Vol. 10, No. 3

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1990, p. 991-999

0270-7306/90/030991-09$02.00/0 Copyright ©D 1990, American Society for Microbiology

Binding of a Liver-Specific Factor to the Human Albumin Gene Promoter and Enhancert MONIQUE FRAIN,.12t* ELIZABETH HARDON,' GENNARO CILIBERTO,'§ AND

JOSIt M. SALA-TREPAT2

European Molecular Biology Laboratory, 6900 Heidelberg, Federal Republic of Germany,' and Laboratoire d'Enzymologie du Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France2 Received 2 October 1989/Accepted 11 November 1989

A segment of 1,022 base pairs (bp) of the 5'-flanking region of the human albumin gene, fused to a teporter directs hepatoma-specific transcription. Three functionally distinct regions have been defined by deletion analysis: (i) a negative element located between bp -673 and -486, (ii) an enhancer essential for efficient albumin transcription located between bp -486 and -221, and (iii) a promoter spanning a region highly conserved throughout evolution. Protein-binding studies have demonstrated that a liver trans-acting factor which interacts with the enhancer region is the well-characterized transcription factor LF-B1, which binds to promoters of several liver-specific genes. A synthetic oligodeoxynucleotide containing the LF-Bl-binding site is sufficient to act as a tissue-specific transcriptional enhancer when placed in front of the albumin promoter. The fact that the same binding site functions in both an enhancer and a promoter suggests that these two elements influence the initiation of transcription through similar mechanisms. gene,

(bp) immediately upstream from the cap site (17, 38). However, additional sequences may be required for transcriptional control during development, since an enhancer located at -10 kilobases (kb) from the cap site is essential for high-level expression of the albumin gene in transgenic mice (40). Comparison of the rodent albumin 5'-flanking sequences with the human counterpart demonstrates more than 90%o sequence conservation up to position -250, indicating that these promoter sequences are recognized by evolutionarily conserved proteins (7, 13). The characterized DNA-binding proteins interacting with these cis-acting sequences in the rat albumin promoter (2, 7) and in the mouse promoter (28, 29) include the liver-specific LF-Bl/HNF1 (1Sa) and C/EBP (26), and the ubiquitous CTF/NF1 (39, 45) and NFY (43). Our footprinting studies show that in human albumin NF1 binds at two regions: -136/-107 and -102/ -79 (39) and that LF-B1 recognizes the -66/-50 region in the promoter (22). In the work described in this paper, we analyzed a sequence of 1,022 bp immediately upstream from the cap site of the human albumin gene by transient-expression experiments. We show that this fragment is capable of directing tissue-specific expression in transfected Hep3B cells. Upon further analysis, we can distinguish three domains: a negative cis-acting element, a tissue-specific enhancer, and the promoter region. We also demonstrate that a trans-acting factor which interacts with the enhancer region is the same factor, LF-B1, that we have shown previously to interact with the promoter.

Gene activity in higher eucaryotes is regulated largely at the level of transcription initiation (see references 30 and 53 for reviews). This cis-acting regulatory sequences involved in the activation of transcription are usually distinguished in promoters and enhancers. The former is made up of a cluster of cis-acting elements located immediately upstream of the initiation site and is generally defined as the minimal DNA segment capable of directing transcription. Enhancers are defined as DNA sequences able to activate transcription over and above that observed with the promoter alone and can exert their effect in a position- and orientation-independent fashion. Recently, however, the distinction between promoters and enhancers has become less sharp. Both are modular units containing common and/or distinct short DNA motifs which are binding sites for trans-acting factors and have a specific role in tissue specificity or a general enhancement of transcription (see references 24 and 36 for reviews). Serum albumin is the major protein synthesized by liver cells, and its concentration increases from low levels early in fetal development to a high plateau level in adulthood. Other organs such as the thyroid gland and the gastrointestinal tract produce trace amount of albumin. High levels of albumin are also found in the yolk sac during fetal life. The human albumin gene, which is expressed in a tissue-specific and temporally regulated manner during development, represents a good model for studying cell-specific gene control. The tissue specificity of albumin synthesis appears to be regulated mainly at the level of transcription initiation (9, 41). Transient-expression experiments and in vitro transcription assays on rodent albumin promoters have shown that the DNA sequences necessary and sufficient for hepatocytespecific transcription are located within the 150 base pairs

MATERIALS AND METHODS Isolation and sequencing of a human albumin genomic clone. Two human genomic libraries propagated in the cosmid vector pTCF were kindly provided by F. G. Grosveld (20) and screened with 32P-labeled human albumin cDNA clones (27) by the method of Hanahan and Meselson (21). A positive clone was isolated. The positive clone, cHSA1, contains a region of genomic DNA that encompasses the first eight exons of the human albumin gene (33, 52) and 24 kb of 5'-flanking sequence. A 3.6-kb HindIIlEcoRI fragment containing the cap site was subcloned into

Corresponding author. t This article is dedicated to the memory of J. M. Sala-Trepat, *

who died during the course of this work. t Present address: Laboratoire de Gdndtique Moldculaire, Ecole Normale Superieure, 75230 Paris, France. § Present address: Dipartimento di Biochimica e Biotechnologie Mediche, University di Napoli, 2nd Medical School, 80131 Naples, Italy. 991

992

FRAIN ET AL.

M13 derivatives mpl8 and mpl9 (32). About 1.7 kb immediately upstream of the cap site was sequenced by the dideoxy method (44). Plasmid constructions. A SacI-BstEII fragment from cosmid cHSA1 spanning -1103 to +47 of the albumin sequence was treated with BAL 31 exonuclease to delete the albumin initiation codon at its 3' end. The resulting DNA (-1022 to -1) was cloned into the Hindll site of pEMBL8 (12) and sequenced. The chloramphenicol acetyltransferase (CAT) construct 5' A-1022 was obtained by inserting the Hindlll-HindlIl CAT cassette from pUC19CAT2 (10) downstream of the albumin fragment into the HindlIl site of the polylinker of pEMBL8. (i) 5' end deletions. The 5'A-1022 albumin-CAT construct was linearized by BamHI and incubated at different times in the presence of BAL 31. The resulting BAL 31-XbaI fragments containing shortened albumin sequences (all ending at -1) linked to the CAT gene were first transferred and sequenced in M13mpl9 and then inserted into pUC19, leading to the albumin-CAT constructs 5'A-673, 5'A-486, 5'A-221, 5'A-147, and 5'A-56. (ii) 3' end deletions. The 5'A-1022 albumin-CAT construct was digested with Hindlll and treated with BAL 31. The resulting EcoRI-BAL 31 fragments containing the albumin sequence shortened at its 3' end were cloned into M13mpl9 and sequenced. The corresponding BamHI-BamHI fragments containing shortened albumin sequences (all starting at -1022) were inserted into pUC19CAT2, upstream of the simian virus 40 (SV40) promoter leading to the albumin-CAT constructs 3'A-33, 3'A-73, 3'A-193, 3'A-269, and 3'A-435. This vector contains the SV40 TATA box and the 21-bp repeats up to position 179 of the SV40 DNA physical map (51). The longer 3' albumin deletion was inserted into the BamHI site of pEMBL8CAT. Cell cultures, DNA transfections, and CAT assays. Human hepatoma cells Hep3B and HepG2 (25) were cultured in F12 medium supplemented with 10% fetal calf serum. HeLa cells were cultured in Dulbecco modified Eagle medium with 10% fetal calf serum. Cells were plated out at a density of 0.5 x 105 cells per 6-cm dish 24 h before transfection. DNA transfections were performed by the calcium phosphate precipitation technique (18). Cells were transfected with 2.5 pmol (ca. 10 ,ug) of plasmid DNA. In cotransfection, each precipitate contained an additional 0.025 pmol of pSV0-globin (54) used as an internal marker. The precipitate was removed 8 h later, and cells were refed with fresh medium. Cells were harvested 48 h after transfection for measurement of CAT activity and 20 h after transfection for RNA extraction. The CAT assays were performed by the method of Gorman et al. (16). Reaction mixtures containing 20 [l of cell extract (ca. 100 pLg of total protein), 0.5 pCi of ['4C] chloramphenicol, and 0.44 mM acetyl coenzyme A were incubated for 1 h at 37°C. The reaction products were separated by thin-layer chromatography. To quantitate the conversion of labeled chloramphenicol to the acetylated derivatives, we cut the radioactive spots from the thin-layer plates and counted them in a liquid scintillation counter. Si nuclease mapping. The uniformly labeled singlestranded probes were obtained as previously described (37). For transcripts from albumin-CAT fusions a SacI-EcoRI fragment of 5'A-147 construct was cloned into M13mpl9. For transcripts from pSV2CAT and the plasmids in which the albumin promoter was substituted with that of SV40, a BamHI-EcoRI fragment of pSV2CAT was cloned into M13mpl9. For transcripts from the pSV-,-globin construct, a BamHI-BamHI fragment was cloned into M13mpl9. The

MOL. CELL. BIOL.

probes Alb-CAT and SV40-p-globin were labeled to a specific activity of 3 x 10' cpm/[.g, and the probe SV-CAT was labeled to a specific activity of 1 x 108 cpm/>lg. SI mapping was performed by the methods of Berk and Sharp (3) and Ciliberto et al. (8) with the following modifications. About 2 x 105 cpm of probe was dried down with 20 ,ug of total RNA. The pellet was suspended in 30 ,ul of 80% formamide-0.4 M NaCl-1 mM EDTA-40 mM piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES; pH 6.4). The mixture was denatured at 85°C for 10 min and hybridized at 46°C for 12 h. Then the S1 digestion was performed at 24°C by addition of 270 ,ul of 0.28 M NaCl, 50 mM sodium acetate (pH 4.6), and 4.5 mM zinc acetate, containing 880 U of S1 nuclease (Bethesda Research Laboratories, Inc.). After 1 h of incubation, the RNA-DNA hybrids were precipitated with ethanol and analyzed on a 6% acrylamide-7 M urea sequenc-

ing gel. Nuclear extracts. Liver nuclear extracts from 3-month-old

Sprague-Dawley rats were prepared by the method of Gorski et al. (17). Nuclear extracts from Hep3B and HeLa cells were prepared by the method of Dignam et al. (14). Rat liver and Hep3B nuclear extracts were loaded onto a heparinSepharose CL-6B column equilibrated with buffer E (20 mM N-2 -hydroxyethylpiperazine -N' -2 -ethanesulfonic acid [HEPES; pH 7.9], 0.2 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol) containing 200 mM KCl. The column was eluted with a linear gradient of KCl (200 to 600 mM) in buffer E. Gel mobility shift and methylation interference assays. For mobility shift DNA-binding assays, a few micrograms of each nuclear extract was preincubated in a 20-,ul reaction containing 20 mM Tris (pH 7.6), 8% Ficoll, 25 mM KCl, 4 mM MgCl2, 4 mM spermidine, 1 mM EDTA, 0.5 mM dithiothreitol, 3 ,ug of poly(dI-dC), and 50 ng of salmon sperm DNA. After 10 min, 8,000 cpm of the end-labeled DNA fragment was added and the incubation was continued for 10 min at room temperature. Free DNA and DNAprotein complexes were resolved on a 5% polyacrylamide gel in 0.5x TBE (45 mM Tris borate, 45 mM boric acid, 2 mM EDTA). For competition experiments, a 5- to 700-fold molar excess of cold double-stranded oligonucleotides was added. For methylation interference assays, the DNA probe was partially methylated with DMS as described previously (31) before being used in preparative-scale binding reactions. After electrophoresis, both the free DNA and the DNAprotein complexes were excised from the gel and treated as described by Hardon et al. (22), except that the DNA was purified over DE52 resin and extracted twice with phenolchloroform before precipitation. DNase I footprint assays. Protein fractions were preincubated in a 10-p.l reaction containing 20 mM HEPES (pH 7.9), 25 mM

KCI,

4 mM

MgCI2,

4 mM

spermidine,

0.5 mM

EDTA, 12 ng of pUC19, and 3 p.g of poly(dI-dC). After 10 min at 4°C 5,000 cpm of end-labeled probe was added, and

the incubation was continued for 10 min at room temperature. DNase 1 (2 pl) freshly diluted to a final concentration of 30 to 200 p.g/ml in water was added, and the digestion was allowed to proceed for 90 s at room temperature. Digestion was stopped by the addition of 100 p.1 of phenol-chloroform and 50 p.l of 2.5 M ammonium acetate. The DNA was extracted once with phenol-chloroform, precipitated with 2.5 volumes of ethanol, suspended in 80% formamide, and

electrophoresed on a 6% acrylamide-7 M urea gel.

VOL. 10, 1990

A. -1022

THE HUMAN ALBUMIN GENE

5' Deletion Abumin 5' fanking region

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FIG. 1. Deletion analysis of the albumin 5'-flanking region. The open bars indicate the albumin sequence fused to the CAT gene. (A) Progressive 5' deletions of the albumin fragment from -1022 to -1 cloned in pUC19CAT. (B) Progressive 3' deletions of the same fragment fused to the SV40 early promoter (the 21-bp repeats and TATA box) of the plasmid pUC19CAT2 or without the SV40 promoter in the plasmid pEMBL8CAT. The precise endpoints of each deletion, determined by DNA sequencing, are indicated in base pairs relative to the albumin cap site. The CAT activities obtained after transfection of each deletion in Hep3B and HeLa cells are expressed relative to that of pSV2CAT (as 100%o) and are given with the standard deviation. pEMBL8CAT and pUC19CAT2, which are inactive in Hep3B or HeLa cells, were used as negative controls. The results in panel A are the mean of four independent transfections in Hep3B and in HeLa cells, and the results in panel B are the mean of three independent experiments.

RESULTS Expression of the cloned human albumin gene in human cell lines. To characterize the sequences responsible for specific transcription of the human albumin gene in liver, a DNA segment spanning nucleotides (nt) -1022 to -1 of this gene was fused upstream to the coding region of the bacterial CAT gene (16). The resulting construct, 5'A-1022, was transfected into the human hepatoma cell line Hep3B, which expresses the albumin gene (25), and into the human epithelial cell line HeLa, in which the albumin gene is not active. Gene expression was inferred by an enzymatic CAT assay and determined by Si mapping of the CAT transcripts. As a negative control, we used the plasmid pEMBL8CAT, which contains the coding sequence of the CAT gene without a eucaryotic promoter and is not transcribed in Hep3B or HeLa cells. The positive control, pSV2CAT (16), which contains the SV40 promoter-enhancer sequences inserted upstream of the CAT gene, is expressed at high levels in both cell types. The CAT activity from this construct is taken as 100% in our assays. The 5'-flanking region (-1022 to -1) of the human albumin gene exhibits some cell type specificity (Fig. 1A), since it directs a fivefold-higher CAT expression in the hepatoma Hep3B cells than in HeLa cells (5.4 and 1%, respectively, relative to pSV2CAT). Considering that the very low relative CAT activity found in HeLa cells is close to the background level of pEMBL8CAT, it is likely that the specificity of expression has been underestimated. However, by analogy with the mouse (40), it is possible that there is a far-upstream element that was not analyzed in this work. Three different regulatory elements are present in the

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FIG. 2. Analysis by Si mapping of transcripts in transfected Hep3B cells. Total RNA (20 jLg) was extracted from Hep3B cells, cotransfected with some of the 5' and 3' deletion plasmids described in Fig. 1 and the SV-,-globin plasmid as an internal control, and subjected to Si protection analysis. The position of the protected products of RNA initiating at the SV40 cap site (SV-CAT and ,3-globin) and at the albumin cap site (Alb-CAT) are indicated. The fainter signals observed for the SV-CAT transcripts reflect a threefold-lower specific activity of the SV40-CAT probe compared with the Alb-CAT probe. In the lower panel, a schematic drawing shows the M13 single-stranded DNA probes used (FII) and the length of the corresponding protected band ( ) with respect to the CAT and ,B-globin transcription units.

albumin 5'-flanking region. To delineate the regulatory elements contained in the albumin 5'-flanking region, we generated a set of 5' and 3' end deletions with BAL 31 (Fig. 1). The 5' end deletion mutants were cloned into pUC19CAT, a transient-expression vector which carries a polylinker in front of the CAT gene. The relative CAT activities obtained after transcription of the different constructs are shown in Fig. 1A. Deletion of the 5' sequence from -1022 to -673 (5'A-673) did not significantly affect the expression of the test gene. In contrast, further deletion of 187 bp, to position -486 (5'A-486), led to a 3.5-fold increase in CAT activity in Hep3B cells and to a smaller increase in HeLa cells. A strong decrease in expression occurred upon deletion of sequences between -486 and -221 (5'A-221). Further deletions of the highly conserved albumin 5' sequence from -221 to -56 resulted in a stepwise drop of CAT activity, leading to a construct (5'A-56) that is almost inactive in any cell type. The transcriptional activity of these 5' deletion constructs was also monitored by Si mapping (Fig. 2, lanes 1 to 4). In this case, these constructs were cotransfected with pSV,-globin, a plasmid carrying the rabbit ,B-globin gene driven by the SV40 promoter-enhancer sequences (54). Protection of a 267-nt fragment indicates accurate initiation of the Alb-CAT transcripts at the natural albumin start site (52). CAT mRNA levels of 5' deletions in Hep3B cells were

994

FRAIN ET AL.

MOL. CELL. BIOL.

TABLE 1. CAT mRNA levels measured by densitometric analysis of the Si-protected bands Construct

5'A-1022 ....................................

5'A-673 ............ ........................ 5'A-486 ..................................... 5'A-221 ..................................... 3'A-32 ..................................... 3'A-73 .................................... 3'A-435 ....................................

CAT mRNA (%)U in Hep3B cellslevel

3.64

3.80 25.80 2.24 18.80 7.74 2.30

a The CAT mRNA level for each construct was normalized according to the ,-globin expression and is given relative to that of pSV2CAT, which is taken as 100%.

measured by densitometric analysis of the Si-protected bands and normalized according to the ,B-globin expression (Table 1). The values obtained for each construct are expressed relative to that of pSV2CAT as 100% and are in agreement with the CAT assays, suggesting the existence of a negative and a positive element upstream of the promoter. However, the larger difference in expression observed among the various constructs in Si mapping could be explained by the fact that the values obtained in the CAT assays often include some readthrough transcription from upstream (vector-derived) promoters. The 3' end deletion mutants, which result in removal of the TATA box, were fused upstream of the SV40 early promoter (21-bp repeats and TATA box) of plasmid pUC19CAT2 (10). The shortest 3' deletion (3'A-32) was efficiently expressed in Hep3B and HeLa cells, although the vector pUC19CAT2 itself is inactive in both cell lines (Fig. 1B). However, the same 3' deletion lacking the SV40 promoter is expressed only in Hep3B cells. The deletion of albumin flanking sequences between -32 and -73 (3'A-73) led to a 50% decrease in CAT activity in Hep3B cells, with only a slight decrease in HeLa cells. Further deletion to position -193 (3'A-193) restored tissue specificity by abolishing activity in HeLa cells. A possible explanation for this result is that the well-characterized ubiquitous factor NF1 binding in the albumin promoter (39) might be involved in an interaction with Apl and Spl, which are present in the SV40 promoter, thus generating an active but non-liver-specific promoter. The stepwise decrease of activity in Hep3B cells that followed the progressive deletion from -32 to -435 underlines the presence of multiple regulatory elements in the albumin promoter. These data were confirmed by Si analysis (Fig. 2, lanes 5 to 7; Table 1). Initiation of transcription of the Alb-SVCAT constructs occurred mainly at the late start sites in the SV40 promoter as indicated by a protected fragment of 355 nt. The lower SV-CAT transcript levels calculated for 3' deletions reflect a threefold-lower specific activity of the SV40-CAT probe compared with the Alb-CAT probe and therefore correlate well with the CAT activities. These data, together with those obtained from the 5' end deletions, suggest that the albumin 5'-flanking region analyzed is composed of at least three regulatory elements: an upstream negative element (-673 to -486), an upstream positive element (-486 to -221), and a promoter element, each of which exhibits some cell type specificity. The albumin upstream positive element interacts with a liver-specific factor. The transient-expression assays have demonstrated the necessity of a close upstream positive element for efficient albumin transcription. We have previously reported the interaction of the ubiquitous nuclear

factor NF1 and of the liver-specific factor LF-B1 with promoter sequences of the human albumin gene (22, 39). To study the trans-acting factor(s) binding to the upstream positive region, we performed gel mobility shift, methylation interference, and DNase I footprinting experiments (Fig. 3). The end-labeled fragment spanning the region from -481 to -193 was incubated with nuclear extracts from the cell lines Hep3B and HeLa and from rat liver, in the presence of nonspecific competitors of DNA-binding proteins, poly(dIdC) and salmon sperm DNA (Fig. 3A). Two protein-DNA complexes were observed with Hep3B (lane 2) and HeLa (lane 3) nuclear extracts, whereas only the slower-migrating complex was present with rat liver nuclear extracts (lane 4). The critical contact points for the binding of the protein(s) involved were determined by methylation interference experiments (48). This procedure identifies guanine residues whose methylation interferes with the binding of the protein to the probe. Within the positive regulatory protein, methylation of only three guanines (at positions -352, -356, and -357) on the coding strand interfered with the binding of a nuclear factor that is present in Hep3B cells (Fig. 3B, lanes 7 and 8) and in rat liver (lane 10), but not in HeLa cells (lane 9; only the analysis of the faster-migrating complex is shown; the result obtained with the second complex was the same). The corresponding noncoding strand did not yield any protected G residues with any extract (data not shown). To further delineate the exact binding site of the factor(s) present in hepatic cells on the albumin upstream region, we performed DNase I protection analysis (Fig. 3C). A fraction (0.35 to 0.4 M KCl) of a rat liver extract fractionated on heparin-Sepharose was incubated with a probe spanning -481 to -277 of the albumin flanking sequence. The protected sequences map to the region that was found to be sensitive to dimethyl sulfate methylation and extend from -363 to -338 on the coding strand (Fig. 3C, lanes 14 and 15) and from -366 to -342 on the noncoding strand (lanes 19 and 20). The binding site of the liver-specific factor interacting with the albumin upstream region contains an imperfect palindrome (six of nine nucleotides), 5'-TTGGTTAGT/ AATTACTAA-3', centered between nucleotides -359 and -342 (Fig. 3D). The liver-specific factor LF-B1 interacts with the albumin upstream positive region. Comparison of the nucleotide sequence of the protected site of the albumin upstream region with the target DNA sequences of known eucaryotic transacting factors revealed a good match with the consensus recognition site described for the LF-B1/HNF1 factor: 5'-TG GTTAATNATTA(A/C)CA(A/G)-3' (6, 11, 15a, 22, 46). To examine whether the albumin upstream sequence binds LFBi or a related factor, we carried out mobility shift competitions in the presence of increasing amounts of competitor oligodeoxynucleotides containing an LF-B1-binding site (Fig. 4A). The sequences of the oligodeoxynucleotides used as competitors are represented at the bottom of the figure. The end-labeled albumin fragment (-481 to -193), when incubated with a heparin fraction (0.35 M) of a Hep3B cell nuclear extract, gave rise to only the slower-migrating complex (lane 0). The observation of a minor faster protein-DNA complex with the nonfractionated Hep3B extract (Fig. 3A, lane 2) could be due to a protein which elutes at a different place on the gradient or does not bind the heparin-Sepharose column. The addition of increasing amounts of unlabeled Alb upstream oligo, which span the region protected in the albumin upstream sequence, abolished complex formation (Fig. 4A, lanes 1 to 4). Similarly, the two oligodeoxynucleotides Alb promoter oligo and alAT B oligo, which contain

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THE HUMAN ALBUMIN GENE

VOL. 10, 1990

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FIG. 3. Analysis of DNA-binding protein interactions with the albumin upstream positive region. (A) Gel mobility shift assay. The 3'-end-labeled fragment from -481 to -193 was incubated with 1 ,u1 of various crude nuclear extracts, and the resulting complexes were analyzed on a nondenatuning polyacrylamide gel. Lanes: 1, no extract; 2, Hep3B extract (2.7 ,ug); 3, HeLa extract (3.4 ,ug); 4, rat liver extract (3.6 ,ug). F indicates the free DNA, and arrows show the bound protein-DNA complexes. (B) Dimethyl sulfate methylation interference analysis. The 5'-end-labeled fragment spanning -481 to -193 (coding strand) was partially methylated and used as probe in a gel mobility shift assay with the previously indicated nuclear extracts as detailed in Materials and Methods. Lanes: 5, G+A chemical cleavage ladders of the probe; 6, free DNA; 7 and 8, DNA recovered from the top and bottom Hep3B complexes, respectively; 9, DNA recovered from the bottom HeLa complex; 10, DNA recovered from the rat liver complex. Protected G residues and their positions in the recognition sequence are indicated. The corresponding 3'-end-labeled fragment (noncoding strand) did not yield any protected G residues and is not represented. (C) DNase I footprint analysis. The fragment Sphl-NcoI (-481 to -277) was radiolabeled at the NcoI site with the Klenow fragment or with polynucleotide kinase. The two resulting probes (coding and noncoding strands) were incubated with 2 of a 10-mg/ml heparin fraction (0.35 to 0.4 M) of a crude rat liver nuclear extract (lanes 14, 15, 19, and 20). The control pattern of DNase I digestion obtained in the absence of any added protein is displayed in lanes 12, 13, 17, and 18, along with the purine-specific sequence markers (lanes 11 and 16). Lanes alongside the autoradiography delineate the sequences protected from DNase I digestion. (D) Summary of the results of methylation interference and DNase I footprinting experiments on the human albumin upstream region. The DNA sequence of the relevant protein-binding region of the albumin flanking region is shown. The brackets indicate the sequences that are protected from DNase I cleavage on both the coding and noncoding strands. Dots indicate the guanine residues whose methylation interferes with binding.

the well-characterized LF-B1 binding site present in the promoter of the human albumin and al-antitrypsin genes, respectively, also prevent complex formation (lanes 9 to 16). As expected, the control oligodeoxynucleotide containing a polylinker sequence did not compete with the labeled probe for complex formation (lanes 5 to 8). These data suggest that LF-B1 may be the factor that interacts with the albumin upstream positive region. This hypothesis was confirmed by DNase I protection analysis with purified LF-Bl from rat liver (1Sa). Purified LF-B1 protects the positive regulatory region in a footprint profile identical with that observed with

the crude liver nuclear extract (Fig. 4B; compare with Fig. 3C). Enhancer properties of the albumin upstream positive region. The necessity of the upstream sequences from -486 to -221 nt for efficient albumin transcription suggested the presence of an enhancer element. One criterion for enhancer activity is the ability to activate transcription independent of orientation (47). In this respect, we have shown that the Alb-CAT construct A-486 in which the bp -486 to -193 region was inverted from its natural orientation, yielded an almost equal level of activity in Hep3B cells to that of Ai486

996

FRAIN ET AL.

MOL. CELL. BIOL.

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FIG. 4. Binding of the purified transcription factor LF-Bl protein to the human albumin upstream positive region. (A) Gel mobility shift competition by oligodeoxynucleotides with or without the LF-B1-binding site. The 3'-end-labeled fragment spanning -481 to -193 (10 fmol, 10,000 cpm) was incubated with 2 ,ul of a 2-mg/ml heparin fraction (0.35 M) of a crude Hep3B nuclear extract in the absence or presence of unlabeled competitor oligodeoxynucleotides. The retarded complex is indicated by an arrow; F is the free probe. Lane 1 contains no competitor; the lanes contain 700-, 250-, 50-. and 5-fold molar excess of cold competitor corresponding to albumin upstream oligo (lanes 1 to 4), unspecific pUC18 polylinker (lanes 5 to 8). albumin promoter oligo (lane 9 to 12). or a1-antitrypsin B oligo (lanes 13 to 16). In the lower panel the sequence of the top strand of the oligodeoxynucleotides containing an LF-B1-binding site (underlined) used in this study are shown. Numbers indicate the position of the nucleotide relative to the cap site of the albumin or al-antitrypsin. (B) Footprint of purified LF-Bl in the albumin upstream region. The coding and noncoding strand probes used in Fig. 3C were incubated in the absence (lanes -) or presence (lanes +) of 1.2 ng of purified LF-B1 protein (>95%; 15a ). G+A chemical cleavage ladders of each probe were used as markers. The relative position of the footprint of purified LF-B1 is indicated.

(Fig. 5), confirming that the upstream region could function as an enhancer.

Our previous binding studies established that the liverspecific factor LF-BI interacts with this functionally defined enhancer element. It was important to determine whether the LF-Bl-binding site per se was sufficient to mimic the transcriptional activation of the enhancer. One or a few copies of the Alb upstream oligo (Fig. 4A) spanning the LF-Bl-binding site were inserted into the plasmid 5'A-221, which contains the albumin promoter-CAT gene fusion. Plasmids lacking an enhancer (pEMBL8CAT) or containing the full complement of the two enhancer elements of SV40 (pSV2CAT) were used as controls. The data in Fig. 5 show that one copy of the synthetic LF-B1-binding site cloned in front of the albumin promoter increases CAT activity to 62% of that of the plasmid containing the albumin enhancer (Fig. SB, compare Xl with A-486). Multiple copies are considerably more active than the enhancer (Fig. SB, X3 and X5), thus indicating that LF-Bl is a major if not the only factor interacting with the enhancer. DISCUSSION In this report, we defined three distinct regulatory regions in the 5'-flanking region of the human albumin gene which are involved in liver-specific expression of the gene in transient-expression experiments: (i) a negative regulatory

region spanning nt -673 to -486; (ii) an enhancer spanning nt -486 to -221 that interacts with the liver-specific transcription factor LF-BI; and (iii) the promoter region, composed of multiple regulatory elements. The 5'-flanking region (nt -1022 to -1) of the human albumin gene is able to direct CAT expression in the hepatoma cell line Hep3B at a level 5- to 10-fold higher than in HeLa cells (Fig. 1, 5'A-1022 and 5'z-486). This specificity of expression is probably underestimated, since the CAT activity is close to the background level of pEMBL8CAT (1% of that of PSV2CAT). However, we cannot rule out the possibility that other elements further upstream also enhance transcription and confer a stronger cell specificity, as in the case of other specific genes (40, 55). The stepwise decrease in promoter activity in Hep3B cells by progressive deletions at its 3' and 5' ends suggests the presence of multiple activating elements (Fig. 1). Thus, a 3' deletion from -32 to -73 diminished CAT activity to 50% in Hep3B cells. This significant drop in activity includes a region (nt -66 to -50) which binds the liver-specific transcription factor LF-B1 (22), suggesting an important role for this factor in albumin expression. The presence of the strong SV40 promoter, to which transcription factors Apl and Spl bind, in the albumin promoter deleted of its own TATA box induces CAT activity in HeLa cells (3'S-32 and 3'A-72). This lack of tissue specificity possibly involves the interaction of

THE HUMAN ALBUMIN GENE

VOL. 10, 1990

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spanning -486 to -193 was either cloned in the inverrse orientation in front of the albumin promoter (5' Xji6) or replace(d by one (X1), three (X3), or five (X5) copies of the 40-bp synthetic L,F-B1 site: Alb upstream oligo represented in Fig. 4A. The stru ctures of the different plasmid constructs are shown in panel B. p'SV2-CAT and pEMBL8-CAT were positive and negative controls, respectively. The positions of [14C]chloramphenicol (CM) and acetyelated forms of chloramphenicol (ACM) are shown. (B) Relative CAT activities of albumin-CAT constructs in Hep3B cells. The CAT a determined by averaging two independent experimients and are expressed relative to that of pSV2CAT (as 100%). The standard deviation values for each construct are included.

either Apl or Spl with some ubiquitous factor( s) binding in the region spanning nt -193 to -73 of the albunfnin promoter because the further deletion (3'A-193) prese nts a liverspecific pattern of expression. It is interestilng that two binding sites for the ubiquitous transcription factor NF1 spanning nt -136 to -107 and nt -102 to -7'2 have been identified in this region (39). Also, the tissue speccificity of the human albumin promoter can be totally overcoir ie by cloning the SV40 enhancer upstream. This enhancer ac,tivates transcription of the albumin promoter in Hep3B aind in HeLa cells to a level comparable to that of pSV2C/ NT (data not shown). A negative element between nt -673 and -4,86 repressed CAT expression in Hep3B cells and, to a lesse r extent, the residual activity found in HeLa cells. This dowin regulation

997

operates on accurately initiated transcription from the AlbCAT fusion gene (Fig. 2). However, more work is required to substantiate the role of this negative element in albumin gene expression. The only documented case in which albumin synthesis is subject to negative control is during the acute-phase reaction (34). It would be interesting to establish whether the negative element plays a role in this process. A similar case of tissue-specific down regulation has been found in the rat a-fetoprotein gene (35). These authors postulate that the binding of a repressor to the negative regulatory element could be responsible for the shutoff of the at-fetoprotein after birth. Analysis of the nucleotide sequence does not reveal any homology with the negative regulatory element described for other liver-specific genes. Transient-expression assays have shown that the albumin 5'-flanking region between nt -486 and -221 is able to stimulate the expression of a linked CAT gene in Hep3B hepatoma cells (Fig. 1). The stimulation occurs mainly in albumin-producing hepatoma cells, indicating that this activating element exhibits some cell type specificity. Its orientation-independent activity (Fig. 5) relative to the direction of transcription indicates the presence of an enhancer. By gel retardation analysis, we observed a major complex with Hep3B extracts, comigrating with a similar complex obtained with HeLa cell extracts (Fig. 3). However, on the basis of the methylation interference results, we show that the Hep3B complex is due to a liver-specific factor. The comigration of the HeLa complex and the Hep3B complex is therefore fortuitous. We have not characterized the HeLaprotein complex further. The binding site of the liver-specific protein in the enhancer was further delineated by footprint analysis and revealed a high degree of homology with the consensus described for the liver-specific transcription factor LF-B1/HNF1. Competition experiments and footprint assays with the purified protein allow us to infer that the protected region of the enhancer is indeed a binding site for the LF-B1 protein (Fig. 4). Functional analyses have demonstrated that the LF-Bl-binding site per se reconstitutes most (62%) of the transcriptional activation obtained with the enhancer (Fig. 5). Thus, LF-B1 appears to be crucial for the enhancer activity. However, we cannot exclude the possibility that other proteins are involved. In particular, we note the presence of two CAAT motifs at positions -422 and -413 that could interact with the CAAT-binding factor C/EBP (19, 23). LF-B1 is known to interact with several different promoter elements (6, 11, 22, 46). In this paper, we have shown that it also interacts functionally with its binding site in an enhancer to stimulate transcription. Recently, Wirth and Baltimore (56) have demonstrated that the KB site of nuclear factor NF-KB functions as both an enhancer and a promoter element. Similarly, the transcription factor Spl binds to a variety of promoter elements and to the enhancer of U2 snRNA (1, 5, 50). For some other specific protein-binding sites such as the octamer motif, a CCAAT-related motif, and the Apl-binding site, it is not clear that the same protein binds to either enhancer or promoter elements, since more than one protein might interact with the same sequence (4, 15, 49). However, the fact that the LF-B1 site is bifunctional indicates that enhancers and promoters may regulate the initiation of transcription through common mechanisms. It is believed that positive trans-acting factors work through the direct interaction of an activation domain with components of the transcriptional apparatus. If the same interaction takes place with the transcription apparatus when the same protein binds to the promoter and to the enhancer, it is likely that

998

FRAIN ET AL.

this interaction occurs via a looping mechanism as proposed by Ptashne (42). ACKNOWLEDGMENTS We especially thank R. Cortese for his invaluable help and encouragement. We thank I. W. Mattaj, G. Grimaldi, and M. Price for stimulating discussions and critical comments on the manuscript. We thank P. Stevenson for technical assistance and H. Seifert for help in the preparation of the manuscript and figures. This work was supported in part by grant BAP-0115-D from the European Economic Community. LITERATURE CITED 1. Ares, M., Jr., J.-S. Chung, L. Giglio, and A. M. Weiner. 1987.

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