Galarneau et al., 1996; Apergiset al., 1998), and is also required for HNF3-mediated repression,but does not bind HNF3. Thus, the most likely targets of HNF3-mediated repression would therefore be FTF or nkx-2.8.

HNF3 has been shown to negatively regulate the adolase Ppromoter and the a-1-microglobulin/bikunin enhancer (Gregoriet al., 1994; Rouet et al., 1995). These two examples involvedirect binding of HNF3 to DNA, and are thus likely to be dif-ferent than repression of the AFP promoter in HepG2 cells. Sev-eral explanations could account for HNF3-mediated repressionof the AFP promoter via indirect mechanisms. HNF3 may sim-ply inhibit promoter activity by nonspecific squelching wherebyHNF3 overexpression titrates other general transcription fac-tors (Natesan et al., 1997). However, D44–lacZ, when fused tothe transthyretin promoter HNF3 binding sites, was activatedby HNF3 in HepG2 cells; this would argue against a nonspe-cific squelching mechanism. A second possibility is that HNF3could regulate the transcription of other transcription factorsthat control AFP promoter activity. HNF3 is known to regulateseveral known liver-enriched transcription factors includingHNF1a, HNF1b, and HNF4a (Costa, 1994; Kaestner, 2000);HNF1 regulates the AFP promoter through a site centered at2120 (Feuerman et al., 1989; McVey et al., 1993), but thereis no evidence that these factors bind sites within the AFP pro-

HNF3 AND REPRESSION OF AFP PROMOTER ACTIVITY 567

FIG. 6. In vivo synthesized HNF3b-DBD truncated proteinbinds the transthyretin promoter HNF3 site but does not bindthe AFP promoter “A” fragment. EMSAs were performed withradiolabeled probes corresponding to the rat transthyretin pro-moter HNF3 binding site (HNF3) or the AFP promoter “A”fragment extending from 2205 to 2137 (A). Probes were in-cubated with no extract (free probe, F.P.; lanes 1–2), or extractsfrom Hela cells that were transfected with the empty vector(E.V.; lanes 3–4), HNF3a vector (lanes 5–6), HNF3b expres-sion vector (lanes 7–8), 3b-DBD vector (lanes 9–10) or extractsfrom untransfected HepG2 cells (lanes 11–12). The asterisk cor-responds to the band described in the legend to Figure 4.

moter “A” fragment. In addition, it seems unlikely that this typeof indirect regulation would be detected in a transient assay.

A third possibility, which we favor, is that HNF3 repressesAFP promoter activity by modulating the binding or activity ofother transcriptional regulators that bind to the “A” fragment.Recent studies have demonstrated that transcription factors canbind to other factors and influence their transactivation poten-tial. For example, the glucocorticoid receptor (GR) can controltranscription of some genes by a “tethering” mechanism thatallows GR to bind other transcription factors and regulate theirtransactivating potential (Yamamoto et al., 1998). GR can re-press NF-kB-mediated activation of the ICAM-1 and IL-8 promoters by such a mechanism; GR tethers to the relA/p65component, which interferes with NF-kB-dependent phospho-rylation of the RNA Polymerase II C-terminal domain (Nissenand Yamamoto, 2000). Alternatively, HNF3 binding elsewherein the AFP control region could influence nucleosome posi-tioning and therefore modulate the accessibility of factors tosites within the “A” fragment. Elegant studies by Zaret and col-leagues have shown that HNF3 binding to the albumin enhancercan alter nucleosome positioning (Shim et al., 1998). By thismechanism, HNF3 could alter chromatin in such a way to re-duce the binding of FTF or nkx-2.8 to their target sites at 2165of the AFP promoter, leading to a reduction in AFP promoteractivity. In this regard, it is interesting that 150–lacZ andD44–lacZ were slightly transactivated by HNF3, suggesting thepresence of HNF3 binding sites in these plasmids. A predictionof the nucleosome positioning model would be that an expres-sion vector containing the AFP promoter “A” fragment, butlacking any other HNF3 binding sites, would not be repressedby HNF3 in HepG2 cells.

The AFP gene is dramatically repressed within the first sev-eral weeks after birth. The basis for this repression is not wellunderstood, and specific trans-acting factors that govern thisrepression have not been identified. Transgenic mouse studieshave shown that repression is mediated, at least in part, by thepromoter (Peyton et al., 2000). In addition, a mutation in theoverlapping HNF1/NF1 site at 2120 in the AFP promoter isassociated with the hereditary persistence of AFP in humans(McVey et al., 1993). Our studies raise the possibility thatHNF3 may also be involved in postnatal AFP repression, pos-sibly by blocking the activity or binding of other liver-enrichedfactors to the AFP promoter. A more thorough understandingof the factors that control AFP promoter activity, and the anal-ysis of these factors during liver development, will be neededto further understand this control.

ACKNOWLEDGMENTS

We thank Rob Costa for providing HNF3 and HNF6 ex-pression vectors, David Peyton for production of the 3b-DBDexpression vector; Martha Peterson for critically reading themanuscript. These studies were supported by Public Health Ser-vice Grants GM45253 and DK51600.

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Address reprint requests to:Brett T. Spear, Ph.D.

Department of MicrobiologyImmunology & Molecular Genetics

University of Kentucky College of Medicine800 Rose Street

Lexington, KY 40536-0298

E-mail: bspear@uky.edu

Received for publication February 5, 2002; received in revisedform and accepted May 6, 2002.

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