MOLECULAR AND CELLULAR BIOLOGY, Nov. 1994, p. 7086-70940270-7306/94/$04.00+0Copyright C 1994, American Society for Microbiology

Genetic Analysis of a Transcriptional Activation Pathwayby Using Hepatoma Cell Variants

GARY A. BULLAt AND R. E. K. FOURNIER*

Department of Molecular Medicine, Fred Hutchinson Cancer Research Center,Seattle, Washington 98104

Received 11 March 1994/Returned for modification 5 July 1994/Accepted 1 August 1994

A hierarchy of liver-enriched transcription factors plays an important role in activating expression of manyhepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encodingHNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathwaygenetically, we prepared somatic cell variants that were deficient in expression of the liver-specific a1-antitrypsin (alAT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was

accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers underalAT promoter control were prepared; second, variant sublines that could no longer express either transgenewere isolated by direct selection. In this report, we demonstrate that the variants contain defects in theHNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was

affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variantclasses could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1.Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forcedHNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to eitherHNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alAT,aldolase B, and a-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes

were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence ofan autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4.

The genetic basis for the specification of tissue identity inmammals is not well understood. One useful model system forstudying cellular differentiation has been the hepatocyte: thebiochemistry and molecular biology of the liver is well ex-plored, and many liver genes and liver-enriched transcriptionfactors have been isolated and characterized. Considering thelarge number of genes that are specifically expressed in theliver, it is reasonable to expect that some liver genes might becoordinately controlled by common regulatory pathways. In-deed, as an increasing number of liver genes have been studied,common binding sites for several liver-enriched transcriptionalactivators have been found. In particular, hepatocyte nuclearfactor 1 (HNF-1) and HNF-4 seem to play critical roles inactivating expression of many liver genes (reviewed in refer-ences 25 and 40).HNF-1, one of the first liver-specific transactivators to be

defined (1, 4), seems to play a central role in maintenance ofthe hepatic phenotype. The promoters of more than 20 differ-ent liver genes contain HNF-1 binding sites (9, 40), andtransfection tests have generally shown that these sites activateexpression in liver cells 20- to 100-fold (2, 13, 17, 22). Thetarget genes that are regulated by HNF-1 encode productsinvolved in a number of different liver functions, includingcarbohydrate metabolism, detoxification, and the productionof serum proteins.Another liver-enriched transactivator, HNF-4, was recently

shown to bind not only to several liver gene promoters but also

* Corresponding author. Mailing address: Fred Hutchinson CancerResearch Center, A2-025, 1124 Columbia St., Seattle, WA 98104.Phone: (206) 667-5217. Fax: (206) 667-6522.

t Present address: Division of Cellular and Developmental Biology,Pediatric Research Institute, St. Louis, MO 63110.

to the promoter of the HNF-1 gene itself (39). Promotermutagenesis and gene transfer experiments have demonstratedthat HNF-4 is the major activating factor of the HNF-1promoter (24, 39). In addition, the silent HNF-1 genes of thededifferentiated rat hepatoma line H5 could be activated byectopic expression of HNF-4 (24). This finding providedevidence for the existence of a hierarchical pathway of tran-scriptional activation that involves HNF-4 and HNF-1. Thus,these two transactivators play a central role in activatingexpression of a large number of liver genes.HNF-4, a member of the steroid/thyroid/retinoid receptor

superfamily (37), and HNF-1, a homeodomain-containing pro-tein (19), are both expressed at high levels in the liver, butlow-level expression can be detected in the kidney, intestine,and pancreas (25, 26). HNF-4 and HNF-1 are also expressed inmany differentiated hepatoma cell lines, but they are absentfrom dedifferentiated hepatoma variants and extinguishedsomatic cell hybrids (2, 5). These observations suggest thatexpression of HNF-4 and HNF-1 is required for maintenanceof the liver phenotype, but direct data supporting this conten-tion have not yet been obtained.Rat hepatoma cell lines derived from H4IIEC3 have been

useful tools for studying factors involved in liver-specific geneexpression (2, 4, 5, 7, 12, 23, 24). Two other cell types,dedifferentiated hepatoma variants and somatic cell hybrids,have also provided insight into control of the liver phenotype(reviewed in reference 20). Rare, dedifferentiated sublines ofH4IIEC3 were isolated previously on the basis of their nonhe-patic morphology (12); these variants typically fail to expressmost liver genes, including the HNF-1 and HNF-4 genes (1, 5).Although the rescue of HNF-1 expression by ectopic HNF-4expression in these cells has been reported (24), the inability torestore expression of other liver genes suggests that these

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TRANSACTIVATION IN HEPATOMA CELL VARIANTS 7087

variant cells harbor multiple genetic or epigenetic lesions.Furthermore, these variant phenotypes generally behave asdominants in genetic crosses with differentiated hepatomacells.

Intertypic somatic cell hybrids formed by fusing hepatomacells with fibroblasts also fail to express liver functions, aphenomenon termed extinction (11). As in the dedifferentiatedhepatoma variants, the hybrid cells fail to express eitherHNF-4 or HNF-1 (2, 5). In both variants and cell hybrids, lossof the hepatic phenotype is reversible, either by rare reversionevents (in variants) or by segregation of fibroblast chromo-somes (in cell hybrids) (7, 12). In either case, expression ofHNF-1 and HNF-4 is restored, suggesting that this activationpathway is required to maintain the liver phenotype.We recently devised a selection scheme for isolating hepa-

toma cell variants with trans-acting defects in expression ofspecific liver genes, and we used this approach to isolatevariant lines deficient in otl-antitrypsin (alAT) gene expres-sion (3). The human alAT promoter, which requires bothHNF-1 and HNF-4 for activity (2, 13, 27), was fused to twoselectable markers, the murine adenine phosphoribosyltrans-ferase (APRT) gene (aprt) and the bacterial xanthine-guaninephosphoribosyltransferase gene (gpt), and the transgenes werestably introduced into APRT- hypoxanthine phosphoribosyl-transferase-deficient (HPRT-) rat hepatoma cells. Subsequentselection against expression of both transgenes resulted in theisolation of clonal variant lines that failed to express bothtransgenes as well as their chromosomal alAT alleles. Theexpression of other liver genes was also affected in many of thevariants. Here we report the characterization of several variantlines that appear to have specific defects in the pathway ofHNF-4/HNF-1 transactivation, and we use these lines to assessthe relative role of this pathway in maintaining liver geneexpression in living cells.

MATERIALS AND METHODS

Cell lines and culture conditions. Fado-2 is an APRT-HPRT- rat hepatoma line derived from H4IIEC3 (23). Fg-14cells were derived from Fado-2 by two rounds of transfection;they contain stably integrated plasmids pAT-aprt and pAT-gpt,as described previously (3). Fg-14 cells were maintained inmedium containing adenine-aminopterin-thymidine (AAT) toselect for AT-aprt transgene expression. Hepatoma variantlines were isolated from Fg-14 cells by selection in 2,6-diaminopurine (DAP) plus 6-thioxanthine, and the variantswere maintained in medium containing DAP, as describedpreviously (3). Somatic cell hybrids were generated by polyeth-ylene glycol-mediated fusion (23) followed by selection inG418 (500 ,ug/ml) and hygromycin B (1 mg/ml) or by selectionin hypoxanthine-aminopterin-thymidine (HAT). All cells weremaintained in 1:1 Ham's F12 medium-Dulbecco's modifiedEagle's medium plus 5% fetal bovine serum (Gibco). Antibi-otics were not used, and all cells were free of Mycoplasmacontamination as judged by staining with Hoechst 33258 (6).

Plasmid constructs. Recombinant plasmids contained in theFg-14 parental line are shown in Fig. 1. Plasmids pAT-aprt andpAT-gpt contain the -640 to -2 bp human alAT promoterfragment fused to the mouse aprt gene (14) and the bacterialgpt gene (31), respectively, as described previously (3).

Plasmid pKOneo contains the neomycin phosphotransferasegene (neo) driven by the simian virus 40 early promoter.pKOneoBl contains a second transcription unit in which theRous sarcoma virus (RSV) long terminal repeat drives expres-sion of HNF-1 cDNA (19). pKOneoSM is identical to pKO-neoBl except that the HNF-1 coding sequence contains two

alAT PromoterHNF-1

HNFt4

-640 En En} l-2TATA

Fado-2

x1 AT- aprt I AAT

Fap(pool)

a 1AT- gpt 4HAT

Fg-14

| DAP/6TX

Hepatoma VariantsM29, HI1, etc.

| AATor HAT

M29B, HI IB, etc

PhenonpeAPRT

APRT+

APRT, GPT

APRT , GPT

APRT, GPT

FIG. 1. Derivation of hepatoma variant lines used in this study.Plasmids pAT-aprt and pAT-gpt, containing the -640 to -2 bp alATpromoter fragment shown, were introduced into Fado-2 hepatomacells to generate the Fg-14 cell line as described previously (3).Negative selection against expression of both aprt and gpt transgeneswas used to generate the hepatoma variants (M29, Hi1, etc.), whicharose at a frequency of approximately 10-6. Subsequent counterselec-tion of hepatoma variants for reexpression of either the AT-aprt orAT-gpt transgene (in AAT or HAT, respectively) resulted in back-selectant clones (M29B, H11B, etc.) that arose at frequencies rangingfrom 10-3 to <10-7, depending on the particular variant that wascounterselected. Binding sites for the HNF-1 and HNF-4 transactiva-tors in the alAT promoter are shown at the top, as are putativeenhancer (En) regions. 6TX, 6-thioxanthine.

frameshift mutations which inactivate the protein's DNA bind-ing domain (19). pHNF4neo was prepared by replacing theHNF-1 expression cassette of pKOneoBl with an RSV-HNF4expression cassette derived from pRSVHNF4. pRSVHNF4was constructed by replacing the HNF-1 cDNA of pKOneoBlwith 3.4 kb of HNF4 cDNA from plasmid pLNE (37). PlasmidpCMVHytk (28) contains a hygromycin phosphotransferase-thymidine kinase fusion gene (HyTK) driven by the cytomeg-alovirus major intermediate-early promoter.DNA transfections. Exponentially growing cells were har-

vested and suspended to 1.2 x 107 cells per ml of ice-coldphosphate-buffered saline, and 30 ,ug of NdeI-linearized plas-mid DNA was added. The cells were electroporated (8) at 960,uF and 300 V, using a Bio-Rad Gene Pulser. The cells wereincubated in nonselective medium for 48 h, and selectivemedium was added. After 3 weeks, clones were pooled orpicked individually and expanded.RNA analysis. RNA was extracted from nearly confluent

monolayers in 100-mm-diameter dishes by washing the cells

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7088 BULLA AND FOURNIER

twice with saline, harvesting the cells by scraping, and pelletingthe cells in a microcentrifuge at 12,000x g for 10 s. Cells werelysed in Nonidet P-40 and extracted twice with phenol-chloro-form as described previously (2). RNA (5,ug) was denatured in50% formamide at65°C for 5 min and loaded onto 1%agarose-2.2 M formaldehyde gels (29). Gels were run at 7V/cm for 4 h, and RNA was transferred to nylon membranes(Zetabind; Cuno, Inc.) overnight. The blots were placed inhybridization solution (50% formamide, 1% bovine serumalbumin [BSA], 5% sodium dodecyl sulfate [SDS],1 mMEDTA, 0.5 M NaHPO4, 0.08 mg of yeast tRNA per ml [pH7.2]) for at least 30 min at42°C. Probe was added in the samehybridization solution, and the filters were incubated overnightat42°C. The filters were washed twice for5 min each time in2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)at room temperature and then for 30 min in 0.1x SSC-0.5%SDS at52°C and exposed to film for1 to 5 days. For theaclATprobe, filters were prehybridized and probed at 65°C in amixture containing 50% formamide,5x SSPE (1 x SSPE is 150mM NaCl, 1 mM EDTA, and 10 mM sodium phosphate [pH7.4]), 1% SDS,Sx Denhardt's solution (lx Denhardt's solu-tion is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02%BSA), and 10,ug each of poly(A) and poly(C) (Pharmacia) perml. Filters were probed overnight, washed at 65°C in 2xSSC-0.5% SDS and then at 65°C for 30 min in 0.1x SSC-0.5%SDS, and exposed to film for 1 to 3 days. Cloned DNAsequences fromao-tubulin (Ka-1 [10]), aldolase B (pHL413[36]), HNF-1 (pRSVB1 [19]), a-fibrinogen (pRoffib [1]), ty-rosine aminotransferase (TAT; pCTAT [34]), albumin(pRSA57 [33]), and alcohol dehydrogenase (ADH; pZK6-6[15]) genes were labeled with [32P]dCTP by the randomhexamer primer method (18). The otlAT probe was a 500-nucleotide [32P]UTP-labelled riboprobe from linearizedpAT500.2 (gift of K. Krauter, Albert Einstein College ofMedicine, New York, N.Y.).To detect HNF-4 expression, an RNase protection assay was

used (29). A 179-nucleotide PvuII-NheI (+613 to +802 bp)fragment of rat HNF-4 cDNA was blunt ended with T4 DNApolymerase and subcloned into SmaI-digested pBSKS(-)(Stratagene); the resulting plasmid was linearized with EcoRIand transcribed with T7 RNA polymerase. Ten micrograms oftotal cellular RNA was incubated with 5 x 106 cpm of probeand hybridized overnight at 52°C. The mixture was digestedwith RNase, and protected fragments were resolved on an 8%polyacrylamide gel. As an internal control for RNA integrityand product recovery, a labeled 145-nucleotide actin-specificriboprobe (16) was included in each reaction mix.

Gel shift assays. Nuclear extracts were prepared (35) andincubated with the following 32P-end-labeled, double-strandedoligonucleotides: HNF1, CCTTGGTTAATATTCACC; andOct-1, GGGGGTAA'lT17GCAT'IlCTAAGGG. Each bind-ing reaction mixture included S to 10 jig of nuclear extract in7.5% glycerol-60 mM KCl-5 mM MgCl2-0.1 mM EDTA-0.75mM dithiothreitol-0.5% aprotinin-2 mM benzamidine-0.3 jigof leupeptin per ml-1 jig of antipain per ml-1 mM phenyl-methylsulfonyl fluoride-2,ug of poly(dI-dC)-25 mM N-2-hy-droxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH7.6). The samples were incubated at room temperature for 30min, loaded onto 4% polyacrylamide gels, and run at 15 V/cmfor 3 to 4 h. The gels were dried and exposed to film for 1 to5 days.

RESULTS

Transgene selection alters the hepatic phenotype. Fg-14hepatoma cells contain two selectable transgenes under oulAT

114- m mr- N cn

6) M87C M29 N

cHNFIcHNFlSM

HNF1_

al-RT

Tubulin

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H1 l- HS2

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FIG. 2. Ectopic HNF-1 expression in hepatoma variant lines. Vari-ant lines M87, M29, Hll, and HS2 were transfected with expressionplasmids containing the RSV long terminal repeat driving expressionof either HNF-1 (labeled cHNF-1) or mutant HNF-lsm (labeledcHNF-lsm) cDNAs. The SM mutant construct contains two compen-sating, in-frame mutations that inactivate its DNA binding domain.For each variant line, 20 to 100 transfectant clones were pooled andexpanded, and RNA was extracted. M87B, M29B, and H11B arepolyclonal (50 to 200 clones) APRT+ counterselectants derived fromthe respective variant lines. Hllneo is a control line transfected withpKOneo alone. Cytoplasmic RNA was size fractionated on a 1%agarose gel, transferred to a nylon membrane, and sequentially probedwith atlAT, HNF-1, andao-tubulin probes as described in Materials andMethods. Two HNF-1 transcripts, 3.6 and 3.4 kb, were expressed inFg-14 parental hepatoma cells, whereas only the 3.6-kb transcript wasproduced from the transfected HNF-1 cDNA. FR is a rat hepatoma(FTO-2B)x rat fibroblast somatic cell hybrid line (2).

promoter control: AT-gpt and AT-aprt (3). Variant lines wereobtained by selecting APRT- GPT- subclones of Fg-14 (Fig.1). As described previously (3), most of the variant clonesisolated in this manner failed to express both transgenes as wellas their chromosomal otlAT alleles. Thus, the variants harbordefects that operate in trans. As a further test of whether thetransgenes and the endogenous ao1AT genes were regulatedcoordinately, APRT+ counterselectants were isolated from thehepatoma variants by selection in AAT. RNA blot hybridiza-tion demonstrated that the counterselectants reexpressedalAT mRNA (Fig. 2), indicating that the transgenes andendogenous genes were responding to common regulatorysignals. One variant line, HS2, failed to yield APRT+ or GPT+revertants (frequency of <10-7). We reasoned that determin-ing the nature of the defects that resulted in the loss of otlATgene expression in the variants might provide insight into transregulation of the liver phenotype.

Antitrypsin-deficient variants fail to express HNF-1. Themajor transactivators of the aclAT promoter are HNF-1and HNF-4. In rat hepatoma cells, HNF-1 is the majortransactivator of the aolAT promoter, as mutations in theHNF-1 binding site reduce promoter activity 100- to 500-fold (2). Mutations in the HNF-4 binding site also resultedin a significant (5- to 10-fold) decrease in promoter activity(2). Therefore, the HNF-4/HNF-1 transactivation pathwayis required for high-level aclAT gene expression. We there-fore determined whether HNF-1 was expressed in thealAT- variants. As shown in Fig. 2, HNF-1 mRNA couldnot be detected in the hepatoma variant M87, M29, Hll, orHS2. Variant H10 also failed to express HNF-1 mRNA (notshown).

MOL. CELL. BIOL.

TRANSACTIVATION IN HEPATOMA CELL VARLANTS 7089

alAT expression is rescued by ectopic HNF-1 expression inH11 variant cells. The lack of HNF-1 expression in the variantlines and the strong requirement for HNF-1 activation of theotlAT promoter suggested that the variants might containdefects in the HNF-4/HNF-1 transactivation pathway. To testwhether ectopic expression of HNF-1 was able to rescue thevariant phenotypes, we introduced a neo expression vectorcontaining cloned HNF-1 cDNA driven by the RSV longterminal repeat into four variant lines. As controls, paralleltransfections with a mutant form of HNF-1 (designated HNF-lsm) were performed. G418r clones were pooled, expanded,and analyzed by RNA blot hybridization. Introduction of theHNF-1 cDNA expression cassette, but not the neo vector aloneor the HNF-lsm mutant cassette, rescued alAT expression inthe H1l variant but not in the three other variant clones tested(Fig. 2).We also determined whether the AT-aprt and AT-gpt trans-

genes were reexpressed in the HNF-1 transfectants. Again,only Hil-derived transfectants reactivated transgene expres-sion, as assessed by their ability to survive in AAT or HATselective medium (data not shown). These results suggestedthat transgene expression was regulated coordinately withendogenous otlAT gene expression in the variant lines and thatthe primary defect in Hll cells was their inability to expressHNF-1.

Antitrypsin-deficient variants fail to express HNF-4. Thelack of HNF-1 expression in the hepatoma variant lines couldbe due to a number of possibilities, such as mutated or deletedHNF-1 gene sequences, altered HNF-1 mRNA stability, oraltered upstream regulation. Since previous reports indicatedthat HNF-4 activates HNF-1 expression (24, 39), we deter-mined whether HNF-4 expression was affected in the variantlines. Using an HNF-4-specific riboprobe in an RNase protec-tion assay, we found that all variant lines tested lackeddetectable HNF-4 mRNA (Fig. 3A). A -y-actin probe includedin the RNase protection assay served as an internal control forRNA loading and integrity.To test whether the lack of HNF-4 expression was the

primary cause of the HNF-1-deficient phenotype of the hepa-toma variants, an HNF-4 expression cassette in a neo vectorwas stably transfected into each variant line. Control transfec-tions were performed with neo vector DNA alone. HNF-4mRNA was readily detected in the pooled HNF-4 transfectantsbut not in cells transfected with the neo vector alone (Fig. 3A).Furthermore, ectopic expression of HNF-4 in variant linesH10, Hll, and M29 rescued expression of their chromosomalHNF-1 genes, although not to wild-type levels (Fig. 3B).HNF-1 mRNA expression was not rescued by HNF-4 in theHS2 variant.We next measured otlAT mRNA levels in the HNF-4-

transfected hepatoma variants. In two of the four variant linestested, otlAT mRNA expression was detected after HNF-4transfection (H1O and Hll; Fig. 3B), although not to wild-typelevels. HNF-4 did not rescue HNF-1 or alAT expression invariant HS2, and it rescued HNF-1 but not alAT expression invariant M29. This variant clone seems to harbor defects bothupstream and downstream of HNF-4.To determine whether the HNF-1 protein expressed in the

HNF-4-transfected hepatoma variants could bind to DNA,nuclear extracts from the transfectants were incubated with anHNF-1-specific oligonucleotide. HNF-1 binding activity wasdetected in H1l, H10, and M29 transfectants but not intransfectants derived from HS2 (Fig. 3C). The ubiquitousOct-1 binding activity was detected in all of the variant extracts(Fig. 3D).These results suggest that the primary defects in H10 and

H11 cells appear to be in their ability to express HNF-4mRNA. Clones M29 and HS2, however, appear to harboradditional defects, as neither the endogenous otlAT allele northe AT-gpt or AT-aprt transgene could be activated by ectopicexpression of HNF-4. The additional defects in M29 cells arenot likely to be nonfunctional aolAT alleles, as these genescould be activated in APRT+ counterselectants (Fig. 2).Rather, the additional defects in M29 appear to act in trans, asneither the AT-gpt nor AT-aprt transgenes was activated in theHNF-4 transfectants (data not shown). Finally, clone HS2appears to contain multiple defects in the HNF-4/HNF-1pathway, as evidenced by the inability of ectopic HNF-4expression to restore expression of HNF-1 (Fig. 3B).The variant phenotypes are recessive in somatic cell hy-

brids. The dedifferentiated hepatoma variants previously iso-lated and characterized by Deschatrette et al. were recognizedby virtue of their altered cellular morphology. The phenotypesof these lines are pleiotropic-few, if any, liver genes areexpressed, and the dedifferentiated phenotype appears domi-nant in cell hybrids (12). In contrast, the variants describedhere were selected for inactivation of a liver-specific promoter,and their morphologies were indistinguishable from that oftheir differentiated hepatoma parent. Therefore, we testedwhether the defects in these hepatoma variants were dominantor recessive in cell hybrids. To do this, the variants weretransfected with a plasmid containing the hygromycin B resis-tance gene. Pooled transfectants were then fused with neo-marked Fado-2 rat hepatoma cells (FAn), and whole-cellhybrids were selected in G418 plus hygromycin B. CytoplasmicRNAs prepared from each hybrid population contained readilydetectable levels of otlAT mRNA (Fig. 4). Hybrids formed byfusing wild-type hepatoma cells (FAn) with the H1l and HS2variants expressed alAT mRNA at levels comparable to thatof their wild-type FAn parent. The H10 x FAn and M29 xFAn crosses yielded hybrid populations that expressed alATmRNA, but at reduced levels. As all four crosses yieldedalAT-expressing hybrids, the variant phenotypes behave asgenetic recessives. The M29, and possibly H10, phenotypemight be considered to be partially dominant.To determine whether the AT-aprt and AT-gpt transgenes

were activated in these hepatoma x variant hybrids, thehygromycinr G418r hybrids were challenged with mediumcontaining either AAT or HAT. H11 x Fado-2 hybrids showed>90% survival in both AAT and HAT, indicating that expres-sion of both aprt and gpt transgenes was activated in thehybrids. This finding was in agreement with the conclusion thatthe Hll variant phenotype was recessive (see above). H10 xFado-2 hybrids survived in AAT but not HAT, possibly be-cause of loss of gpt sequences from the hybrids via chromo-some segregation (reversion of H10 to Gpt+ was <1-0 percell per generation). Interestingly, hybrid pools from fusions ofM29 or HS2 to Fado-2 cells failed to reactivate transgeneexpression (<1% survival in either HAT or AAT). It is notclear why transgene expression was not rescued in thesehybrids, whereas their alAT genes were expressed. Deletion,rearrangement, segregation, or underexpression of transgenesequences may have occurred in these cells.These experiments suggest that the primary defects of H10

and H1l cells are in expression of the HNF-4 gene itself or inexpression of upstream genes that are required for HNF-4expression. On the basis of their phenotypic responses toectopic expression of HNF-4 and/or HNF-1 (Fig. 2 and 3), M29and HS2 seem to contain multiple genetic or epigeneticdefects. To determine whether the different variants containeddistinct defects, the variants were fused with each other, andotlAT mRNA levels were assayed in the resulting hybrid

VOL. 14, 1994

7090 BULLA AND FOURNIER

Hepatomavariants

I_- ° 4N

< I I

cHNF4 L- -- - + - + - +

B.cHNF4

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al-AT

Tubulin

Hepatomavariants

- N

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C.

cHNF4

HNF1 -_

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0en XN,.

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FIG. 3. Ectopic HNF-4 expression in hepatoma variant lines. (A) RNase protection analysis of HNF-4 expression in variant lines stablytransfected with either pKOneo (- lanes) or the HNF-4 expression vector pRSVHNF4 (+ lanes). The Hll, H10, M29, and HS2 variants wereelectroporated with pKOneo or pRSVHNF4 as described in Materials and Methods, and G418-resistant clones were selected and pooled.Cytoplasmic RNA (10 ,ug) from the transfected cells was hybridized with a labeled, antisense riboprobe specific for HNF-4 (179 nucleotides) orwith a 145-nucleotide actin-specific riboprobe. RNase digestion products were separated on an 8% polyacrylamide gel. The gels were dried andexposed to film for 1 day (actin) or 1 week (HNF-4). (B) RNA blot hybridization analysis of HNF-1 and otlAT mRNA expression in HNF-4transfectants. Cytoplasmic RNA (5 ,ug) from the transfectants shown in panel A was size fractionated on an agarose gel, transferred to a nylonmembrane, and probed sequentially with HNF-1, oxlAT, and ot-tubulin probes as described in Materials and Methods. HNF-1 mRNA expressionwas activated in three of the four HNF-4-transfected variants, and two of those transfectants also reactivated expression of aelAT. (C and D)HNF-1 binding activity in the HNF-4 transfectants. Nuclear extracts from the transfected variant lines were incubated with end-labeledoligonucleotides containing binding sites for either HNF-1 (C) or the ubiquitous factor Oct-1 (D), and DNA-protein complexes were resolved on4% polyacrylamide gels. For the HNF-1 binding assays, the extracts were preheated at 65°C to eliminate interfering bands due to binding of vHNF1(4). - lanes are no-extract controls. RATlneo cells are pKOneo-transfected Rat-1 fibroblasts, and FT0-2B cells are rat hepatoma cell controls.F marks the position of free, uncomplexed oligonucleotides.

populations. Each variant line was marked with either neo orhygromycin B' plasmids, and the marked variants were fusedwith each other in all combinations. G418' hygromycin B'hybrids were selected, and their phenotypes were assayed byRNA blot hybridization. None of the variant combinationstested resulted in restoration of aclAT mRNA expression (Fig.4). Therefore, these variants were noncomplementing. Thisresult suggests that the four variant lines contain a commongenetic or, more likely, epigenetic defect that precludes dxlATgene expression. As the variants displayed different pheno-types in response to ectopic expression of HNF-4 and/orHNF-1, some of the variant lines (e.g., M29 and HS2) seem tocontain multiple defects. Other hepatoma variants (3) arebeing tested for complementation in a similar manner.

Activation of liver gene expression in Hll transfectantsstably expressing HNF-1. The existence of hepatoma variantsthat are specifically deficient in the HNF-4/HNF-1 transacti-vation pathway provides an opportunity to assess the contri-bution of HNF-1 to the activation of chromosomal liver-specific genes in living cells. To do this, we assayed expressionof several liver-specific mRNAs in HNF-1-deficient Hit cellsand in Hit transfectants that stably expressed HNF-1. pKO-neoBi was electroporated into Hit cells, and G418r transfec-tants were selected. Reactivation of the previously silentAT-aprt transgenes in these transfectants was assessed bychallenge in AAT selective medium (Table 1). Individualclones were isolated, and 50 to 200 cells of each were platedinto triplicate wells containing G418 alone, G418 plus AAT, orG418 plus DAP. Of 16 G418r clones isolated from the HNF-1transfection experiment, 9 contained significant numbers ofAPRT+ cells as determined by survival in AAT. In contrast,none of the G418r clones from a control experiment in whichthe HNF-lsm mutant cDNA was transfected were able tosurvive AAT selection (Table 1).

(xl RT

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FIG. 4. Analysis of hepatoma x variant and variant x variant cellhybrids. Each variant line was transfected with either pKOneo orpCMVHyTk, and transfectants were selected in G418 or hygromycinB, respectively. In the hepatoma X variant crosses, pCMVHyTk-marked transfectant pools were fused with neo-marked Fado-2 hepa-toma cells (FAn), and hybrids were selected by using G418 plushygromycin B. The hybrid cells were pooled (>100 clones per pool)and expanded, and alAT mRNA was analyzed by blot hybridization.In the variant X variant crosses, neo- or HyTK-marked cells of eachtype were fused by using polyethylene glycol, and hybrids were selectedin G418 and hygromycin B. Hybrid pools (20 to 200 clones per pool)were isolated and expanded, and RNA was extracted. CytoplasmicRNA was fractionated on a 1% agarose gel and probed with alAT andax-tubulin probes as described in the legend to Fig. 2. FAn is apKOneo-transfected derivative of Fado-2. The suffixes "h" and "n" inthe variant line designations indicate cells that were marked withpCMVHyTk or pKOneo, respectively.

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TRANSACTIVATION IN HEPATOMA CELL VARIANTS 7091

TABLE 1. Relative survival of G418' Hll transfectantsin DAP and AAT selective mediaa

No. of resistant clonesTransfectant designation

G418 G418 + DAP G418 + AAT

HNF-1 transfectedHllBl-l 100 15 60HllBl-2 200 200 4HllBl-3 19 0 22HllBl-4 100 100 1HllBl-5 200 200 20HllBl-6 100 100 50HllBl-7 100 36 3HllBl-8 200 200 0HllBl-9 28 12 1HllBl-10 100 0 36HllBl-12 100 30 100HllBl-13 100 100 1HllBl-15 200 27 100HllBl-16 100 50 100HllBl-17 100 100 30HllBl-18 100 100 0

HNF-lsm mutant transfectedH11SM-1 54 75 0H11SM-2 75 75 0H11SM-3 100 100 0H11SM-4 100 100 0H11SM-5 200 200 0H11SM-6 100 100 0H11SM-7 100 100 0H11SM-8 30 30 0H11SM-9 200 200 0H11SM-10 100 100 0

a Each clone was trypsinized and plated (approximately 200 cells per well) inmedium containing G418 alone, G418 plus DAP, or G418 plus AAT. Theapproximate number of clones surviving selection (rounded to the nearest 100 if>100) is shown.

To determine whether ectopic HNF-1 expression couldactivate expression of liver-specific genes in Hll cells, twotransfectant clones (Bl-1 and B1-16) that expressed HNF-1mRNA at hepatoma-typical levels were isolated and charac-terized. Clones transfected with mutant HNF-lsm (SM-2and SM-3) were used as controls. One transfectant (SM-2)expressed a truncated form of HNF-lsm mRNA, as seenpreviously in other HNF-1 and HNF-lsm transfectant clones(2), and the other (SM-3) expressed very low levels of HNF-lsm RNA. However, the phenotypes of these two cloneswere otherwise similar to those of HNF-lsm transfectantsthat expressed hepatoma-typical levels of HNF-lsm RNA(data not shown). RNA blot hybridizations demonstratedthat alAT mRNA expression was fully restored in the HNF-1-expressing transfectants but not in clones transfectedwith HNF-lsm (Fig. 5). Expression of three other genes thathave HNF-1-responsive promoters was also tested. All threegenes, encoding albumin, aldolase B, and a-fibrinogen, werestrongly activated by ectopic HNF-1 expression, although notto wild-type levels. Interestingly, aldolase B and at-fibrinogenwere activated to higher levels in B1-1 than in B1-16, despitethe fact that the two transfectants expressed similar levels ofHNF-1 mRNA. This finding suggests that other factors may belimiting expression in B1-16 cells. Two other liver genes,encoding TAT and ADH, were not strongly affected by thepresence or absence of HNF-1, their expression being withinthe limits of clonal variation for these hepatoma cell lines ineach transfectant population (Fig. 5). The observations thatthe TAT and ADH genes, as well as the phosphoenolpyruvate

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stably transfected with HNF-1. Cytoplasmic RNA was prepared fromrat hepatoma cells (Fg-14), fibroblasts (Ratl), the Hll variant, andHll cells transfected with either HNF-1 (B1-1 and B1-16) or the SMmutant form of HNF-1 (SM-2 and SM-3) cDNA. Triplicate RNAsamples, 5 p,g each, were fractionated on 4% agarose gels, transferredto nylon filters, and probed for expression of liver-specific mRNAs asdescribed in the legend to Fig. 3. The RNA blots were probed forexpression of HNF-1 mRNA and for expression of the HNF-1-responsive genes encoding a1AT (alAT), serum albumin (ALB),aldolase B (AldB), and a-fibrinogen (a-fib), as indicated. Expression oftwo other liver genes that are not activated strongly by HNF-1,encoding ADH and TAT, was also assessed. The HNF-1-expressingtransfectant clones B1-1 and B1-16 were chosen on the basis of theirsurvival in AAT (Table 1). A twofold titration of Fg-14 RNA isincluded for signal quantitation. a-Tubulin mRNA (Tubulin) served asa loading and transfer control.

carboxykinase and argininosuccinate synthetase genes (38),were highly expressed in Hit cells (data not shown) demon-strate that these genes do not require HNF-1 activation in vivo.These data also support the view that Hit cells express ahepatic phenotype that lacks only the HNF-4/HNF-1 activationpathway.The relative contribution of HNF-1 toward driving expres-

sion of chromosomal liver-specific genes in vivo can be esti-mated by comparing RNA blot hybridization signal intensitiesin Hit cells with or without cloned HNF-1 expression withintensities of serial dilutions of wild-type hepatoma cell RNA(Fig. 5). For example, alAT RNA levels were reduced by>64-fold in the Hit variant but were fully rescued in theHNF-1 transfectants. Therefore, a strong requirement forHNF-1 is observed, consistent with results from oclAT pro-moter mutagenesis experiments (2, 13, 27, 30). Similarly,albumin, aldolase B, and aL-fibrinogen mRNA expression wasstrongly dependent on the presence of HNF-1, as suggested byprevious studies (1, 21). Albumin mRNA levels, reducedapproximately 64-fold in Hit, were only modestly affected byexogenous HNF1 expression. Interestingly, although the TATpromoter has been reported to bind HNF-4 (32), TAT mRNAlevels were not affected by the loss of HNF-4 expression in the

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VOL. 14, 1994

7092 BULLA AND FOURNIER

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FIG. 6. Ectopic expression of HNF-1 rescues HNF-4 expression inH 1I variant cells. RNase protection analysis of HNF-4 mRNA expres-sion was performed with RNA from Hi1 cells stably transfected witheither the HNF-1 expression vector (pKOneoBl) or the mutantHNF-lsm expression vector (pKOneoSM). The Hi1 cells were elec-troporated as described in Materials and Methods, and G418r cloneswere selected and pooled (>40 clones or pool), or individual clones(B1-1 and B1-16) were isolated. Cytoplasmic RNA (10 pLg) from thecells was hybridized with a labeled antisense riboprobe specific forHNF-4 (179 nucleotides) or with a 145-nucleotide actin-specific ribo-probe. RNase digestion products were separated on an 8% polyacryl-amide gel. The gels were dried and exposed to film for 1 day (actin) or1 week (HNF-4). Fg-14 is the parental hepatoma cell line from whichHll cells were derived. B1-1 and B1-16 are H1l transfectant clonesexpressing HNF-1.

hepatoma variants. This finding suggests either that HNF-4does not contribute to activation of the TAT gene in vivo orthat other factors compensate for HNF-4 binding to facilitateTAT transcription in the variants. In either case, these resultssuggest that transient transfection experiments should beinterpreted with caution when one is attempting to extrapolateto the in vivo regulation of gene expression.HNF-4 expression is rescued by ectopic HNF-1 expression in

Hl1 variant cells. The pathway of HNF-4/HNF-1 transactiva-tion is thought to be sequential: HNF-4 can activate the HNF-1promoter (24, 39), and HNF-1 activates many liver genes (9,40). The phenotypes of our hepatoma variants are largelyconsistent with this model. However, to test the postulatedsequential aspect of the transactivation pathway, we assayedHNF-4 expression in Hll variant cells that had been trans-fected with HNF-1. To do this, we used an RNase protectionassay to measure HNF-4 mRNA levels in H1 1 cells transfectedwith neo vectors containing an expression cassette encodingeither authentic HNF-1 or the HNF-lsm mutant protein.Surprisingly, expression of HNF-1 cDNA in H1l cells restoredHNF-4 expression, but the HNF-lsm mutant cDNA had noeffect (Fig. 6). Furthermore, HNF-4 mRNA expression wasactivated in the two HNF-1-expressing H11 transfectant clones(B1-1 and B1-16) whose liver gene phenotypes are shown inFig. 6, with B1-1 expressing more HNF-4 mRNA than B1-16(Fig. 6). These results suggest that HNF-1 can activate HNF-4gene expression, resulting in an autoregulatory loop in whichHNF-1 activates its own transcription through HNF-4.Whether HNF-1 activates HNF-4 expression directly or indi-rectly is not yet known.

DISCUSSION

Genetic analyses of tissue-specific gene expression in mam-malian cells have been hampered by the difficulty in generatingmutant or variant lines with specific alterations in expression ofcell-specific genes. Current research relies heavily on the

behavior of isolated DNA fragments from tissue-specific genestransfected into different cell types. While these approacheshave been useful for identifying and isolating many trans-actingfactors involved in cell-specific transcription, they may notadequately assess the roles of those factors in activatingchromosomal genes in vivo. Furthermore, identifying targetgenes for individual transactivators requires a detailed knowl-edge of each gene promoter.To overcome some of these limitations, we devised a strat-

egy for isolating cells that are defective in transactivating atissue-specific promoter (3). The results described in thisreport show that hepatoma variants obtained by selectionagainst a_1AT promoter activity are generally deficient in theHNF-4/HNF-1 transactivation pathway, and they comprisedifferent phenotypic classes. Variant HS2 contained multipledefects, effectively blocking each step of the transactivationcascade. Other variants displayed simpler phenotypes: in H10and Hi1 cells, ectopic HNF-4 expression activated expressionof the chromosomal HNF-1 allele, and the oalAT gene wasexpressed. Forced HNF-1 expression also rescued cxlAT geneexpression in these cells. In contrast, transfection of the M29variant with HNF-4 cDNA resulted in activation of the endog-enous HNF-1 gene, but the oclAT genes remained inactive.Superficially at least, this response is similar to that of thededifferentiated hepatoma cells of Deschatrette et al. (12), inthat the silent HNF-1 gene could be activated by ectopicHNF-4 expression (24), but downstream genes were not ex-pressed.Our hepatoma variants have allowed us to identify a feature

of the HNF-4/HNF-1 transactivation pathway that was notpreviously known. Using transfection tests to complement thevariant lesions, we identified an autoregulatory loop in theHNF-4/HNF-1 cascade. As expected from the current model,HNF-1 rescued aLlAT expression in Hi1 variant cells that aredeficient in both HNF-1 and HNF-4. Surprisingly, HNF-4expression was also activated in response to HNF-1. Bothactivation phenotypes were specific, as they did not result fromexpression of a nonfunctional form of HNF-1. These observa-tions suggest that HNF-1 plays a central role in this pathway ofhepatic gene activation, contributing to its own activation aswell as that of downstream genes. Whether the activation ofHNF-4 by HNF-1 is a direct or an indirect effect is not clearfrom current data.

It is interesting that each variant line failed to expressHNF-4. Previous studies of the alAT promoter by transienttransfection have shown that the HNF-1 binding site is crucialfor basal promoter activity, since mutations in this site reducedcell-specific expression 100- to 500-fold (2, 13). Therefore, itwould have been reasonable to expect some of our variants tobe defective only in HNF-1 expression or activity. As all of thevariants tested failed to express HNF-4, and as HNF-4 rescuedHNF-1 expression in three of four variant lines tested, theseresults suggest that the most common mechanism by whichHNF-1 is down-regulated in these cells is by loss of HNF-4. AsHNF-4 is thought to activate the HNF-1 gene directly (24, 39),this may indicate simply that there are multiple steps upstreamof HNF-4, including HNF-1, each potentially a target forinactivation. Little is known of the regulatory pathway up-stream of HNF-4; therefore, further studies of the variantphenotypes might provide information about HNF-4 geneactivation. It is also possible that variant lines with moremodest reductions in otlAT mRNA expression (3) are defec-tive only in HNF-1 expression.Although loss of HNF-4 and HNF-1 could potentially ex-

plain the phenotypes of all of our variant lines, two of thevariants contained additional genetic or epigenetic defects.

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TRANSACTIVATION IN HEPATOMA CELL VARIANTS 7093

These additional defects precluded alAT gene expressioneven in the presence of HNF-1 and/or HNF-4. This phenotypecould be explained by assuming that some variants (e.g., H10and Hll) are deficient only in HNF-4 activation, whereasothers (e.g., M29) contain a defect that is responsible both forHNF-4 repression and for the failure of HNF-1 to activate thecalAT gene. However, although the variant phenotypes wererecessive to wild type, none of the variants complemented eachother. This finding suggests that variant lines like M29 harbormultiple lesions in the pathway of gene activation. Both thefrequencies with which variants arise in these populations(approximately 10-6) and the observation that some variantscontain multiple defects suggest that the variant phenotypesarise from epigenetic events, such as somatic imprinting. Inspite of this, the variant phenotypes are sufficiently stable toallow for molecular and genetic analysis, as documented here.The variants described in this report are qualitatively differ-

ent from the dedifferentiated hepatoma variants of Descha-trette et al. (12), such as H5. Most of our variant linesdisplayed a hepatoma morphology indistinguishable from thatof their parental hepatoma cells. In contrast, the distinctive,epithelial morphology of H5 and its relatives was the basisupon which these dedifferentiated hepatoma lines were origi-nally recognized and isolated. Furthermore, our variants re-verted to the parental hepatoma phenotype at frequencies(10-3 to 10-6) that were much higher than the frequency(10-8) of dedifferentiated-differentiated reversion. More nota-ble, however, is that our variants were defective only in thepathway of HNF-4/HNF-1 transactivation: liver genes thatrequire these specific transactivators were down-regulated as aresult of lack of transactivation, but expression of other livergenes was unaffected. For example, expression of vHNF-1(HNF-1p), a liver-enriched binding factor that recognizes thesame DNA sequence as HNF-1 (HNF-ia) itself, was notaffected in any of our variant clones (data not shown). Incontrast, H5 cells display a pleiotropic phenotype that affectsall or nearly all liver functions, and this variant phenotype isdominant in cell hybrids. Our variant lines behaved as geneticrecessives, consistent with simple loss-of-function lesions, andthus are likely to be useful in complementation assays designedto restore HNF-1/HNF-4 function.Numerous studies have shown that HNF-1 binding is re-

quired for activation of many different liver promoters, asassessed by transient transfection experiments (2, 13, 17, 21,22, 27, 30). As the Hit variant line appears to be specificallydeficient in HNF-4/HNF-1 transactivation, and as the pathwaycan be rescued by using cloned transactivators, this provides auseful system to test the contributions of the HNF-4/HNF-1cascade on expression of chromosomal liver genes in vivo. Inour experiments, genes whose promoters have shown a re-quirement for HNF-1 transactivation in transient assays (e.g.,the alAT, albumin, a-fibrinogen, and aldolase B genes) wereexpressed at low levels in HNF-1-deficient Hit cells, butexpression could be rescued by ectopic HNF-1 expression. Onthe other hand, genes with HNF-4/HNF-1-independent pro-moter (e.g., the TAT, ADH, and argininosuccinyl synthetasegenes) were expressed at high levels in Hi1 cells. Therefore,these results show the effects of HNF-1 depletion in anotherwise unaffected hepatic environment, and they show thatHNF-1 plays an important role in maintaining the liver phe-notype. Hit cells will be useful for assessing the role ofHNF-4/HNF-1-transactivation on any liver function.

It is interesting that expression of the TAT gene, reported tobe regulated by HNF-4 (32), was unaltered in Hit cells thatlacked detectable HNF-4 mRNA. This finding suggests thatthe ability of a factor to bind to a promoter in vitro does not

necessarily correlate with the role of that factor in driving geneexpression in vivo. It would be interesting to test whether theHNF-4 binding site of the TAT promoter is occupied in Hitcells and whether other factors might bind in the absence ofHNF-4.

It should be possible to use the variant lines described hereto obtain information concerning genetic regulation of theHNF-4/HNF-1 cascade. For example, it might be possible tocomplement the variant phenotypes by introducing singlechromosomes from wild-type hepatoma cells and selecting fortransgene rescue. This would allow for the identification ofupstream genes in the HNF-4/HNF-1 cascade and for studiesof their function.

ACKNOWLEDGMENTS

We thank Linda Breeden and Adam Geballe for critically readingthe manuscript.These studies were supported by grant GM26449 from the National

Institute of General Medical Sciences.

REFERENCES1. Baumhueter, S., G. Courtois, and G. R. Crabtree. 1988. A variant

nuclear protein in dedifferentiated hepatoma cells binds to thesame functional sequences in the 3-fibrinogen gene promoter asHNF-1. EMBO J. 7:2485-2493.

2. Bulla, G. A., V. DeSimone, R. Cortese, and R. E. K. Fournier. 1992.Extinction of al-antitrypsin gene expression in somatic cell hy-brids: evidence for multiple controls. Genes Dev. 6:316-327.

3. Bulla, G. A., and R. E. K. Fournier. 1992. Direct selection ofhepatoma cell variants deficient in cal-antitrypsin gene expression.Somatic Cell Mol. Genet. 18:361-370.

4. Cereghini, S., M. Blumenfeld, and M. Yaniv. 1988. A liver-specificfactor essential for albumin transcription differs between differen-tiated and dedifferentiated rat hepatoma cells. Genes Dev. 2:957-974.

5. Cereghini, S. M., M. Yaniv, and R. Cortese. 1990. Hepatocytededifferentiation and extinction is accompanied by a block in thesynthesis of mRNA coding for transcription factor HNF1/LFB1.EMBO J. 9:2257-2263.

6. Chen, T. R. 1977. In situ detection of mycoplasma contaminationin cell culture by fluorescent Hoechst 33258 stain. Exp. Cell Res.104:255-262.

7. Chin, A. C., and R. E. K. Fournier. 1987. A genetic analysis ofextinction: trans-regulation of 16 liver-specific genes in hepatoma-fibroblast hybrid cells. Proc. Natl. Acad. Sci. USA 84:1614-1618.

8. Chu, G., H. Hoyakawa, and P. Berg. 1987. Electroporation for theefficient transfection of mammalian cells with DNA. Nucleic AcidsRes. 15:1311-1326.

9. Courtois, G., S. Baumhueter, and G. R. Crabtree. 1988. Purifiedhepatocyte nuclear factor 1 interacts with a family of hepatocyte-specific promoters. Proc. Natl. Acad. Sci. USA 85:7937-7941.

10. Cowan, N. J., P. R. Dodner, E. V. Fuchs, and D. W. Cleveland.1983. Expression of alpha-tubulin genes: interspecies conversionof 3' untranslated regions. Mol. Cell. Biol. 3:1738-1745.

11. Davidson, R. L., B. Ephrussi, and K. Yamamoto. 1966. Regulationof pigment synthesis in mammalian cells as studied by somatichybridization. Proc. Natl. Acad. Sci. USA 56:1437-1440.

12. Deschatrette, J., E. E. Moore, M. Dubois, and M. C. Weiss. 1980.Dedifferentiated variants of a rat hepatoma: reversion analysis.Cell 19:1043-1051.

13. De Simone, V., G. Ciliberto, E. Hardon, G. Paonessa, F. Palla, L.Lundberg, and R. Cortese. 1987. Cis- and trans-acting elementsresponsible for the cell-specific expression of the human arl-antitrypsin gene. EMBO J. 6:2759-2766.

14. Dush, M. K., J. M. Sikela, S. A. Kahn, J. A. Tischfield, and P. J.Sambrook. 1985. Nucleotide sequence and organization of themouse adenine phosphoriboslytransferase gene: presence of acoding region common to animal and bacterial phosphotrans-ferases that has a variable intron/exon arrangement. Proc. Natl.Acad. Sci. USA 82:2731-2735.

15. Edenberg, H. J., K. Zhang, K. Fon, W. F. Bosran, and T. K. Li.

VOL. 14, 1994

7094 BULLA AND FOURNIER

1985. Cloning and sequencing of a cDNA encoding the completemouse liver alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA82:2262-2266.

16. Enoch, T., K. Zinn, and T. Maniatis. 1986. Activation of thebeta-interferon gene requires an interferon-inducible factor. Mol.Cell. Biol. 6:801-810.

17. Feuerman, M. H., R. Godbout, R. S. Ingram, and S. M. Tilghman.1989. Tissue-specific transcription of the mouse ox-fetoproteingene promoter is dependent on HNF-1. Mol. Cell. Biol. 9:4204-4212.

18. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiola-belling restriction endonuclease fragments to high specific activity.Anal. Biochem. 132:6-13.

19. Frain, M., G. Swart, P. Monaci, A. Nicosia, S. Stampfli, R. Frank,and R. Cortese. 1989. The liver-specific transcription factor LF-B1contains a highly diverged homeobox DNA binding domain. Cell59:145-157.

20. Gourdeau, H., and R. E. K. Fournier. 1990. Genetic analysis ofmammalian cell differentiation. Annu. Rev. Cell Biol. 6:69-94.

21. Gregori, C., A. Kahn, and A.-L. Pichard. 1993. Competitionbetween transcription factors HNF1 and HNF3 and alternativecell-specific activation by DBP and C/EBP contribute to theregulation of the liver-specific aldolase B promoter. Nucleic AcidsRes. 21:897-903.

22. Herbomel, P., A. Rollier, F. Tronche, M.-O. Ott, M. Yaniv, andM. C. Weiss. 1989. The rat albumin promoter is composed of sixdistinct positive elements within 130 nucleotides. Mol. Cell. Biol.9:4750-4758.

23. Killary, A. M., and R. E. K. Fournier. 1984. A genetic analysis ofextinction: trans-dominant loci regulate expression of liver-specifictraits in hepatoma hybrid cells. Cell 38:523-534.

24. Kuo, C. J., P. Conley, L. Chen, F. M. Sladek, J. E. Darnell, Jr., andG. R. Crabtree. 1992. A transcriptional hierarchy involved inmammalian cell-type specification. Nature (London) 355:457-461.

25. Lai, E., and J. E. Darnell. 1991. Transcriptional control inhepatocytes: a window on development. Trends Biochem. Sci.16:427-430.

26. Lazzaro, D., V. De Simone, L. Magistris, E. Lehtanen, and R.Cortese. 1992. LFB1 and LFB3 homeoproteins are sequentiallyexpressed during kidney development. Development 114:469-479.

27. Li, Y., R.-F. Shen, S. Y. Tsai, and S. L. C. Woo. 1988. Multiplehepatic trans-acting factors are required for in vitro transcriptionof the human al-antitrypsin gene. Mol. Cell. Biol. 8:4362-4369.

28. Lupton, S. D., L. L. Brunton, V. A. Kalberg, and R. W. Overell.1991. Dominant positive and negative selection using a hygromy-cin phosphotransferase-thymidine kinase fusion gene. Mol. Cell.Biol. 11:3374-3378.

29. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

30. Monaci, P., A. Nicosia, and R. Cortese. 1988. Two differentliver-specific factors stimulate in vitro transcription from thehuman old-antitrypsin promoter. EMBO J. 7:2075-2087.

31. Mulligen, R. C., and P. Berg. 1981. Factors governing the expres-sion of a bacterial gene in mammalian cells. Mol. Cell. Biol.1:449-459.

32. Nitsch, D., M. Boshart, and G. Schultz. 1993. Extinction oftyrosine aminotransferase gene activity in somatic cell hybridsinvolves modification and loss of several essential transcriptionalactivators. Genes Dev. 7:308-319.

33. Sargent, T. D., J. R. Wu, J. M. Sala-Trepat, R. B. Wallace, A. A.Reyes, and J. Bonner. 1979. The rat serum albumin gene: analysisof cloned sequences. Proc. Natl. Acad. Sci. USA 76:3256-3260.

34. Scherer, G., W. Schmid, C. M. Strange, W. Rowekamp, and G.Schultz. 1982. Isolation of cDNA clones coding for rat tyrosineaminotransferase. Proc. Natl. Acad. Sci. USA 79:7205-7208.

35. Shapiro, D. J., P. A. Sharp, W. W. Wahli, and M. J. Keller. 1988.Laboratory methods: a high-efficiency HeLa cell nuclear transcrip-tion extract. DNA 7:47-52.

36. Simon, M.-P., C. Besmond, D. Cottreau, A. Weber, P. Chaumet-Riffaud, J.-C. Dreyfus, J. S. Trepat, J. Marie, and A. Kahn. 1983.Molecular cloning of cDNA for rat L-type pyruvate kinase andaldolase B. J. Biol. Chem. 258:14576-14584.

37. Sladek, F. M., W. Zhong, E. Lai, and J. E. Darnell, Jr. 1990.Liver-enriched transcription factor HNF-4 is a novel member ofthe steroid hormone receptor superfamily. Genes Dev. 4:2353-2365.

38. Surh, L. C., S. M. Morris, W. E. O'Brien, and A. L. Beaudet. 1988.Nucleotide sequence of the cDNA encoding the rat argininosuc-cinate synthetase. Nucleic Acids Res. 16:9352.

39. Tian, J., and U. Schibler. 1991. Tissue-specific expression of thegene encoding hepatocyte nuclear factor 1 may involve hepatocytenuclear factor 4. Genes Dev. 5:2225-2234.

40. Tronche, F., and M. Yaniv. 1992. HNF1, a homeoprotein memberof the hepatic transcription regulatory network. Bioessays 14:579-586.

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