hepatocyte nuclear factor-3d promoter regulation involves...

Vol. 8, 69-82, Janua,y 1997 Cell Growth & Differentiation 69

Hepatocyte Nuclear Factor-3d Promoter Regulation InvolvesRecognition by Cell-specific Factors, Thyroid TranscriptionFactor-I , and Autoactivation”

Richard S. Peterson, Derek E. Clevidence,Honggang Ye, and Robert H. Costa2

Department of Biochemistry, University of Illinois at Chicago, Chicago,

Illinois 60612-7334

AbstractThe hepatocyte nuclear factor-3a (HNF-3a) and -31�proteins share homology in the winged helix/fork headDNA binding domain and regulate cell-specifictranscription in hepatocytes and respiratory epithelium.In this study, we used transfection assays todemonstrate that the -520 nucleotides upstream ofthe rat HNF-3a gene were sufficient for cell-specificexpression. We identified binding sites for a liver andkidney-enriched nuclear factor and a kidney-enrichedprotein that recognizes two distinct promoterelements. We showed that the rat HNF-3a promoterbinds the HNF-3 protein isoforms, which may serve anauto- and/or cross-regulatory role. Furthermore, weshowed that cotransfection of the thyroid transcriptionfactor-I expression vector enhanced HNF-3a promoteractivity. We discuss these results with respect to thetranscriptional induction of the HNF-3a gene inrespiratory epithelium during embryogenesis. Becausethe HNF-3a promoter region bound nuclear factors inkidney extracts, we used in situ hybridization todemonstrate that it was expressed in the urothelium ofthe renal pelvis in aduft and embryonic kidney. We alsoreport on a novel expression pattern of HNF-3a in theepithelium of the urinary bladder, penile urethra, andthe prostate gland, and show that its expression in theintestinal epithelium increases from the proximalduodenum to distal ileum. We also demonstrate thatHNF-3a is abundantly expressed in the colonicepithelium. Furthermore, we use the HNF-3 DNAbinding consensus sequence to identify putative targetgenes in the renal pelvis and gut epithelium.

IntroductionThe liver functions as a physiological focal point by virtue ofits anatomic relation to digestive and circulatory pathwaysand its metabolic specialization for nutritional, hormonal, andstress responses (1). This capacity for response results fromthe hepatocyte-specific expression of numerous enzymes

for carbohydrate management and circulatory carrier pro-teins, the synthesis of which are regulated at the transcrip-

tional level (2). Functional dissection of numerous hepato-cyte-specific promoter and enhancer regions has revealed

their structural complexity. They consist of multiple DNA

binding sites recognized by distinct families of liver-enriched

transcription factors (3). The combinatorial action of thesefactors on multiple DNA sites is required for the activation oftranscription and the maintenance of hepatocyte-specificgene expression (4). Structural similarities in the DNA bindingand/or dimerization domains define five distinct families ofliver-enriched factors: (a) the winged helix/fork head familymembers HNF3-3a, HNF-3�, and HNF-3’y proteins (5, 6); (b)the steroid hormone receptor family members HNF-4 (7) and

apolipoprotein Al regulatory protein-i (8); (c) the POU-home-

odomain containing HNF-i a and HNF-1 p (9, 1 0); (d) the bZIPfamily members C/EBPa (1 1 , 12), C/EBP�, and C/EBPS (13-18); and (e) the proline and acidic amino acid-rich bZIPsubfamily members albumin D-box binding protein (19),chicken vitellogenin gene-binding protein/mouse thyrotrophembryonic factor (20, 21), and hepatic leukemia factor (22,23).

The HNF-3 proteins were originally identified as factorsmediating hepatocyte-specific transcription of the transthy-retin (encodes a serum carrier protein of thyroxine and vita-mm A) and ai -antitrypsin genes (24). The HNF-3 proteins

share homology within a 100-amino acid winged helix DNAbinding domain (6, 25), which directs monomeric recognitionof the DNA consensus sequence A (APr)TRTT(GIflRY1Y (26).

Subsequent studies have illustrated the importance ofHNF-3 recognition elements in the promoters of numerousliver-specific genes, the expression of which is necessary forliver function (reviewed in Ref. 4). In support of the role ofHNF-3 in regulating the expression of these hepatocyte-specific genes, a hepatoma cell line that expresses a dom-

Received 9/25/96; revised 10/30/96; accepted 11/6/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This work was supported by Public Health Service Grant AOlGM43241-07 from the National Institute of General Medical Sciences.R.H.C. is an Established Investigator of the American Heart Association/Bristol-Myers Squibb.2 To whom requests for reprints should be addressed, at Department ofBiochemistry (M/C 536), The University of Illinois at Chicago, College ofMedicine, 1819 West Polk Street, Chicago, IL 60612-7334. Phone: (312)996-0474; Fax: (312) 413-0364; E-mail: [email protected].

3 The abbreviations used are: HNF, hepatocyte nuclear factor; bZIP, basicregion leucine zipper; C/EBP, CCAAT/enhancer binding protein; BF, brainfactor; TTF-1 , thyroid transcription factor-i ; CAT, chloramphenicol acetyl-transferase; CMV, cytomegalovirus; EMSA, electrophoretic mobility shiftassay; NF, nuclear factor� KF, kidney-enriched factor� HFH-4, HNF-3/forkhead homologue-4; UF1-H3f3 and UF2-H3f3, ubiquitous factors 1 - and2-HNF-3�, respectively; SP-l , specificity protein-i; Tg, thyroglobulin;SPB, surfactant protein B; Fabpi, intestinal fatty acid binding protein;PDGF A, platelet-derived growth factor A; pGEM, Promega plasmid; mut.,mutant; POU, Pit-i Oct-i Unc-86.

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70 Cell-specific Regulation of the HNF-3a Promoter

HNF-3cz Genomic Structure

XhoiE��

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Fig. 1 . HNF-3a genomic structure and HNF-3a reporter construct. Genomic organization of the rat HNF-3a clone. The figure is a schematic representationof the two HNF-3a exons (black boxes), 3’ untranslated region (stippled box), one intron (caret), and various restriction enzyme cleavage sites of the ratgenomic clone. B, Bglll; H, Hindlll; P, Pst; X, Xbal. Also shown is the reporter construct containing the -4-kb HNF-3a upstream sequences driving CATreporter gene expression.

inant-negative HNF-3 mutant extinguished transcription ofseveral HNF-3 target genes (27). Furthermore, in vivo foot-printing studies of the - 1 0-kb albumin enhancer region havedemonstrated that the HNF-3 proteins are involved in orga-nizing the nucleosome architecture of the albumin enhancer

in hepatocytes (28).

Mammalian HNF-3 (5, 6) and the Drosophila homeotic

gene fork head (29) are the first members of a large family oftranscription factors that share homology in the winged helixDNA binding domain (reviewed in Refs. 4 and 30). Accumu-lating evidence demonstrates that the winged helix transcrip-tion factors are involved in differentiation of diverse cellular

lineages. The HNF-3a and HNF-3f3 genes are expressedduring the gastrulation stage of embryogenesis (31-34) andare induced during retinoic acid differentiation of the F9embryonic carcinoma cell line (35, 36). Targeted disruption ofthe HNF-3� gene in homozygous knockout mice causes an

embryonic lethal phenotype that exhibits defects in the for-mation of notochord, neurotube, somites, and gut endoderm(37, 38). Furthermore, ectopic hindbrain expression of theHNF-313 gene in transgenic mice mediates the conversion ofhindbrain to floorplate as evidenced by activation of theendogenous HNF-3a and HNF-3f3 genes, as well as severaladditional floorplate marker genes (39). The winged helixtranscription factor BF-1 is involved in morphogenesis of the

telencephalon, and mice homozygous for a BF-1 -null mutantgenotype die at birth and exhibit a dramatic reduction in thesize of their cerebral hemispheres (40). Targeted disruptionof the BF-2 gene in mice demonstrated inhibited induction ofrenal mesenchyme into tubular epithelium and branching ofthe ureter and renal collecting system (41). Furthermore, arecent study has demonstrated that disruption of the wingedhelix gene (whn) is responsible for the nude mouse mutation

(42). Taken together, these studies indicate that the wingedhelix protein family plays a critical role in cellular differentia-tion and development.

To determine the transcriptional mechanism restrictingHNF-3a gene expression, we showed that the proximal-520 nucleotides upstream of the rat HNF-3a gene weresufficient for cell-specific expression and were recognized by

several cell-specific factors. An autoregulatory HNF-3 bind-ing element was identified in the distal region, which mayaccount for HNF-3a promoter activation upon ectopicHNF-3f3 gene expression in transgenic mice (39). In respira-tory epithelium, we demonstrated that TTF-1 recognizes twosites in the HNF-3a regulatory sequences and activatedHNF-3a promoter expression in cotransfection studies. Wediscuss these results with respect to the transcriptional in-

duction of the HNF-3a gene in bronchiolar epithelium duringembryogenesis.

ResultsCharacterization and Isolation of HNF-3a Rat GenomicClone. The HNF-3a cDNA was used to screen a HarlanSprague Dawley rat genomic A Dash II library (Stratagene),and several HNF-3a genomic clones containing the 5’ pro-moter sequence were isolated. Further characterization ofthese genomic clones by Southern blot and PCR analysis(data not shown) demonstrated that the HNF-3a protein wasencoded on two exons separated by a 2.3-kb intron (Fig. 1).The HNF-3a gene consists of a first exon of 129 bp encoding23 amino acids and a second exon that contains the remain-

der of the coding sequence (Fig. 1). We determined that themRNA start site did not extend farther than the published 5’sequence (5) by analyzing 5’ rapid amplification of cDNAends (RACE; Life Technologies) fragments prepared fromliver cDNA (data not shown; Fig. 2A, underlined nucleotide).The rat HNF-3a genomic structure is consistent with thatreported for the mouse HNF-3a gene, which also lacks adiscernible TATA box sequence (43).

Characterization of HNF-3a Promoter Region. To de-fine the minimal HNF-3a promoter, various 5’ truncations ofHNF-3a sequences located upstream of the transcriptionalinitiation site were joined to the CAT gene (starting from -4kb and extending to +43; see “Materials and Methods”). Theconstructs were transfected into human hepatoma (HepG2)cells, and protein extracts prepared 48 h after DNA trans-fection were used to determine CAT enzyme activity (Fig.2B). A CMV promoter-driven 13-galactosidase construct was

- 3 8 5 GGCGGCTGGA CTGGCGGGCG GCCGCCTCAC AGGTGCACCT C000CTTTGT AGGTGCGAGC

CCGCCGACCT GACCGCCCGC CGGCGGAGTG TCCACGTGGA GCCCGAAACA TCCACGCTCG

� TTF-l-294 � ________

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-2 6 5 ATCCACATCA GCCCAACTGC ACTCGCTTCG CACAGGCCGC CAGCTCACTT CCCGCGGAGG

TAGGTGTAGT CGGGTTGACG TGAGCGAAGC GTGTCCGGCG GTCGAGTGAA GGGCGCCTCC

-205 CGCTGCCGGG CGCGGCTCCG CGGCCGCCTC CTGTCCCCGG CGCTGCCCCC TCCCGCCGCG

GCGACGGCCC GCGCCGAGGC GCCGGCGGAG GACAG000CC GCGACG0000 AGGGCGGCGC

- 1 4 5 CCGCCGCCGC CGCCGCCGCC GCCGCCGCCG CCGCGCACGC CGCGCCCCGC AGCGCCGGGC

GGCGGCGGCG GCGGCGGCGG CGGCGGCGGC GGCGCGTGCG GCGC0000CG TCGCGGCCCG

� KF TrF-1-74 � �j [�9�3�s -38�

- 8 5 TTCCTCCTCG c�CCGOQTOGC QCTGAGCCcT CG&.OCGCTCC GGTGACC�CA OC000TCCGCAAGGAGGAGC dGOCCCACCG CG&CTCQGG� QCTCQCG�.GG CCACTGG�QT CGCCG&GGCG

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Cell Growth & Differentiation 71

HNF-3a Promoter Construct % Wild-type Promoter Activity

A-625 TCTAGAGCCC TGGCCTGTAC TCCAGGCACC GCCTATTTTT CTTTTTCTTT TCTTTCTTTT

AGATCTCGGG ACCGGACATG AGGTCCGTGG CGGATAAAAA GAAAAAGAAA AGAAAGAAAA

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-565 TTTTTTCCCC CTTTACTAAA GGGGGTCACA GATACACCCG CCCCAT�TTC TTCTTTCCCT

AAAAAAG000 GAAATGATTT CCCCCAGTGT CTATGTGGGC G000TAIIIAAQ

KF HNF-3 Fl H-�I -485��’ -_ -456 _______

- 5 0 5 AGCC000000 CTTA&ACCAA �r�CACTGCT TTGTAAACAA AGTG&QGO��C AOGTTTG000

TCGGCGCCCC GAATTTQGTT Ak1�GTGJLCGA AACATTTGTT TCkCTCCCc�Q TCCAAACCCC

UF1-H3� SP-1 � -483 �_j -457

-44 5 G�GGGGATGG C000A0000C GC�GGGGGCG CGCGGGCTGG CGCGGC0000 CGGGAGGCGC

CTCCCCTACC GCCCTCCCCG CG�CCCCCGC GCGCCCGACC GCGCCGCCCC GCCCTCCGCG

�J -423

Fig. 2. Analysis of HNF-3a promoterby transfections. A, nucleotide se-quence of the HNF-3a promoter and po-sition of the transcription factor bindingsites. Positioned on the HNF-3a up-stream sequence (-625 to +43) are thetranscription factor binding sites identi-fled in this study. They include a kidneyand liver-enriched factor (K/LF), UF1-H3f3, SP-1, NF-1, KF, JTF-1, and aHNF-3 autoregulatory site. Also shownby arrows are the positions of the oligo-nucleotides used in the gel mobility shiftassays to identify the aforementionedbinding proteins. Note that the nucleo-tide sequence of these protein bindingsites is conserved between the mouse(43) and rat HNF-3a promoter regions.B, identification of the HNF-3a promoterregion. CAT reporter constructs drivenby HNF-3a upstream sequences withvarious 5’ boundary truncations weretransfected into human hepatoma(HepG2) cells and analyzed for CAT ox-pression levels. The figure shows the re-suits of this promoter analysis ex-pressed as percentage of wild-typeactivity (-542 construct) derived from atleast three distinct transfection experi-ments. + 3 6

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Fig. 3. The HNF-3a promotercontains two distinct binding sitesfor a KF and a putative NF-l bind-ing site. Nuclear protein extractsprepared from liver, kidney, orlung tissue were used for EMSAwith the HNF-3a -5181-483 or-74/-46 promoter oligonucleo-tides and competed with a 100-fold molar excess of unlabeled 01-igonucleotide containing theindicated binding sites or selfcompetition. The kidney-enrichedfactor recognizes both proximaland distal HNF-3a promoter re-gions,whereas the NF-l proteinbinds to the proximal HNF-3a-74/-46 sequences.

included with the HNF-3a promoter constructs as an internal

control to normalize for differences in transfection efficiency

(44, 45). Analysis of several HNF-3a promoter 5’ deletionsdemonstrated that removal of sequences between -4 kb

and -542 bp produced no significant decreases in CATreporter activity. Removal of sequences extending to -430nucleotides of HNF-3a promoter sequences resulted in 60%

reduction in expression levels, and deletion of sequences

beyond the - 125-bp region eliminated detectable promoter

activity (Fig. 2B). Furthermore, the -542/+43 HNF-3cr pro-moter construct was not active when transfected into the

human epithelial HeLa cell line (data not shown). Thus, cell-

specific expression of the rat HNF-3a promoter requires

upstream -542/-430 and -125/+43 nucleotides, regions

that exhibit nearly identical sequence homology with the

mouse promoter (43).

Identification of a Putative NF-1 Binding Site and Bind-ing Sites for a Kidney-enriched Factor. Preliminary DNaseI footprinting analysis of the functional HNF-3a promoter

region using liver and kidney nuclear extracts identified sev-

eral protein binding sites (data not shown). To further char-

acterize the proteins recognizing these DNase I protected

regions, double-stranded oligonucleotides were then syn-

thesized to these sequences and used for EMSAs with tissue

nuclear extracts. The HNF-3� -74/-46 proximal site pos-

sessed sequence similarity to the NF-1 consensus-binding

sequence (TTGGCNNNNNGCCAA). DNA binding assays

demonstrated liver and lung nuclear protein-formed com-

plexes that were effectively inhibited with either a NF-1 bind-

ing site or self-competition (Fig. 3, Lanes 1-7), but they were

not diminished with an Octi binding site (Fig. 3, Lane 4).

These complexes were not disrupted by an antibody di-

rected to the distinct nuclear factor Y protein, which recog-

nizes a subset of NF-1 recognition sequences (data not

shown). These binding data suggest that the HNF-3a -74/

-46 sequence is putatively bound by the NF-1 protein.

The distal HNF-3a -51 8/-483 promoter sequences

bound a kidney-enriched protein in EMSA (Fig. 3, Lanes13-16), which also was inhibited by the HNF-3a -74/-46

oligonucleotide (Fig. 3, Lane 1 7). This kidney-enriched com-

plex was distinct from NF-1 because it was not inhibited by

the NF-1 binding site (Fig. 3, Lane 18), and the HNF-3a

-518/-483 was not able to compete for NF-i recognition

derived from liver nuclear extracts (Fig. 3, Lane 8). Consistent

with this observation, the HNF-3a -74/-46 site formed two

specific complexes with kidney nuclear extracts, one of

which remains after removal of the NF-1 complexes via NF-1

binding-site competition (Fig. 3, Lane 10, black bar). These

binding data suggest that the HNF-3a -74/-46 site puta-

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Fig. 4. A HNF-3 binding site is present in the HNF-3a -485/-457 se-quences. Antibody supershift studies demonstrate that the HNF-3n-485/-457 sequences bind three HNF-3 isoforms in liver nuclear ex-tracts. Antibodies specific to each of the HNF-3 isoforms (a, f3, and y)disrupted the corresponding protein-DNA complex with the HNF-3a-485/-457 sequence, whereas the HNF-4 antibody did not affect proteincomplex formation with liver nuclear extracts (top portion of the gel isshown). The slowest migrating protein-DNA complex is due to HNF-3abinding and is visible when the HNF-313 complex is removed by antisera(a, Lane 3). The middle protein-DNA complex is due to HNF-3f3 recogni-tion and is visible when the HNF-3a complex is removed by antisera (b,Lane 2). The fastest moving complex is due to HNF-3y binding, which isdisrupted by HNF-3y antisera (g, Lane 4). Partially degraded HNF-3 pro-tein-DNA complexes are not disrupted by antisera (indicated by white dot)because they lack N-terminal sequences, which are recognized by theHNF-3 antisera. Also included were competitions with strong (Lanes 8 and10) and weak (Lane 9) affinity HNF-3 binding sites.

tively binds the NE-i protein and a KE protein that is similar

to that which binds the HNE-3a -5i8/-483 sequence.

Distal HNF-3a Promoter Sequences Are Recognizedby the HNF-3 Isoforms. Transgenic studies have demon-

strated that ectopically expressed HNE-3f3 protein inducesexpression of the endogenous HNF-3a and HNF-3f3 genes

(39). We identified a putative HNF-3 binding site within the

transcriptionally important -476/-465 HNE-3a promoter re-

gion (Fig. 2A; TTTG1TrACAAAG). DNA binding assays withthe HNF-3a -485/-457 oligonucleotide demonstrated pro-

tein-DNA complexes with liver nuclear extracts that were

competed with high-affinity HNE-3 sites (Fig. 4, Lanes 8 and

10), but not with a low-affinity binding site (Fig. 4, Lane 9).

Each of the corresponding protein-DNA complexes was dis-

rupted by HNF-3 isoform-specific antisera (Fig. 4, Lanes

1-4), but not with a HNF-4-specific antibody (Lane 5). These

experiments identify a HNF-3 binding site that is recognized

by all three of the HNF-3 isoforms in the liver.

Identification of a Liver and Kidney-enriched ProteinBinding to the HNF-3a Proximal Promoter Region. DNA

binding assays with HNF-3a -2/+31 sequences demon-strated specific protein-DNA complexes with liver and kidney

nuclear extracts, but not with lung extracts (Fig. 5; Lanes 1-2

and Lanes 1 1-14). The formation of this protein-DNA com-

plex was not inhibited with binding sites recognized by a

HNF-3� promoter-binding factor, UF2-H313 (Fig. 5, Lane 3),

the retinoic acid receptor (Lane 5), and the activator protein-i

(Lane 7). Additional competition studies (Fig. 5) showed that

binding sites for the liver-enriched transcription factors

HNF-4 (Lane 4), HNF-3 (Lanes 6 and 10), HNF-1 (Lane 8), and

Fig. 5. Identification of a kidney and liver-enriched factor binding toHNF-3r proximal sequences. The HNF-3� first exon is recognized by afactor in liver and kidney nuclear extracts. The figure shows EMSAS withthe HNF-3a -2/+31 oligonucleotide and either liver, kidney, or lungnuclear extracts. A 1 00-fold molar excess of unlabeled competitor oligo-nucleotides was added to the binding reaction and included UF2-H3f3(47), HNF-4 (7), retinoic acid receptor responsive element (84), HNF-3(transthyretin promoter and HFH-1 #3; Refs. 24 and 26), HNF-l (9), C/EBP(1 7), and activator protein-i (85).

C/EBP (Lane 9) did not reduce complex formation. These

binding studies identified a liver and kidney-enriched factor

that recognizes the HNF-3a -2/+31 sequences and is not

competed by binding sites for several known transcription

factors.

Proximal and Distal Sequences Are Recognized bySP-1 and UFI-H313 Proteins. The proximal and distal

HNF-3a promoter regions were found to contain potential

binding sites for the SP-i protein (46) (KRGGMGKRRY) and

the UF1 -H3f3 protein (GCCCTACCCCCACC), a ubiquitous

DNA-binding protein known to be functionally important for

HNF-3f3 promoter activity (47). Indeed, DNA binding assays

confirmed the formation of two specific complexes with the

HNF-3a -38/-8 oligonucleotide and HepG2 nuclear ex-

tracts (Fig. 6A, left). The SP-1 protein is found in the slower

moving protein-DNA complex, which was competed by a

SP-1 binding site (48) and partially supershifted by a SP-1 -

A Extract

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:H3�_� � UF1�H3)l* #{248}�ii -�Promoter UF1-H3f Sequence

calcineurin (-8261

Interleukin-6 (-149)

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Mouse HNF-3(, (-2891

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74 Cell-specific Regulation of the HNF-3� Promoter

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Fig. 6. Proximal binding sites forthe SP-1 and UF1-H3(3 proteins are involved in repressing HNF-3a promoter activity. A, HNF-3a -381-8 and -456/-423sequences contain binding sites for the SP-i and UF1-H3(3 proteins. EMSA with HepG2 nuclear extracts and labeled HNF-3a -38/-8 and -456/-423oligonucleotides demonstrate protein complexes that are competed by unlabeled SP-l and UF1 -H3f3 binding site sequences. The figure shows the topportion of the gel depicting the protein-DNA complexes and the position of protein complexes competed by the SP-l and UF1 -H3� binding siteoligonucleotides. Antibodies specific to the SP-i protein (cr-SP-l ; Santa Cruz) were used in EMSA with HepG2 nuclear extracts and HNF-3a -38/-8binding site. The SP-i antibody partially altered the migration or supershifted the SP-i -containing protein-DNA complex (black dot) but not the UF1 -H3pcomplex. As a control, we showed that the SP-i antibody supershifted (black dot) an equivalent amount of the protein-DNA complex with a SP-l bindingsite derived from the SV4O promoter (SP-l). Note that the distal sequences possess weak binding affinity for SP-l protein. B, alignment of putative UF1 -H3�3binding sites from various promoter regions. Alignment of UF1 -H3� binding sites from the HNF-3 promoters (43, 47), and the calcineurin (49) andinterleukin-6 (50) promoters allowed us to compile a consensus �Fl -H3f3 DNA binding sequence. Note that the transcriptional initiation site for the mouseHNF-3a promoter was determined by primer extension assay of mouse stomach cDNA (43), which occurs 150 nucleotides upstream of the rat liver initiationsite. The figure also shows the sequence of the mutant HNF-3cr -38/-8 oligonucleotides; mutant A disrupts SP-l and uF1-H3� binding sites, and mutantB disrupts only the UF1 -H3f3 binding site. C, EMSA with HepG2 nuclear extracts and wild-type or mutant HNF-3a -38/-8 oligonucleotides. The figureshows the top portion of the gel depicting the SP-l and UF1 -H3[3 protein-DNA complexes. 0, disruption of the SP-l and UF1 -H3�3 binding sites increasesHNF-3a promoter activity. Wild-type or mutant A (Mut A) HNF-3a promoter constructs (-542 or - 1 85) were compared in parallel by transfection in HepG2cells, and (3-galactosidase normalized extracts were analyzed for CAT enzyme activity. Expression levels were expressed as percentage of the wild-type- 542 HNF-3c� promoter construct and represent an average of three separate experiments.

specific antibody (Fig. 6A). The faster-migrating HNF-3a

-38/-8 protein complex was competed by the UF1-H3p

oligonucleotide derived from the HNF-3j3 promoter, but not

by a SP-i binding site (Fig. 6A). Again, SP-1 and the UF1-

H3� protein recognition of the HNF-3a -456/--423 se-

quences (Fig. 2A) was suggested by competition with both

the corresponding unlabeled binding sites and the HNF-3a

-38/-8 oligonucleotide (Fig. 6A). The HNF-3a -456/-423

sequences differed from the proximal sequences because

they exhibited weak binding affinity for the SP-1 protein

family (Fig. 6A). These results suggest that both proximal and

distal HNF-3a regulatory sequences possess binding sites

for the SP-1 protein family and a UF1 -H3�-related protein.

The Proximal SP-1 and UFI -H3� Binding Sites RepressHNF-3a Promoter Activity. Alignment of UF1 -H3p binding

sites from the HNF-3 promoters (43, 47) and the calcineurin

(49) and interleukin-6 (50) promoters allowed us to compile aconsensus-binding sequence shown in Fig. 6B [GCCCC(A/

T)CCCCC(G/A)CC]. In the HNF-3c� -25/- 1 1 sequences, the

Sp-1 and UF1 -H3p DNA binding sequences are partially

overlapping (see Fig. 14). To examine these binding so-

quences further, we made two HNF-3a -38/-8 oligonucleo-

tides containing nucleotide substitutions within these bind-

ing sites; mutant site A was designed to prevent recognition

by SP-1 and UF1 -H3/3 proteins, whereas mutant site B was

designed to disrupt only UF1 -H3f3 protein recognition (Figs.2A and 6B). As expected, mutant A was no longer able tocompete for complex formation with the wild-type HNF-3a

-38/-B sequences and it did not form any complexes with

HepG2 nuclear extracts (Fig. 6C). Mutant B oligonucleotide

lost its ability to compete for UF1 -H3�3 protein recognition,

but these nucleotide changes also resulted in decreased

affinity for the SP-i protein (Fig. 6C, black dot).

Site-directed mutation of the UF1 -H3�3 recognition se-

quence in the HNF-3� promoter resulted in a significant

decrease in promoter activity (47). We introduced mutant A

sequences (-25 to -12 bp) in the context of the -542

HNF-3a promoter, anticipating a similar contribution of UF1 -

H3f3 binding on HNF-3a promoter activity. Mutant A and

wild-type -542 HNF-3a promoter constructs were trans-


fected into HepG2 cells and compared in parallel for tran-sient CAT expression levels. Unlike the HNF-3f3 promoterregion, disruption of the SP-i and UF1 -H3�3 binding resultedin a 2-fold increase in promoter expression of the -541HNF-3a construct, suggesting that they are involved in re-pression of HNF-3a promoter activity (Fig. 6D). It is interest-ing that when mutant A sequences were introduced in thecontext of the - 185 HNF-3a promoter region, its activity wasequivalent to that of the -542 mutant A promoter construct.However, removal of the distal UF1 -H3� and HNF-3 bindingsites caused reduction in HNF-3a promoter activity (Fig. 2B).These results suggest that the HNF-3a distal sequences(-542/-430) may participate in overcoming the SP-i- andUF1 -H3f3-mediated repression of HNF-3a promoter activity.

TTF-1 Binds to the HNF-3a Promoter and Activates ItsExpression. Analysis of promoter regions regulating liver-enriched transcription factors has revealed that their expres-sion is mediated by cross-regulation via one or more unre-lated liver-enriched transcription factors (47, 51-53). To testthe hypothesis that respiratory epithelium used this cross-regulatory mechanism, we analyzed the sequence for ele-ments that might confer respiratory epithelial control. Com-puter-assisted searches of the rat HNF-3a upstreamsequences identified two putative TTF-i DNA binding sites(GNNCACTCAAG; 54-56) in the HNF-3a promoter region

(-285/-272 and -61/-SO), which were also conserved inthe mouse HNF-3a promoter (43). To determine whetherTTF-i binds to these promoter sequences, the H44i bron-chiolar epithelial cell line, which expresses the transcriptionfactors HNF-3a and TTF-i (54, 57-59), was used as thesource for nuclear protein extracts for EMSA. DNA bindingassays with the distal HNF-3a -268/-294 oligonucleotidedemonstrated two distinct protein-DNA complexes withH44i nuclear extracts (Fig. 7A, dHNF-3a). The faster-migrat-ing protein-DNA complex is due to TTF-i recognition be-cause its formation is competed by TTF-i binding sites (Fig.7A) derived from the thyroglobulin (Lane 3) and the surfactantprotein B (Lane 4) promoters. By comparison, the migrationof the TTF-i protein-DNA complexes with the TTF-i .Tg andTTF-i .SPB promoter sites were identical to those of theHNF-3a -268/-294 binding site (Fig. 7A, compare Lanes1-4 with Lanes 5-12). Furthermore, these TTF-i complexeswere cross-competed by the HNF-3a -268/-294 Se-quences (Fig. 7A, Lanes 7 and 1 1), suggesting that it isrecognized by the TTF-i protein. An additional specificHNF-3a -268/-294 protein complex was not competed byany of the TTF-i binding sites; the protein involved in thiscomplex remains unknown (Fig. 7A, arrow). DNA bindingstudies with the HNF-3a -74/-46 sequences demonstrate asecond putative TTF-i binding site that is competed by thestrong-affinity TTF-i binding site (Fig. 7A, Lanes 13-16). Thisproximal HNF-3a promoter site exhibited lower TTF-i bind-ing affinity than the distal site. These binding studies werefurther confirmed using recombinant in vitro-translatedTTF-i protein (Fig. 7B).

To determine whether TTF-i contributed to HNF-3a gene

expression, we performed cotransfection experiments in hu-man hepatoma (HepG2) cells, which lack endogenous TTF-iexpression (data not shown). Two HNF-3a promoter con-

structs that differed in the number of putative 1W-i bindingsites were used for cotransfection assays with the liT-icDNA expression plasmid (Fig. 14). TTF-i cotransfectionwith the - 1 85 HNF-3a promoter construct, which contains asingle low-affinity TTF-i binding site, resulted in little tran-scriptional activation (Fig. 7C). However, cotransfection ofhF-i with the -544 HNF-3a promoter construct containingboth hF-i binding sites resulted in a 9-fold stimulation ofpromoter activity. These studies demonstrate that the TTF-iprotein activates HNF-3a promoter expression and thatcross-regulation is a transcriptional regulatory mechanism inlung just as in liver.

HNF-3a Is Expressed in Collecting Duct Epithelium ofMouse Embryonic and Aduft Kidney. Because we identi-fled several kidney factors binding to the HNF-3a promoterregion and that HNF-3a expression had not been reported inadult or embryonic kidney, we examined its expression pat-tern via in situ hybridization. We used 33P-labeled antisense

HNF-3a RNA probe to hybridize with paraffin sections of16-day post coitum mouse embryos and adult mouse kid-ney. After hybridization, stringent washes, and autoradiog-raphy, dark-field microscopy was used to visualize HNF-3a-expressing cells in the tissues. Fig. BA shows a paraffinsection near the middle of the 1 6-daypost coitum embryonic

abdominal cavity showing that the HNF-3a gene is abun-dantly expressed in the epithelium of the submandibulargland, the lung, and the intestine, whereas lower expressionlevels are observed in hepatocytes (Fig. BA). This HNF-3aexpression pattern is consistent with previous in situ hybrid-ization studies with earlier staged mouse embryos (32).These studies also revealed novel expression patterns for theHNF-3a gene (Fig. 8A-.C) in the urothelium ofthe renal pelvis,the prostate gland, the urinary bladder, and the penile ure-

thra. Continued HNF-3a expression was observed in theneuroepithelium of the spinal chord (Fig. 8C). We also foundthat HNF-3a expression increases from the proximal duode-num to the distal ileum axis of the intestine and displaysintensive hybridization signals in the epithelium lining the

anal canal (Fig. 8, A and C). Examination of adult mousekidney by in situ hybridization showed that the HNF-3a genecontinues to be expressed throughout the urothelium of thecollecting duct in the renal pelvis (Fig. 8D). Although HNF-3ais abundantly expressed in the urothelium of the renal col-lecting duct, these epithelial cells represent only a smallpercentage of total cells of the adult kidney, and thus it is notreadily detectable in total kidney RNA via Northern blot anal-ysis (Fig. 8E). By contrast, HNF-3a is highly expressed in theabundant parenchymal cell-type of the adult prostate glandand therefore is readily detected by Northern blot analysis.Moderate expression levels are observed in adult liver, lung,small intestine, and colon by Northern blot analysis.

DiscussionHNF-3a Is Expressed in the Urothelium of the Renal Pel-vis and the Urinary Tract. HNF-3a was the first memberidentified in the winged helix family of transcription factors,which comprises an extensive protein family regulating dif-ferentiation of distinct cellular lineages. In mouse organogen-esis, HNF-3a and HNF-3p expression is restricted to tissues

A Extract

Probe

Competitor

H441

dHNF-3ct TTF-1 .Tg TTF-1 .SPB pHNF-3a

a�Q-h�1�

,L1_’�‘-�Li�LL�Iw�-i-

J��’:J:

�Q)c?h

I��-,u5U.’;-,L1�’;-

JZu1�JZul-Ju..’�w��.1wx�-lW�LL

�

a)h

I Cl) �

B

Probe

TTF-1 protein

a.Cl)

.

#{182}-

LLI-

IV)I

u_ZI

C�)I

LI�ZI

I- V Q.

TTF-1 -*1

L

+_

-I

TTF-1 -4’�W

i� I IC

!� �

1 2 3 4 5 6 7 8 9 1011 12 13141516

C

Reporter Expression

-544 HNF-3a + CMV TTF-1

-544 HNF-3a + CMV Vector.

-185 HNF-3a + CMV 11�F-1

-185 HNF-3cc + CMV Vector.

H

]I I I I

0.0 2.5 5.0 7.5 10.0 12.5

76 Cell-specific Regulation of the HNF-3c� Promoter

Fold Increase

Fig. 7. TTF-1 binds to the HNF-3a promoter and activates its expression. A, TTF-l protein recognizes two distinct sites in the HNF-3a promoter region.EMSA with nuclear extracts prepared from H441 cells, a bronchiolar epithelial cell line expressing TTF-l (54, 59). Oligonucleotide probes and competitorsused for EMSA are the following: putative 1TF-1 binding sites in the HNF-3c� promoter -268/-294 (dHNF-3a) and -74/-46 (oHNF-3a); TTF-l binding sitesfrom the surfactant protein B (1TF-1.SPB) promoter (54), and the thyroglobulin (1TF-1.Tg) promoter (55, 56). Also indicated is the position of complexesthat are nonspecific (NS) band and disrupted by homologous competition (arrow). B, recombinant TIE-i protein binds to the HNF-3a promoter sites.Recombinant TTF-1 protein (+) was synthesized by in vitro transcription/translation using the transcription/translation coupled reticulocyte lysate system(Promega) and was used for EMSA with the hF-i . SPB, dHNF-3c� and pHNF-3a hF-i binding sites. Reticulocyte lysates that were not programmed forsynthesis with the TTF-1 cDNAtemplate(-) produced no TTF-1 protein-DNA complexes. C, cotransfection ofTTF-l cDNA expression constructs stimulatesthe HNF-3c� promoter activity. The CMV TTF-l expression plasmid was cotransfected into HepG2 cells with HNF-3c� promoter-CAT construct containingeither one (- 185) or two (- 544) hF-i binding sites: protein extracts were prepared 48 h later and analyzed for CAT enzyme activity. The data are presentedas fold increase over the HNF-3a promoter transfected with the CMV vector containing no cDNA insert and are the average of two separate experiments.

derived from the gut endoderm such as hepatocytes and

respiratory and intestinal epithelium (32, 43, 60, 61). In corn-

bination with other cell-specific transcription factors, the

HNF-3 proteins activate the transcription of numerous genes

important for hepatocytic (4) and respiratory epithelial cell

function (57, 60-62). In this study, we use in situ hybridiza-

tion to identify a novel HNF-3a expression pattern in the

urothelium of the renal pelvis of embryonic and adult kidney.

Expression of HNF-3� in the collecting duct urothelium of the

renal pelvis is consistent with our identification of kidney

nuclear factors, which recognize functional sequences in the

HNF-3a promoter region. We also determined that HNF-3a is

expressed in the epithelium of the urinary bladder, penile

urethra, and the prostate gland of 1 6-day post coitum mouse

embryos. The HNF-3a protein therefore may participate with

other cell-specific transcription factors in epithelial cells to

regulate the transcription of genes critical for kidney and

urinary tract function. In support of this hypothesis, putative

HNF-3 binding sites were found in the vasopressin receptor

VIa, E-cadherin, PDGF A, thromboxane receptor, and Na/K

Bright Field Dark Field


Fig. 8. HNF-3a expression in theepithelium of the renal pelvis, theurinary bladder, the penile urethra,and the prostate. A, in situ hybrid-ization of 1 6-day post coitum mouseembryo with 33P-labeled antisenseRNA HNF-3a probe. The figureshows the abdominal body portionof the embryo, in which the dark-field depicts expression in the epi-thelium of the submandibular gland(S), bronchioles of the lung (Lu), theintestine (In), and the hepatocytes ofthe liver (Li). Also indicated on theembryo is the position of the heart(He), gall bladder (G), and vertebrae(Ve), which do not show any specifichybridization. B, enlargement of theembryonic kidney (Ki) showsHNF-3a hybridization in the collect-ing duct urothelium of the renal pel-vis (RP), in hepatocytes of the liver(Li), and the epithelium of the intes-tine (In). C, enlargement of the em-bryonic urinary system showsHNF-3a hybridization in the urothe-hum of the bladder (B!), penile ure-thra (PU), and prostate (Pr) gland, aswell as in the neuroepithelium of thespinal cord (sc), the epithelium liningthe intestine (In), and the anal canal(An). The figure also shows the ver-tebral (ye) column, which displaysno HNF-3cr expression. D, in situ hy-bridization of adult mouse kidneydemonstrates continued expressionin the collecting duct urothelium ofthe renal pelvis (RP). No HNF-3a ox-pression is observed in the papilla(Pa) or the renal cortex (Cx). E,HNF-3a is abundantly expressed inthe adult prostate gland. Northernblots containing 2 j.�g of polyadeny-lated RNA isolated from the mdi-cated human tissues were probedwith the rat HNF-3a cDNA and theglyceraldehyde-3-phosphate dehy-drogenase cDNA as a control.

E�

� � ,�; �

� � � � �

HNF-3a (3.5 Kb)-� #{149}* � � #{149}GAPDH-#{248}-#{149}*S��*. � ..4� -,

78 Cell-specific Regulation of the HNF-3cr Promoter

Table 1 Putative HNF-3 target genes expressed in the urothelium of renal pelvis and in intestinal epithelium

We used the HNF-3 consensus binding sequence to search promoters for genes that are expressed in the urothelium of the renal pelvis or the intestinalepithelium. The table shows the name of the gene, GenBank accession no., position in the promoter, and the putative HNF-3 binding sequence.

Gene ac�t�o. Position Sequence8

Urothelium of renal pelvis�’r. vasopressin receptor VIa D83546 - 1480/- 1468 TGTGTTTGTTTC

r. vasopressin receptor VIa D83546 - 1350/- 1362 GTTATTTGCTCG

m. E-cadherin X60961 -551/-563 TTTGTTTGTTTT

m. E-cadherin X60961 -3081-320 TTTGTTTGCTTA

h. PDGF A M20488 -3791-391 CATATTTGCGGA

h. thromboxane receptor U30503 -9591-571 CTTGTTTGTACA

r. Na/K-ATPase Aiphal X53233 -3031-315 GGTATTTGACTG

r. Na/K -ATPase Alpha2 090049 -832/-820 TTTGTTTGTTTT

r. Na/K -ATPase Beta2 090048 -942/-954 TTTGTTTGTTTT

Intestinal epitheliumcm. sucrase isomaltase S40076 - 142/- 130 TATATTTATATA

h. aminopeptidase N M55523 -9041-916 AATATTTGTATT

h. aminopeptidase N M55523 -509/-521 TTTATTTGTTTT

r. Fabpi Ml 8080 - 101 5/- 1 003 TATATTTGTATG

r. Fabpi M18080 -623/-635 TTTATTGATATC

m. E-cadherin X60961 -5511-563 TTTGTTTGTTTT

m. E-cadherin X60961 -3081-320 TTTGTTTGCTTA

h. intestinal alkaline phosphatase Y00512 -9531-941 GATGTTTGTTCT

m. cdx-2 U00454 +47161+4728 TTTGTTTGTTTG

h. lactase phlorizin hydrolase L04635 -8791-891 TTTGTTTACATA

r. clusterin U02391 -141 11-1399 TTTATTTACATC

h. carbonic anhydrase II M77176 -845/-857 CCTATTGATTTG

h. carbonic anhydrase Ill M29452 -5371-549 TTTGTTTGCCTG

HNF-3 consensus WWTRTTKRYWYD

a The first HNF-3 DNA binding consensus sequence is derived from in vitro DNA binding site selection and HNF-3 sites in liver-enriched promoters (26).

Nucleotide abbreviations of the HNF-3 consensus are as follows: W is A or T, R is A or G, K is G or T, V is C or T, and D is not C.b Genes expresed in the urothelium of the renal pelvis are the following: r. vasopression receptor VIa (63), m. E-cadherin (64), h. PDGF A (65), thromboxane

receptor (66), and Na/K ATPase subunits (67). r., rat; m. mouse; h., human.C.Genes expressed in the intestinal or colonic epithellum are the following: r. Fabpi (68), mouse cdx-2 homeodomain transcription factor (69), E-cadherin

(70), m. Sucrase isomaltase (71), lactase phlorizin hydrolase genes (72), carbonic anhydrase II and III (73), aminopeptidase N (74), intest. alkaline phos., andrat clusterin genes (75). Other abbreviations as in footnote b.

ATPase subunit genes, which are expressed in the urothe-hum of the renal pelvis (Table 1). Furthermore, these studiesreveal that the expression pattern of HNF-3a increases alongthe proximal duodenum to distal ileum axis of the smallintestine and exhibits high expression throughout the entireepithelium lining the colon. Putative HNF-3 binding siteswere also identified in promoter regions of the sucrase iso-maltase, aminopeptidase N, Fabpi, E-cadherin, intestinal al-kaline phosphatase, homeodomain cdx-2, Iactase phlorizinhydrolase, clusterin, and carbonic anhydrase II and III genes,which are transcribed in the intestinal and/or colonic epithe-hum (Table 1). Expression of the Fabpi gene, which containsseveral potential HNF-3 binding sites, parallels that of theHNF-3a gene, exhibiting increased expression along the du-odenal-to-colonic axis (76).

Identification of Cell Type-specific Transcription Fac-tore Involved in Restricting HNF-3a Expression. Analysisof promoters regulating liver-enriched transcription factors

suggests that expression in hepatocytes is maintainedthrough cross-regulation of the promoter by one or more

unrelated liver-enriched transcription factor(s). A cross-talkregulatory loop was discovered during the analysis of theHNF-1 and HNF-4 promoters, in which each of these factorsregulated the transcription of the other promoter (51 , 53). Inaddition to an auto- and/or cross-regulatory binding site, the

HNF-33 promoter region is also bound and activated bymembers of the bZlP and the proline and acidic amino acid-rich bZlP family and the HNF-6 protein (47, 52, 77). Ourcurrent studies identify a liver and kidney-enriched factor and

a KF factor that participate in cell-specific transcription of theHNF-3ct gene (Fig. 9). However, the HNF-3cr promoter regiondoes not contain binding sites for the cell-specific bZlP andHNF-6 proteins, and thus its promoter organization differsfrom that of the HNF-3� gene (77). Furthermore, a HNF-3 sitewas found in the distal HNF-3a promoter sequence, which isrecognized by the HNF-3 isoforms and may serve an auto-and/or cross-regulatory role (Fig. 9). This finding corrobo-rates earlier studies in which it was found that ectopic ex-pression of HNF-3p in the embryonic hindbrain of transgenicmice caused the induction of both endogenous HNF-3a andHNF-3� genes (39).

Cross-regulatory mechanisms may also play a role inmaintaining cell-specific transcription factor expression inthe respiratory epithelium. In situ hybridization studies havedetermined that the winged helix family members HFH-4,HNF-3a, and HNF-3f3, and the homeodomain hF-i proteinsare coexpressed in respiratory epithelium (57, 60-62). Re-cent studies have implicated the HNF-3 proteins in regulatingexpression of hF-i in the respiratory epithelium (78). Ourcurrent study extends these results by identification of two


HNF-3a PROMOTER REGION

KF HNF-3 UF1 SP-1

H3�

� ��:;J:; -4Th -#{149}12�� -300

� Kidney

� Kidney & Liver� Lung

� Lung, Liver, & Kidney- Ubiquitous

TTF-1

Represses

Transcription

NF-1 TTF-1 UF1 SP-1 LIKF

Fig. 9. Summary of the HNF-3a promoter binding factors. Schematic representation of the nuclear factors recognizing the HNF-3a promoter regiondetermined in this study. None of the HNF-3a promoter binding factors was restricted to the liver. Expression of HNF-3a in hepatocytes involves a liverand kidney-enriched factor (L/KF) and an auto- and/or cross-regulatory HNF-3 binding site. Expression of HNF-3a in respiratory epithelium involves twodistinct binding sites for the homeodomain TTF-1 , which is expressed in thyroid and bronchiolar epithelium and the auto- and/or cross-regulatory HNF-3binding site. HNF-3a expression in the urothelium of the renal pelvis involves binding sites for a KF (two sites), a liver and kidney-enriched factor (L/KF),and the HNF-3 isoforms described above. The HNF-3a promoter is also recognized by the widely distributed nuclear proteins NF-1 , SP-1 , UF1 -H3/3, andan uncharacterized ubiquitous factor binding near the TTF-1 binding site. The proximal SP-1 and UF1 -H3j3 binding sites function to repress the HNF-3apromoter activity.

hF-i binding sites, which mediate HNF-3a transcriptional

activation (Fig. 9). In respiratory epithelium, the HNF-3f3

protein therefore may mediate activation of HNF-3a ex-

pression directly via promoter recognition and indirectly

by induction of hF-i . Furthermore, EMSA with recombi-

nant HFH-4 protein demonstrates that this winged helix

protein also binds to the HNF-3 binding site in the HNF-3a

promoter region and may regulate its expression in bron-

chiolar epithelium.4 HNF-3a promoter regulation by HNF-

313, HFH-4, and TTF-1 may therefore provide a mechanismto drive an abundant HNF-3a expression pattern in respi-

ratory epithelium compared with hepatocytes (Fig. 8).

Moreover, mice containing targeted disruption of the

1TF-1 gene exhibit defects in the formation of thyroid,

pituitary, and lung parenchyma and possess only a rudi-

mentary bronchiole tree (79). Our studies identify the

winged helix transcription factor HNF-3a as an important

7TF-1 target gene that participates in regulation of genes

important for respiratory epithelial cell function.

Overlapping Proximal SP-1 and UF1-H3� BindingSites Are Involved in Repressing HNF-3a Promoter Ac-tivity. In addition to several cell-specific factors, the

HNF-3a promoter region binds widely distributed NF-1,

SP-1 , and UF1 -H3�3 proteins, the latter of which are critical

for HNF-3f3 promoter activity (47). Two regions of the

HNF-3a promoter, -38/-8 and -456/-423, were recog-

nized by the UF1 -H3f3 protein, but they differ from the

HNF-3j3 promoter in that the UF1-H3f3 sites are closely

spaced with SP-1 binding sites and the distal sequences

possess weak SP-1 binding affinity. Mutagenesis of the

proximal UF1-H3�/SP-1 binding site resulted in activation of

4 Lim, L., H. Zhou, and R. H. Costa. The winged helix transcription factorHFH-4 is expressed during choroid plexus epithelial development in themouse embryo, submitted for publication.

HNF-3a promoter activity, suggesting that it is involved in

repression. The mechanism of this negative regulation re-

mains unknown but may involve recognition of this site by

the SP-3 isoform, which mediates transcriptional repression

of target genes (80). By contrast, deletion of the distal region

containing the HNF-3, UF1-H3p, and the weak affinity SP-1

binding sites (-544/-430) caused a 60% reduction in pro-

moter activity, suggesting that these sequences contributed

positively to HNF-3co promoter activity. The distal region

therefore may activate HNF-3a expression by overcoming

repression by SP-1/UF1-H3p at their proximal binding sites.

Because of the ability of the HNF-3 proteins to alter chro-

matin structure (28), recognition of the HNF-3a promoter

region by the HNF-3 isoforms, in collaboration with the distal

UF1 -H3�3 protein, may play an important role in its activation

through disruption of the SP-1/UF1-H3f3 inhibitory com-

plexes.

In summary, we report on a novel expression pattern of

HNF-3a in the urothelium of the renal pelvis, urinary bladder,

penile urethra, and the prostate gland of adult and embryonic

tissues and show that HNF-3a expression increases along

the duodenal-to-colonic axis. The HNF-3 consensus se-

quence was used to identify putative target genes, the ex-

pression of which are restricted to the gut and renal pelvis

epithelium. Our promoter studies identified cell-specific

binding factors, which may participate in limiting HNF-3�

promoter expression to hepatocytes and urothelium of the

kidney collecting duct. A HNF-3 binding site was identified,

which is recognized by the HNF-3 isoforms, and which may

participate in auto- and cross-regulation in hepatocytes and

in epithelium of the respiratory bronchioles, intestine, colon,

and renal pelvis. Moreover, we show that the TTF-1 protein

may play a cross-regulatory role in respiratory epithelium by

binding to the HNF-3a promoter region and activating gene

expression.


Materials and MethodsConstruction of HNF-3a Promoter Deletions and UFI-H3p Site-dl-

rected Mutant. A 1.5-kb Xbal HNF-3a rat genomic subclone that

contained the entire first exon was cleaved at the 3’ terminus anddigested with Bal 31 exonuclease (New England Biolabs). The diges-

tion was terminated at regular intervals with phenol/chloroform, blunt-ended with T4 DNA Polymerase (New England Biolabs), cleaved at the

5’ Xbal, and subcloned as an Xbal-Hincll fragment into pGEM-1 . Oneclone containing the region from Xbal (5’ ; -625 bp) to +43 bp wasretained for analysis of promoter activity. The -5kb to +43 construct

was generated through multiple cloning steps, the last of which was

ligation of the genomic 5’ Sall-Xhol fragment with the Xhol to +43fragment. The Xbal (5’ ; -625) to +43 plasmid was deleted from the 5’

end with Bal 31 to generate successive deletions in upstream so-quences as described previously (47).

Site-directed mutagenesis ofthe UF1 -H3p/SP-1 sites was achieved by

a PCR-mediated strategy (81), which replaced the UF1 -H3(3/SP-1 bindingsequence with an EcoRl restriction site. Regions extemal to and contain-ing the binding site region were amplified using external primers in the

pGEM and CAT sequences (SP6 and CAT) and complementary mutantprimers that span the UF1 -H3j3/SP-1 binding site (mut.UF1-H3f3/SP-1 .A

and mut.UF1-H3f3/SP-1 .S). Primer sequences were mut.UF1 -H3f3/SP-1 .A

(5’-ctg cgc ggg tcg gqa aft ctg cgc gga gc-3’), mut.UF1 -H3�/SP-1 .S(5’-gct ccg cgc aga aft ccc gac ccg cgc ag-3’), and CAT (5’-ccg ttg atatat ccc aat ggc atc-3’). These products were digested with EcoRl (under-

lined), ligated, digested again with Sstl and Hindlll, then cloned upstreamof the CAT reporter gene.

Transient Transfection Assays. HepG2 and HeLa cervical carcinomacells were maintained in monolayer cultures as described (82). H441 , a

pulmonary epithelial cell line, was grown in RPMI 1640 (Life Technologies)supplemented with 10% fetal bovine serum (Life Technologies) without

antibiotics. HNF-3a promoter-CAT constructs (40 �tg) and CMV immedi-ate-early promoter-f3-galactosidase construct (0.75 �g) were cotrans-fected into HepG2 or HeLa cells on 100-mm dishes by a modified calciumphosphate coprecipitation method (47, 82). Cellular protein extracts were

prepared from transfected cells 48 h after transfection and analyzed for

CAT and f3-galactosidase enzyme activity as described previously (44,45). Several of these transfection experiments also used the LJpofectinreagent (Life Technologies) according to the manufacturer’s protocol(35-mm plates, 2 �.Lg of CAT construct, 0.1 �sg of CMV-)3-galactosidaseconstruct, and 10 p1 of Lipofectin). Cotransfection studies into HepG2

cells were performed with a CMVTTF-1 cDNA expression plasmid (0.5 �g;a gift from R. Di Lauro, stazione Zoologica Anton Dohm, Naples, Italy) andHNF-3cr promoter CAT reporter construct (2 �.tg) using Lipofectin reagent.

In Situ Hybridization and Northern Blot Analysis. In situ hybndiza-tion of adult tissues was performed with 33P-labeled antisense RNA

probes generated from linearized HNF-3a cDNA templates using SP6RNA polymerase. Mice were perfused first with PBS via intraventricular

catheterization followed by perfusion with 4% paraformaldehyde to fix thetissue. Adult kidney and embryos were rinsed in PBS, refixed in paraform-

aldehyde, dehydrated through a graded ethanol series, and then impreg-nated in paraffin before making sagittal sections as described (83). Anti-

sense �P-labeled RNA probes were hybridized to sagittally sectioneddewaxed tissue and rinsed at high stringency as described (83). After anexposure period to Kodak NTB-2 silver emulsion (2-4 weeks), the slideswere developed and counterstained with H&E. A dark-field condenser

was used to enhance the visualization of the silver grains due to specifichybridization. Northem blots containing human RNA prepared from van-

ous tissues were purchased from Clontech and hybridized with HNF-3aand glyceraldehyde-3-phosphate dehydrogenase cDNA as described bythe manufacturer.

Nuclear Protein Extract Preparation, EMSAs, and Antibody Super-shift Assays. Nuclear protein extracts were prepared from rat tissues

and cell lines as described previously (47, 82). Double-stranded oligo-nucleotides were made to the DNasel protected region and then ra-

diolabeled using [y-32P]ATP and T4 polynucleotide kinase (New Eng-land Biolabs). EMSAs were performed as described previously (44, 45).A 100-fold molar excess of unlabeled double-stranded oligonucleotidewas added to the reaction for competition experiments. The NF-1 andOcti binding site sequences used for competition assays are 5’ -CCT

TTG GCA TGC TGC CAA TATG-3’ and 5’-TGT CGA ATG CAA ATC ACT

AGAA-3’, respectively. In the antibody supenshift assay, labeled DNAprobe was incubated with nuclear extract for 30 mm, and then affinity-purified HNF-3 antibodies were added and incubated for an additional

30 mm before PAGE (44, 45). Generation of antisera specific to ratHNF-3a and HNF-3f3 was described previously (35). The HNF-37 andHNF-4 antisera were gifts from Dr. E. Lai and F. Sladek, (University ofCalifornia, Riverside, CA), respectively. The TTF-1 cDNA pGEM-1 tem-plate was used to synthesize hF-i protein with the transcription!

translation coupled reticulocyte lysate system (Promega) for EMSAswith HNF-3a -268/-294 and -74!-46 oligonucleotides.

AcknowledgmentsWe thank Pradip Raychaudhuri, Robert Storti, Ronald Reichel, UzmaSamadani, David Overdier, and Lorena Um for critically reading thismanuscript. We are grateful to Stephen Duncan for providing us with the

in situ hybridization protocol and Brooks Robey for interpretation of ourkidney in situ hybridization signals. We thank E. Lai and F. Sladek forproviding the HNF-3y and HNF-4 antisera, respectively. We also thankUzma Samadani for the generous gift of tissue nuclear extracts and R. DiLauro for the TTF-1 cDNA expression construct.

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