Proc. Natl. Acad. Sci. USAVol. 89, pp. 7300-7304, August 1992Biochemistry

Hepatocyte nuclear factor la is expressed in a hamster insulinomaline and transactivates the rat insulin I gene

(insulin gene expression/transcription/tissue specificty)

LEISHA A. EMENS*, DIANE W. LANDERSt, AND LARRY GENE MOSS*ttDepartments of *Cell Biology and tMedicine, Baylor College of Medicine, Houston, TX 77030

Communicated by Roger H. Unger, April 24, 1992

ABSTRACT Systematic mutational analysis previouslyidentified two primary regulatory elements within a minien-hancer (-247 to -198) of the rat insulin I promoter that arecritical for transcriptional activity. The Far box (-241 to-232) and the FLAT element (-222 to -208) synergisticallyupregulate transcription and, together, are sufficient to confertissue-specific and glucose-responsive transcriptional activityon a heterologous promoter. Detailed analysis of the FLATelement further revealed that, in addition to the positiveregulatory activity it mediates in tandem with the Far box, itis a site for negative regulatory control. A portion of the FLATelement bears considerable sequence imilarit to the consensusbinding site for hepatocyte nuclear factor la (HNFla; LF-B1),a liver-enriched homeodomain-containing transcription factor.Here we show that the HNFl-like site within the FLAT elementexhibited positive transcriptional activity in both HepG2 andHIT cells and bound similar, but distnguihable, nuclearprotein complexes in the respective nuclear extracts. Screeningof a hamster ulioma cDNA library with a PCR-derivedprobe encompassing the DNA-binding domain of rat HNFlaresulted in isolation of a hmsIrHNFla (hHNFla) cDNAbomolog. Specific antiserum identified the HNFla protein asone component of a specific FLAT-binding complex in HITnuclear extracts. Expression ofthehHNFlacDNA inCOS cellsresulted in transactivation of reporter constructs containingmultimerized segments ofthe rat insulin I minienhanr. Thus,HNFla, one component ofa DNA-binding complex involved intranscriptional regulation of the rat insulln I gene, may play asignificant role in nonhepatic as well as hepatic gene transcrip-tion.

The expression of the insulin gene in the adult mammal isspecifically localized to (3 cells of the pancreatic islet and istightly regulated by blood glucose concentration. Detailedstudies have defined a small region (-247 to -198) in thepromoter of the rat insulin I gene, the FF-minienhancer,capable of conferring both tissue-specific and glucose-responsive transcriptional activity on a heterologous pro-moter (1). It is composed oftwo primary regulatory elements,the Far box (-241 to -232) and the FLAT element (-222 to-208), which interact to strongly upregulate transcription.Further analysis characterized the FLAT element as a clusterof several cis loci that mediates discrete positive and negativeactivities (2). The activity of a positive locus, FLAT-F, isrevealed only upon mutation of the adjacent negative locus,FLAT-E. This functional complexity is reflected by its abilityto specifically bind a number of DNA-binding proteins (2).Notably, the sequence of the FLAT-F element bears signif-icant similarity to the consensus binding site for hepatocytenuclear factor 1 (HNF1; LF-B1), a liver-enriched homeo-domain-containing transcription factor. Such similarity could

suggest that HNF1 itself or related factors may play a role intranscriptional regulation of the rat insulin I gene.HNFla was originally characterized as a liver-specific

transcription factor that binds an A+T-rich sequence presentin the promoters of many genes, including (3-fibrinogen,a1-antitrypsin, and albumin, transcribed primarily in the liver(3, 4). Subsequent work extended the tissue distribution ofHNFla messengerRNA to include the kidney, intestine, andspleen (5). Two characteristics distinguish the protein fromother homeodomain-containing transcription factors; it con-tains an additional 21-amino acid loop within the DNA-binding region (6) and dimerizes via an N-terminal domain(7). A related factor with similar dimerization and DNA-binding motifs, HNF1f3 (LF-B3, vHNF1), displays a tissuedistribution distinct from, but overlapping with, that ofHNFla (8-10). The two factors are thought to comprise partof a system that regulates the differentiation of specializedepithelia.Here we report the expression ofHNFla in the pancreatic

islet 3-cell-derived insulinoma cell line HIT.§ Furthermore,we show that HNFla specifically binds to and transactivatesmultimerized rat insulin I gene enhancers containing a naturalHNF1 site. These observations define an unusual role forHNFla outside of the hepatocyte and suggest that it may beone of a complex array of factors that together maintaintissue-specific and physiologic regulation of insulin genetranscription.

MATERIALS AND METHODSLsolation and Analysis of cDNA Clones. Rat liver poly(A)+

RNA was used to generate a reverse transcriptase PCR-derived probe encompassing the homeodomain region of ratHNFla (LF-B1). This probe was used to screen 500,000plaques from a hamster insulinoma cell (HIT-T15 M.2.2.2)Agtll cDNA library (11) at medium stringency (12). Threepositive clones designated 2, 2.5, and 3.5 showed strongsequence similarity to the known sequence for rat HNFla.Sequence similarity for clone 2.5 was found to extend intoboth 5' and 3' untranslated regions; this cDNA insert wassubcloned into pBluescript and bidirectionally sequenced byusing a series of 5' and 3' exonuclease deletions (13).Phmid Constrcton. Multimers were constructed from

oligonucleotides (see Table 1) synthesized with BamHI andBgl II sites on opposite ends; oligonucleotides were annealedand incubated with T4 DNA ligase in the presence ofBamHIand Bgl II to produce unidirectional ligation and size frac-tionated. Five-copy multimers were subcloned into either theBgl II site ofpTE2Asn [-109 thymidine kinase chloramphen-icol acetyltransferase (CAT)] (14) or the BamHI site of -36

Abbreviations: HNFla, hepatocyte nuclear factor la; hHNFla,hamster HNFla; CAT, chloramphenicol acetyltransferase.tTo whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. M95297).

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prolactin CAT (15) or S8AABam (-85 insulin CAT) (2). ThepBShHNFla and pBJ5hHNFla plasmids were constructedby subcloning the 2.5-kilobase phage insert into the EcoRIsite of pBluescript or the pBJ5 expression vector (16),respectively. The pBJ5 vector, designed for COS cell ex-pression, contains the Simian virus 40 origin of replicationand the SRa promoter (17).

Electrophoretic Mobility-Shift Assays. Nuclear extracts (18,19) were prepared from the hamster insulinoma line HIT-T15M.2.2.2 (20), the human hepatoma line HepG2, and thefibroblast-derived line COS-1. Radioactive probes were pre-pared by labeling one strand in the presence of [.-32P]ATPand T4 DNA kinase, annealing it to an excess of the oppositestrand, and resolving the reaction over G50 Sepharose col-umns. Gel mobility-shift assays were performed with 15 ktg ofnuclear extract in a reaction mixture of 10 mM Hepes, pH7.8/75 mM KCl/2.5 mM MgCl2/0.1 mM EDTA/1 mM di-thiothreitol/3% Ficoll/2 ,ug of poly(dI-dC):poly(dI-dC) (2).Competitions included 100-fold molar excess of unlabeledprobe, while antisera experiments incorporated 1 ,ul of eitherpreimmune or HNFla-specific antiserum. In each case,reaction mixtures were preincubated at room temperature for5 min before addition of labeled probe. After incubation ofcomplete reaction mixtures at room temperature for 15 min,reactions were resolved on 4.5% polyacrylamide gels in 0.5xTBE (lx TBE = 90 mM Tris/64.6 mM boric acid/2.5 mMEDTA, pH 8.3) and autoradiographed.

Cell Lines and Transfections. The HIT cell line was grownas described (14). HepG2 and COS-1 cell lines were grown inDulbecco's modified Eagle's medium with 10%6 fetal calfserum. Cells at a density of 2 x 106 cells per 100-mm dishwere cotransfected with 5 pg of test plasmid and 3 ug ofcytomegalovirus P-galactosidase (CMVf8gal) (21) internalcontrol plasmid by the calcium phosphate technique (22).Transactivation experiments were similarly performed with 2pg of test plasmid, 5 jg of pBJ5hHNFla expression vectoror control DNA [pUC18 or pBJ5 with hamster HNFla(hHNF1a) in the antisense orientation], and 3 pg ofCMVBgalplasmid. CAT activity of extracts prepared 48 hr after trans-fection (22) is expressed in arbitrary units of relative CATactivity normalized to assayed (-galactosidase activity. Dataare given as means of three independent determinations.

RESULTSHepG2 and HIT Cells Contain Similar HNFl-like Binding

Activities. The A+T-rich FLAT element in the 5' flankingregion of the rat insulin I gene bears significant sequencesimilarity to the consensus binding site for the liver-enrichedtranscription factor HNF1 (Table 1). To examine the poten-tial relationship between HNF1-related factors and proteinsinvolved in FLAT-mediated rat insulin I gene transcription,

Table 1. Sequence comparison of HNF1 sites and rat insulin IFLAT elementsf3}Fibrinogen*al-AntitrypsintAlbumintHNF1 consensusRat insulin I FLAT§Rat insulin I FLAT-F§Rat insulin I FLAT-E§Rat insulin I FLAT-M§

GATCTGTCAAATATTAACTAAAGGGGATCTTGGTTAATATTCACCGGATCTTGGTTAGTAATTACTAAG

TGGTTAATNATTAACAAGATCTTGTTAATAATCTAATTACCGGATCTTGTTAATAATCGACTGACCGGATCTTGGT M AATCTAATTACCGGATCTTGGTU&AATCGAQTfACCG

*Rat *-fibrinogen HNF1 site at position -84 (3).tHuman ai-antitrypsin HNF1 site at position -63 (23, 24).tHuman albumin HNF1 site at position -358 (25).§Rat insulin I FLAT element at position -222 (2). Mutated nucleo-tides are underlined. Sequences of the FLAT element derivativesare shown. FLAT-M designates the FLAT element with both F andE sites mutated.

we screened nuclear extracts from hepatoma (HepG2), in-sulinoma (HIT-T15 M.2.2.2), and fibroblast-derived (COS-1)cell lines by gel mobility-shift analysis (Fig. 1). Probesincluded the wild-type FLAT element, the two subelementsFLAT-F (E-site mutant) and FLAT-E (F-site mutant), and aprobe with both the FLAT-F and FLAT-E sites mutated,FLAT-M (Table 1). The quality of the nuclear extracts wasdemonstrated by their ability to bind a CCAAT sequencepreviously shown to interact with proteins from a variety ofcell types (Fig. 1A Left) (26, 27). HepG2 and HIT extractscontained activities (bands I and II) that bound known HNF1sites in the *-fibrinogen, al-antitrypsin, and albumin genes(Fig. 1A Middle). The factors in HepG2 cells were lessabundant or had lower binding affinities for the probes thanproteins in HIT nuclear extracts. These complexes also hadslightly altered migration rates compared to those in HITcells (bands I and II), suggesting that the two cell types maycontain distinct protein complexes that exhibit similar bind-ing specificities. The rat insulin I FLAT element also boundproteins (bands III and IV) present in HepG2 and HITnuclear extracts (Fig. 1A Right). Interestingly, while thecomplex previously defined as representing the FLAT-Factivity in HIT nuclear extracts (band III) (2) was notdetected in HepG2 when probed with the wild-type FLATsite, the specific FLAT-F probe revealed a binding patternsimilar to those seen in HepG2 extracts with probes contain-ing known HNF1 sites. This observation lends further sup-port to the hypothesis that HepG2 and HIT cells may containfactors that display similar but subtly distinct binding spec-ificities. Little binding activity was present in COS cellnuclear extracts.We examined potential differences in binding specificity by

competition with excess unlabeled probe (Fig. 1B). Bindingof proteins to the (3-fibrinogen probe in both HepG2 (bandsI and II) and HIT (band III) nuclear extracts was blocked bycompetition with the 3-fibrinogen HNF1 site, the wild-typeFLAT element, and the FLAT-F site, but not by the FLAT-Eelement or the FLAT-M probe (Fig. 1B Left). A complex inHepG2 extracts binding to the wild-type FLAT element(which comigrates with band V present in HIT extracts) wasblocked by competition with the ,3-fibrinogen probe and allFLAT region probes except the double-mutant probeFLAT-M. Taken together, these data define the rat insulin IFLAT element as a specific HNF1-binding site and identifyHNF1-like binding activity in insulinoma cell nuclear ex-tracts. All binding to the wild-type FLAT element in HITextracts was blocked by competition with the f-fibrinogenHNF1 site; competition using probes derived from the FLATelement reproduced previous data defining distinct specific-ities for the FLAT-F (band IV) and FLAT-E (band V) bindingcomplexes (2) (Fig. 1B Right). These data extend thosefindings by suggesting that the band present in HepG2nuclear extracts that comigrates with band V may representa factor related to the HIT cell FLAT-E repressor (band V).The FLAT-M probe was unable to compete for any bindingactivity in HIT nuclear extracts. An unusual binding activity(band VI) appeared with competition using both f-fibrinogensites and FLAT element probes in HepG2, but not HIT,nuclear extracts. Although it is unclear what this complexrepresents, its binding was unaltered by the addition ofantiserum specific for HNFla (8) to the reaction mixture,suggesting that HNFla was not a part of the complex (datanot shown).The (-Fibrinogen and FLAT-F Elements Are Transcription-

ally Active in HepG2 and HIT Cells. The rat insulin I gene isnormally transcribed only in (3 islet cells and insulinoma celllines, a specificity recapitulated by the ability of the naturalinsulin promoter to upregulate reporter gene activity in HITcells (14) but not HepG2 or COS cells (data not shown).Linker scanning mutational analysis identified the FLAT

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PROBE CCAAT 3FIB ax AT ALB- r I 1

NUCLEAR 0 n 0D n 0 ) a ,nEXTRACT I) I C) )

FLAT FLAT-F FLAT-E FLAT-MI Tl~~~ or-- '-- ---

0 cn 0 ( 0 n 0 co

I I 0 = 10 =110 1 mI

_ .... _ _

_ _ .

inW

.0

:,.;t A.. .*

PROBE

NUCLEAREXTRACT

_~.0* 1*

a a

Pfibrinogen HNF - 1 site

HepG2 HIT

II C

0 0hO&LX E ° I~~~LFL FL

COMPETITOR D- -<eL5L.L LL [ L.L4

I__--__-'-V_

m-11-unv-bor VW

rat insulin I FLAT element

HepG2 HITc- - 1!cm0 LL X Ec IL. !

Fe FD <

uL< <

I L U- 4 w

'Itam

L

z8

oh

FIG. 1. HepG2 and HIT nuclearextracts contain factors that bindknown HNF1 sites and the rat in-sulin I FLAT element. Nuclear ex-tracts prepared from HIT, HepG2,and COS cells were examined forbinding to known HNF1 sites andthe rat insulin I FLAT element. (A)(Left) The quality of the extractswas demonstrated by their ability toretard migration of the controlprobe CCAAT. (Middle) Analysis

-IX with probes containing HNF1 sitespresent in the liver-specific genesp-fibrinogen, ac-antitrypsin, and al-bumin is shown. (Right) Analysiswith rat insulin I FLAT elementprobes is shown. (B) Competitionanalysis of DNA-binding activitiesfound in HepG2 and HIT nuclearextracts. Binding specificity wastested by including x 100 molar ex-cess of unlabeled probe as indi-cated.

element as one of three sites within the rat insulin I promotercritical for transcription of the gene in HIT cells (22). Tofurther examine transcriptional activity and cell-type speci-ficity of the FLAT element, reporter constructs containingunidirectional five-copy multimers of the (3-fibrinogen HNF1site or the various FLAT site derivatives upstream ofthe -36prolactin promoter or the -85 insulin promoter were trans-fected into HepG2, HIT, and COS cells (Table 2). Plasmidscontaining the /-fibrinogen site strongly upregulated tran-scription from the two promoters in both HepG2 and HITcells. Of the various FLAT element derivatives, only theFLAT-F site was capable of upregulating transcriptionalactivity. This site exhibited weak activation compared to the

Table 2. Transcriptional activity of rat insulin I CATreporter plasmids

Regulatory HepG2 cells HIT cells COS cells

element PRL INS PRL INS PRL INS

Control 0.4 2.0 1.3 0.9 0.2 0.23-Fib 34.2 32.4 55.9 36.3 0.7 0.3FLAT 0.2 1.0 0.8 0.7 0.2 0.2FLAT-F 21.6 28.8 16.5 9.9 1.0 1.7FLAT-E 0.2 0.2 1.8 2.0 1.4 0.2FLAT-M 0.3 0.3 0.8 1.0 0.2 0.2

Transcriptional activity of reporter plasmids containing multimersof the *-fibrinogen (*-Fib) HNF1 site or rat insulin I FLAT elementderivatives. lIT, HepG2, and COS cells were transfected by thecalcium phosphate technique with the indicated reporter plasmidscontaining the -36 prolactin promoter (PRL) or the -85 insulinpromoter (INS) constructed as described. CAT enzyme activity wasassayed on cell extracts prepared 48 hr after transfection. Data areexpressed as means of three independent determinations in arbitraryunits of relative CAT activity normalized to assayed (3-galactosidaseactivity.

j3-fibrinogen site in HepG2 and HIT cells and was a weakeractivator in HIT cells than in HepG2 cells. None of thereporter constructs tested exhibited significant transcrip-tional activity in COS cells.

Cloning and Isolation of cDNA Clones. To examine therelationship between HNFl-related factors and proteins thatregulate rat insulin I gene expression through the FLATelement, we screened a HIT-T15 M.2.2.2 Agtll cDNA librarywith a PCR-derived probe corresponding to the DNA bindingregion of rat HNFla (25). Comparison of the deduced aminoacid sequence of the isolated hamster cDNA clone to theputative sequence of rat HNFla protein shows 97% identity(Fig. 2). Two additional amino acids are found in the pre-dicted sequence for hHNFla at residues 359 and 603; thesedifferences in amino acid sequence are located at the sameposition as two ofthree additional amino acids in the deducedsequence for human HNFla (28) (Fig. 2).

hHNFla Is a Component of the FLAT-F Complex. Toexamine whether HNFl-like proteins are part ofthe FLAT-Fcomplex in HIT nuclear extracts, we incorporated preim-mune or HNFla-specific antiserum (8) into gel mobility-shiftassays. Antiserum specific for rat HNFla, but not preim-mune serum, supershifted the (3-fibrinogen binding complexin nuclear extracts from both HepG2 and HIT cells (Fig. 3).The FLAT-F complex was specifically eliminated or super-shifted when bound both to the previously characterizedFF-minienhancer probe and the FLAT-F probe (Fig. 3),thereby demonstrating that the rat insulin I FLAT-F complexfound in native HIT nuclear extracts contains the HNFlaprotein.

hHNFla Transactivates Reporter Constructs in COS Cells.To test whether hHNFla can transactivate reporter con-structs in a heterologous cell type, we cotransfected thepBJ5hHNFla expression vector along with CAT reporter

A

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hum LA EG G G T G 70ham MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLMVGDAPLDKGESCSGSRGELAELPNGLGESRVS 70rat G G G T D T 70

hum E T T E 140ham EDDTDDDGEDFAPPILKELENLSPEEAAHQKAVVESLLQDDPWRVAKMVKSYLQQHNIPQREVVDTTGLN 140rat E 140

hum 210ham QSHLSQHLNKGTPMKTQKRAALYTWYVRKQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPAS 210rat 210

hum 280ham QQILFQAYERQKNPSKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK 280rat 280

hum S G PA P PP T ST 350ham LAMDTYNGPPPRPGPGATLSAHSSPGLPTSALSPSKVHGVRYGQPATSEAAEVPSSSGGPLVTVAAPLHQ 350rat G PA P TTT S 350

FIG. 2. Alignment of deducedhum H A 5 420ham VSPTGLEPSSSLLSTEAKLVSATGGPLPPVSTLTALHNLEQTSPGLNQQPQNLIMASLPGVMTIGPGEPA 420 amino acid sequences of hamster,rat S 419 human, and rat HNF1a. Onlyhum T 490 amino acids that differ from theham SLGPTFTNTGASTLVIGLASTQAQSVPVINSMGSSLTTLQPVQFSQPLHPSYQQPLMPPVQSHVAQSPFM 490 hamster sequence are shown forrat 489 the human and rat proteins. Posi-hum G T S T A 560 tion of an extraamino acid in theham ATMAQLQSPHALYSHKPEVAQYTHTSLLPQTMLITDT=NLSALASLTPTKQVFTSDTEASSEPGLHEPSS 55 9rat = T 558sequencehum Q L V AC A S Q S631 = ;twoadditionalaminoacidspres-ham PATTIHIPSQDPSSIQHLQPAHRLSTSPTVSSSSLVLYQSSDSTNGHSHLLPSNHGVIETFISTQMASSSQ 630 ent in the hamster and human se-rat N * 628 quences are indicated by *.

plasmids containing five-copy multimers of FLAT elementderivatives upstream of the -85 insulin promoter into COScells. hHNFla upregulated transcription through theFLAT-F site, but not the FLAT-E reporter construct (Fig. 4).It also transactivated the multimerized FF-minienhancerregion containing five unidirectional copies of the Far boxand adjacent FLAT element linked to the -109 thymidinekinase promoter (data not shown).

DISCUSSIONInsulin production is under tightly regulated physiologiccontrol, with the level of insulin gene expression rigidlycoupled to blood glucose levels. In the adult mammal, theinsulin gene is also subject to stringent mechanisms ofcell-type-specific control, being expressed only in (3 cells ofthe pancreatic islet. The recently characterized FF-minienhancer recapitulates regulation of the native insulingene as shown by transient transfection into insulinoma celllines (1). It is a short but complex regulatory element com-

EXTRACT HepG2IFi

PROBE Pfibrinogen

HIT

FAR-FLAT FLAT-F

ANTISERUM - P I P I - P I - P I

+J!

FIG. 3. Antiserum specific for HNFla alters HIT FLAT-Fcomplex binding. Gel mobility-shift analysis was used to furtherdefine the components of 0-fibrinogen- and FLAT-F-binding com-plexes in HIT nuclear extracts. Either preimmune (lanes P) orHNFla-specific (lanes I) antiserum was preincubated with thebinding reaction mixtures for 5 min at room temperature beforelabeled probe was added; lanes -, no serum was added to reactionmixture.

posed of the Far box and adjacent FLAT elements, which,individually, represent weak transcriptional activators inca-pable of acting over distance. In tandem, however, the twosites interact synergistically to strongly upregulate transcrip-tion regardless of promoter proximity, possibly throughhigher-order interactions of factors that specifically bind thedifferent elements. The Far box is one of two rat insulin I9-base-pair elements whose sequence is similar to the E-boxmotifs important in immunoglobulin gene transcription (29);these sites and a similar one in the rat insulin II promoter arecritical for insulin gene transcription (22, 29-32). Ins-E2 is awell-characterized activity that binds the elements (33, 34)and is thought to be required for (-cell-specific transcriptionof the insulin gene (27, 31). The Ins-E2 protein complex isthought to contain heterodimers of the ubiquitous helix-loop-helix E2A proteins (35) and other helix-loop-helixfactors that may be restricted to the ( cell (32, 36).Here we identify a protein that functions through binding

the FLAT element of the FF-minienhancer. HNFla, a pro-tein essential for expression of a number of liver-specificgenes, is expressed in the (-cell-derived insulinoma cell lineHIT and is part of the FLAT-F protein complex. Theseresults are consistent with a recent report that demonstratedcoexpression of message for HNFla (LF-B1) and L-typepyruvate kinase, a gene expressed primarily in parenchymal

pBJ5hHNFla - + - +

CATReporterConstruct

FA -EFLAFLAT - E FLAT - F

FIG. 4. Cotransfection ofpBJ5hHNFia transactivates rat insulinI reporter constructs in COS cells. COS cells were cotransfected withpBJ5hHNFla and the indicated rat insulin I reporter constructs bythe calcium phosphate method as described. Cells were harvested 48hr after transfection, and CAT activity was measured in cell extractsafter normalizing to ,B-galactosidase activity. Data shown are repre-sentative of two independent transfections.

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liver cells, in the RINm5F insulinoma cell line (37). Further-more, they suggest that HNFla may play an important rolein transcriptional control of rat insulin I gene expression,broadening the regulatory role of HNF1 beyond those genesexpressed primarily in the hepatocyte.

Despite the observation that HNFla is capable of medi-ating transactivation through the FLAT-F site, hepatomacells expressing significant levels of HNFla are unable totranscribe more complex insulin CAT reporter constructs.This is not surprising in light of the neighboring repressorelement FLAT-E recently characterized in insulinoma cells(2). A simple explanation is that negative regulatory proteinssimilar to those found in insulinoma cells may dominate thiselement in many cell types. However, since we have dem-onstrated activation of the complete FF-minienhancer, butnot the isolated wild-type FLAT element, by expressingHNFla in COS cells, this is probably an oversimplification.Not only could the FLAT-E activity fluctuate from cell tocell, but the complement of helix-loop-helix factors thatinteract with the Far box is known to vary from tissue totissue. An additional level of complexity is implied by theobservation that the HNFla-containing complexes in hepa-toma and insulinoma cells may not be identical. This obser-vation suggests that HNFla protein complexes present inHepG2 and HIT cells may be qualitatively different, poten-tially containing distinct factors that interact with the knowndimerization domain. Thus, while overexpression ofhHNFla is sufficient to activate the FLAT-F site in COScells, it is possible that other components of the FLAT-Fcomplex could be required to reconstitute the functional andcell-type specificity observed in vivo. Taken together, theseobservations suggest that tissue-specific insulin gene tran-scription may result from the interaction of an array oftranscription factors and cis-acting elements, which consti-tutes a regulatory system unique to the (3 cell.The islets of Langerhans and the liver appear to share a

glucose-sensing mechanism thought to involve glucokinase(38), a distinct isoform of hexokinase found in the hepatocyteand the (3 cell, and GLUT-2 (39), a member of the glucosetransporter family found in the ( cell, liver, kidney, andintestine. Despite common expression of the glucokinasegene by liver and a islet cells, its expression is differentiallyregulated by distinct, tissue-specific promoters (38). Theliver-specific glucokinase promoter contains a sequence ho-mologous to the HNF1 consensus binding site (40), whereasno such site has been identified in the (-cell-specific glucoki-nase promoter. The GLUT-2 promoter (L.A.E. and J. Ta-keda, unpublished data) also appears to contain putativeHNF1 binding sites. This, along with the strikingly similarpatterns of expression of GLUT-2 and HNFla, suggests apotential role for HNFla in GLUT-2 gene transcription. Asthe liver and the ( cell play distinct roles in metabolicregulation to meet the requirement for balanced glucosemetabolism, a role for HNFla in such differential but coor-dinated regulation would be consistent with the hypothesisthat it may participate in the differentiated function of spe-cialized epithelia.

We thank Cheryl Parker for invaluable assistance in growing thecell lines. We thank Gerald R. Crabtree for the vectors p(fi28)3-CAT,pBJSmHNF1, and HNF1 antisera; Graeme Bell for the GLUT-2promoter; and James Kelley for adult rat liver poly(A)+ RNA. Thiswork was supported by the facilities of the Molecular BiologyInformation Resource, by Diabetes and Endocrinology ResearchCenter Grant USPH P30DK27685 and Grant USPH R29DK43129 (toL.G.M.) and by the John Redfern Trust.

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