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Biochemical and Biophysical Research Communications 260, 829–834 (1999)

Article ID bbrc.1999.0980, available online at http://www.idealibrary.com on

epatocyte Nuclear Factor 1 Binds to and Transactivateshe Human but Not the Rat CYP7A1 Promoter

ean Chen,* Allen D. Cooper,*,† and Beatriz Levy-Wilson*,†,1

Palo Alto Medical Foundation Research Institute, Palo Alto, California 94301; andStanford University, Department of Medicine, Stanford, California

eceived June 14, 1999

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Cholesterol 7a-hydroxylase (CYP7A1), a liver-pecific enzyme, catalyzes the rate-limiting step in theegradation pathway of cholesterol to bile acids, andhus plays a key role in cholesterol homeostasis. Tolucidate the mechanisms that control hepatic expres-ion of the human CYP7A1 gene, we are studying theromoter region. Initially, we observed that up to 40%f the overall transcriptional activity of the promotern HepG2 cells was associated with DNA sequencesrom 265 to 21 of the human gene. Within this region,binding site for the liver-enriched transcription fac-

or HNF-1 (256 to 249) has been identified. Binding ofNF-1 to this site leads to transcriptional activation of

he human promoter. The corresponding segmentrom the rat CYP7A1 gene does not bind HNF-1; in-tead, it is bound by the orphan receptors ARP-1COUP-TFII) and LXRa, that are implicated in dietaryegulation. © 1999 Academic Press

Cholesterol 7a-hydroxylase (CYP7A1), the rate-imiting enzyme in the major degradation pathway ofholesterol to bile acids, plays an important role inholesterol homeostasis (for review, see [1]). In mam-als, CYP7A1 is encoded by a single-copy gene that

s expressed only in the liver. The overall regulationf the CYP7A1 gene is complex involving both tissue-pecificity and modulations by bile salts [2], hor-ones such as insulin [3], glucocorticoids [4], thyroidormone [5], and cholesterol [6]. In rats and rabbits,xpression of the CYP7A1 gene exhibits a circadianhythm with maximal expression at midnight [7, 8].ontrol of the expression of the human gene is poorlynderstood. One of our goals is to identify all of theey regulatory elements from this gene that are nec-ssary for liver-specific expression in vivo. Initially,e identified a number of regulatory sequences at

1 To whom correspondence should be addressed at Palo Alto Med-cal Foundation Research Institute, 860 Bryant Street, Palo Alto, CA4301. Fax: 650-329-9114. E-mail: blwilson@pamfri.org.

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y liver-specific proteins and participate in promoterctivity [9]. Experiments involving transient trans-ections of various human CYP7A1 promoter-eletion reporter gene constructs, revealed thatbout 300 bp of upstream sequence are sufficient forull promoter activity of the human CYP7A1 gene inultured human hepatoma cells (HepG2). However, aeporter construct containing only 65 base pairs ofpstream sequence (265 CAT) retained about 40% ofhe promoter activity [9]. The high level of promoterctivity exhibited by the 265 CAT constructrompted a search for the DNA sequences and nu-lear proteins involved in this activity.Earlier, we described two DNaseI footprints were

reviously described in the segment from 265 to 21 ofhe human CYP7A1 gene; one between 262 to 254 andhe other at 248 to 235 [9], respectively. The ratromoter displayed similar footprints [10]. The DNAequence of this region of the CYP7A1 gene is partiallyonserved among humans, mice and rats. A putativeinding site for the liver-enriched transcription factorNF-1 is present in the segment from 256 to 238 ofoth the human, and rat [9, 10] CYP7A1 genes (Fig. 1,anel A).In this report we show that HNF-1 binds to the

roximal promoter region of the human CYP7A1 genend contributes to its basal transcriptional activity.owever, HNF-1 does not bind to the corresponding

egion of the rat gene. In contrast, recent work bytroup et al. [11], established that the segment from74 to 255 of the rat CYP7A1 gene is bound by theuclear receptors COUP-TFII (ARP-1) and RXR, andhat binding of these proteins stimulates transcrip-ional activity. A bile acid response protein (BARP) waslso postulated to bind the same segment of the ratene [10]. Additionally, Lehmann et al. [12], havehown that this same segment of the rat promoter islso bound by LXR leading to oxysterol activation ofranscription. We now show that ARP-1 (COUP-TFII)nd LXRa do not bind to the human CYP7A1 promoter

0006-291X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

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egment from 274 to 254. These studies demonstratehat a liver-specific transcription factor binds in theroximal promoter of the human CYP7A1 gene andlays a significant role in the basal tissue-specific tran-criptional activity of the human promoter. In contrast,he same segment of the rat gene binds different tran-cription factors that are implicated in dietary controlf gene expression.

ATERIALS AND METHODS

Plasmid construction, tissue culture, and transient transfectionssays. Construction of the 265 CAT plasmid has been previouslyescribed [9]. Human hepatoma cells (HepG2), were grown as pre-iously described [9]. Transient transfections were performed as

FIG. 1. Binding of HNF-1 to the 267 to 235 oligonucleotide. Panehe human sequence is displayed with the putative HNF-1 site shharacters. The HNF-1 consensus binding site is also shown. Panelf the autoradiograms, as well as the source of the nuclear proteinsndicated.

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escribed previously [9], with 5 mg of the CAT plasmid and 5 mg of annternal reference plasmid (pRSVb-gal). An expression plasmid forNF-1 [13] or control vector was sometimes included. CAT assaysere by the procedure of Gorman et al. [14] and CAT activities wereormalized using the b-galactosidase activities to correct for differ-nces in transfection efficiencies.

Gel mobility shift assays. Nuclear extracts from mouse liver, andepG2 cells, as well as preparation of COS extracts enriched inNF-1, ARP-1, LXRa and RXR was as described before [15]. The

equences of the double-stranded oligonucleotides used were as fol-ows: wt (267 to 235) oligonucleotide: AAC CAA GCT CAA GTT AATGA TCT GGA TAC TAT; HNF-1 oligonucleotide: ACA AAC TGTAA ATA TTA ACT AAA GGG A; rat (267 to 235) oligonucleotide:CT CAA GTT CAA GTT ATT GGA TCA TGG TCC TGT; human

274 to 254) oligonucleotide: CTT TGT CAA CCA AGC TCA AGT;at (274 to 254) oligonucleotide: TTT GGT CAC TCA AGT TCAGT.

DNA sequence of the segment from 267 to 235 of the CYP7A1 gene.n in brackets. The highly conserved 59 half-site is shown in boldhe 32P-labeled oligonucleotides used as probes are indicated on topof the competitor oligonucleotides. Use of HNF-1a antibody is also

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Vol. 260, No. 3, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NF-1 Binds to the Segment from 267 to 235of the Human CYP7A1 Promoter andStimulates Transcriptional Activity

There is DNA sequence similarity in the segmentrom 256 to 249 of the human CYP7A1 promoter andhe binding site for the liver-enriched transcriptionactor HNF-1 (Fig. 1, panel A). To test whether thisegment is indeed bound by HNF-1, gel retardationxperiments were performed using as a probe an oli-onucleotide extending from 267 to 235 of the humanromoter. In Fig. 1, Panel B, lanes 1-7, we observehat the 267 to 235 oligonucleotide binds HNF-1 fromither a COS extract enriched in HNF-1a (lane 1) orrom a HepG2 nuclear extract (lane 2). Binding toNF-1 is competed by a 5 and 10-fold excess of non-

adioactive 267 to 235 oligonucleotide (lanes 3 and 4),s well as by the consensus HNF-1 oligonucleotidelanes 5 and 6). In lane 7, the 267 to 235/HNF-1omplex is supershifted by an HNF-1a antibody. Theseata demonstrate that the segment from 267 to 235 ofhe human CYP7A1 gene is bound by the liver-nriched transcription factor HNF-1a. The affinity forNF-1 binding to the human probe was compared to

hat of the strong HNF-1 binding site from the fibrin-gen promoter used as a consensus HNF-1 oligonucle-tide [13]. The mobility of the retarded complex formedith the consensus oligonucleotide is similar to thatbserved with the 267 to 235 oligonucleotide (lanes 8nd 9). Furthermore, the HNF-1 consensus super-hifted complex (lane 15) resembles that obtained withhe 267 to 235 oligonucleotide (lane 7). In lanes 10 and1, the HNF-1 retarded complex is competed for by a0-fold excess (lane 10) and a 50-fold excess (lane 11) ofnlabeled HNF-1 oligonucleotide. In contrast, a much

arger excess of 267 to 235 oligonucleotide is needed toffectively compete for binding to the consensus HNF-1robe (lanes 12–14). We estimate that the affinity ofinding of the 267 to 235 CYP7A1 probe to HNF-1ay be 100-fold lower than that exhibited by the

trong HNF-1 binding site of the fibrinogen promoternd is similar to the binding affinity of HNF-1 to arucial site in the apolipoprotein B second intron en-ancer [15].Results in Fig. 1 demonstrated that HNF-1 binds

o the segment of the human CYP7A1 promoter from67 to 235. An oligonucleotide harboring mutations

n the HNF-1 conserved 59 half-site was made andested for binding in gel retardation assays. It didot bind HNF-1 (data not shown). Cotransfection ofhe wild-type construct with the increasing amountsf an HNF-1 expression vector stimulated the tran-criptional activity of the wild type 265 CAT con-truct (Fig. 2). In contrast, HNF-1 was unable totimulate transcription of a 265 CAT construct thatncorporated a mutation in the HNF-1 binding site

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data not shown). Taken together, the data in Figs. 1nd 2 establishes HNF-1 as playing an importantole in the basal promoter activity of the humanYP7A1 gene.

he 267 to 235 Segment from the Rat GeneIs Not Bound by HNF-1

The sequence conservation between the human andhe rat 267 to 235 segment, shown in Panel A ofig. 3, suggested that HNF-1 may bind to the ratYP7A1 267 to 235 segment. Panel B illustrates bind-

ng of HNF-1a to the human probe (lanes 1–4) and tohe rat probe (lanes 5–8). The human probe forms thexpected HNF-1 retarded complex, that is competed fory itself (lane 2) as well as by the consensus HNF-1ligonucleotide (lane 3). Addition of a large excess (300old) of the unlabeled rat oligonucleotide competes forinding of the human probe, as predicted by the se-uence conservation. However, HNF-1 does not bind tohe rat 267 to 235 sequence (lane 5). This was con-rmed in experiments with a mouse liver nuclear ex-ract. It is evident that the human 267 to 235 probeormed a retarded complex (lane 9) with the same

obility as the HNF-1 complex of lane 1, and that theat 267 to 235 probe did not. The rat probe, however,as bound by a protein(s) distinct from HNF-1 (lanes1 and 12).

he Segment from 274 to 254 of the Rat CYP7A1Gene Is Bound by ARP-1 and LXRa, but the SameSegment of the Human Gene Is Not

Work by others has established that the segmentrom 274 to 254 of the rat CYP7A1 gene binds to theranscription factors ARP-1 (COUP-TFII), RXR [11],

FIG. 2. Effect of HNF-1 upon the transcriptional activity of the65 CAT construct. Transfections into HepG2 cells were as de-

cribed under Materials and Methods in the presence of increasingmounts of an HNF-1a expression vector (pGEM.HNF-1a) as indi-ated in the abscissa.

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nd LXR [12]. The sequence similarities betweenhe human and rat genes (illustrated in Panel A ofig. 4), prompted us to ask whether these regulatoryroteins may also bind to the human CYP7A1 pro-oter. We first tested binding of ARP-1 to the human

equence from 274 to 254 (Panel B, lanes 1–4). ARP-1id not bind to the human 274 to 254 probe (lane 1),ut it bound to the rat oligonucleotide (lanes 5–7).ven a 200 fold excess of the human oligonucleotide

ompetitor did not disrupt binding of ARP-1 to the ratrobe. Binding of the oxysterol receptor LXRa to theegment from 274 to 254 of the human and ratYP7A1 genes was then tested. LXRa does bind to the

at promoter (Panel C of Fig. 4, lanes 5–8) but not tohe corresponding segment of the human gene (lanes–4). Our results suggest important differences in theranscriptional control of the human and rat CYP7A1enes.

FIG. 3. HNF-1 does not bind to the rat 267 to 235 sequence. Pano 235 segments, with the HNF-1 binding site shown on top by a bracanel B shows gel retardation assays with the human and rat 267

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ISCUSSION

Recent studies of the human CYP7A1 promoter re-ion revealed that about 40% of the transcriptionalctivity of the promoter was retained by a reporteronstruct containing only 65 base pairs of upstreamequence [9]. It was of interest, therefore, to identifyhe DNA sequence elements and trans-acting factorsesponsible for this significant portion of the activity ofhe human CYP7A1 promoter. We have now estab-ished that the segment from 256 to 249 of the humanYP7A1 promoter is bound by liver-enriched tran-cription factor HNF-1, leading to transcriptional acti-ation.HNF-1, also named HNF-1a/LFB1, is a liver-

nriched transcription factor that recognizes a 15 bponsensus sequence containing an inverted repeatGTTAATNATTAAC(A/C) (for review see [16, 17]). Se-

A shows the DNA sequence of the human (top) and rat (bottom) 267. Conserved sequences between the human and rat genes are boxed.35 probes. The layout is similar to that in Fig. 1.

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uences recognized by HNF-1 usually harbor a well-onserved half-site, GGTTAAT, although the secondalf-site often degenerates from the consensus. HNF-1inds to DNA as a dimer. A variant form of HNF-1xists, named HNF-1b or vHNF-1, having the sameNA binding specificity as does HNF-1a. HNF-1 func-

ions mainly in terminally differentiated cells whileHNF-1 operates early in differentiation. HNF-1 par-icipates in the regulation of many liver-specific genes.

match to the HNF-1 half-site consensus is foundrom 256 to 249 of the human CYP7A1 promotershown by a bracket in Fig. 1A); the second half-site isore divergent. Our data in Figs. 1 and 2 establishes

hat HNF-1a binds to the human CYP7A1 promoternd enhances its transcriptional activity.The finding that HNF-1 does not bind to the segment

rom 256 to 249 of the rat promoter is consistent withhe fact that instead, the rat 272 to 257 segment is

FIG. 4. ARP-1 and LXR bind to the rat but not to the human 27YP7A1 genes in the region from 274 to 254. The homologous segmites are indicated. Panel B shows binding of ARP-1 to the rat (lanes 5he binding reactions between the human probe and an LXRa-enriche

833

ound by the orphan receptor ARP-1 (COUP-TFII),ither as a homodimer or as heterodimers with RXR11]. The physiological significance of the ARP-1 bind-ng to the rat CYP7A1 promoter is unclear [11]. ARP-1nd RXR recognize the direct repeat AGTTCA, sepa-ated by 4 nucleotides (DR4). The human gene shows 2ismatches to the ARP-1 binding site in the direct

epeat regions. On the other hand, the rat sequenceas a mismatch with the human gene in the center ofhe conserved HNF-1 59 half-site. These mismatchesost likely are responsible for the differences in bind-

ng observed between the human and rat promoters.It has recently been reported that the same sequence

f the rat promoter that binds ARP-1 (and RXR), alsoinds LXR, and this may mediate oxysterol-dependentransactivation of the CYP7A1 gene [12]. Our resultsn Panel C of Fig. 4 demonstrate that the oxysteroleceptor LXR does not bind to the 274 to 254 segment

o 254 sequence. Panel A shows the sequence of the human and ratts between the two species are boxed. The ARP-1 and LXR bindingand human (lanes 1–4) oligonucleotides. Lanes 1–4 of Panel C showOS extract, and lanes 5–8 show binding of the rat sequence to LXRa.

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he human gene is regulated by oxysterols at the tran-criptional level, as it has been suggested [2], the sitet which the LXR receptor binds is different to thatmployed in rats (and mice).To date, the in vivo roles of all of the proximal promoter

equences in the regulation of the human gene remain toe elucidated. Results from transgenic mice experimentssing either the natural human CYP7A1 gene (Goodartt al., manuscript in preparation) or the rat promoteregion linked to a reporter gene [18], suggest that regionsf the gene other than the promoter alone, are requiredor liver expression. Transgenic mice lines that expresshe human CYP7A1 gene in the liver are being developedn our laboratory and will allow us to test the in vivoontributions of the regulatory sites described here byntroducing mutant constructs and measuring changes inxpression levels in comparison to those of mice carryingild-type transgenes.

CKNOWLEDGMENTS

We thank Dr. David J. Mangelsdorf for the LXRa expressionlasmid and Rick Cuevas for his help in preparing the manuscript.his work was supported by the National Institutes of Health grantL-54775 (BLW).

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