cell-specific expression of mouse albumin promoter · 9172 tissue specificity of mouse albumin...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 16, Issue of June 5, pp. 9171-9179, 1989 Printed in (1. S. A.

Cell-specific Expression of Mouse Albumin Promoter EVIDENCE FOR CELL-SPECIFIC DNA ELEMENTS WITHIN THE PROXIMAL PROMOTER REGION AND CIS-ACTING DNA ELEMENTS UPSTREAM OF -160*

(Received for publication, January 20, 1989)

Michael G. IzbanS and John Papaconstantinouj From the Department of Human Biological Chemistn, and Genetics, University of Texas Medical Branch, Galueston, T&x 77550 ’

Regulation of albumin gene expression is believed to be mediated by multiple nuclear factors that interact with cis-acting DNA sequences within the first 160 base pairs (bp) of the promoter. The minimal promoter sequence required to generate tissue-specific expression has not been clearly defined. We have constructed a series of transient expression vectors containing progressive deletions of the mouse albumin gene 5”flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and include the Moloney murine leukemia viral (Mo-MuLV) enhancer. Pro- moter activity was determined in mouse hepatoma and fibroblast cell lines by chloramphenicol acetyltransferase and S1 nuclease analyses. All constructions were compared with -623 Albcat-Mo-MuLV which contains all the sequence homology between the rat and mouse promoters. Low levels of expression were observed with -60 Albcat-Mo-MuLV (10%) in hepatoma but not fibroblast cells. Addition of promoter sequence to -208 bp progressively increased activity to 190% in the hepatoma cells, while -308 and -1612 Albcat-Mo- MuLV had activity similar to the -623 Albcat-Mo- MuLV level, and -3000 Albcat-Mo-MuLV showed a 2- fold reduction in transcriptional activity. The inclusion of promoter sequences upstream of -60 generated low levels of expression in the fibroblasts. We also show that factors from mouse liver nuclear extracts protect at least five regions of the albumin promoter upstream of -160. Our results indicate that tissue specificity is established within the proximal promoter region and that additional cis-acting elements that may have a functional role in the efficiency of albumin gene expression are located upstream of -160 bp.

A variety of cell-specific genes has been analyzed using

* This investigation was supported by United States Public Health Service Grants CA3147204 and CA17701-09 awarded by the National Cancer Institute, Department of Health and Human Services (to J. P. and to the University of Texas Medical Branch Cancer Center, respectively) and by the Zelda Zinn Casper Foundation Grant (awarded to J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence($ reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) J04738.

ML 522, 231 Bethesda Ave., Cincinnati, OH 45267. $ Present address: University of Cincinnati, College of Medicine,

8 To whom correspondence should be addressed Dept. of Human

Branch, F-43, Galveston, TX 77550. Biological Chemistry and Genetics, University of Texas Medical

transient expression assays to define cis-acting elements involved in determining transcription initiation. In some cases, e.g. the rat insulin (1, 2), rat and mouse al-antitrypsin (3, 4), and protein factor B genes ( 5 ) , the enhancers are located adjacent to the promoters, and tissue-specific expression is lost when the enhancers are deleted and the majority of the promoter region remains intact. In other instances, the enhancer is uncoupled from the promoter and located either downstream, e.g. the immunoglobulin gene (6), or upstream of the promoter, for example the mouse a-fetoprotein (7, 8), transthyretin (9), and albumin genes (10). Progressive deletion of the transthyretin 5”flanking sequence between -151 and -108 bp’ results in loss of tissue-specific promoter function in the presence or absence of the homologous enhancer. Therefore, regions required for tissue specificity and promoter function can be localized relatively far upstream of the core promoter TATA box element.

Determination of the effects of deletions on the albumin promoter using transient expression assays is complicated because of the lack of availability of the albumin enhancer and the resultant low levels of expression of enhancerless constructions. To generate higher levels of promoter function in hepatoma cells, the SV40 enhancer has been included in expression vectors. However, this enhancer presents other complications because it also elevates promoter activity in non-hepatic cell types (11, 12). The human adenoviral E1A enhancer has been used successfully to generate high levels of tissue-specific function of the rat albumin promoter (13). In our laboratory, the Moloney murine leukemia viral (Mo- MuLV) enhancer has been used successfully to direct tissue- specific expression of the mouse a-fetoprotein gene promoter (8). In these studies, we used the Mo-MuLV enhancer in expression vectors to generate high levels of tissue-specific function of the mouse albumin gene promoter.

Studies to determine the location of regulatory regions within the mouse and rat albumin promoters using enhancerless constructions indicate that cis-acting elements are within the region between -55 and -160 relative to the start site of transcription (11,14). However, the effects of similar deletions of these promoters on the levels of expression differ when determined by cell-free transcription for the mouse (14) or by transient expression assays for the rat (11). In the former, progressive promoter deletions gradually decreased transcription, indicating that the region between -160 and -60 contains cis-acting elements required for tissue-specific promoter function. In the latter, however, deletions within the region

The abbreviations used are: bp, base pair(s); Mo-MuLV, Moloney murine leukemia virus; HEPES, 4-(2-hydroxyethyl)-l-piperazineeth- anesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; HNFI, hepatocyte nuclear factor I; APF, albumin proximal factor; AFP, a-fetoprotein.

9171

9172 Tissue Specificity of Mouse Albumin Promoter

between -151 and -93 result in complete loss of promoter function. For example, a deletion to -122 decreased promoter function 2-fold in the cell-free transcription system, whereas a similar deletion decreased promoter function 16-fold in the transient expression experiments. In neither of these studies is it demonstrated that tissue-specific function can be un- linked from promoter function.

Both mouse and rat albumin gene promoters contain at least six potential cis-acting elements in the region extending from -34 t o -160 as determined by DNase I protection assays using nuclear proteins from albumin-expressing and nonexpressing cell types (14-19). Sequence analysis of the 5’- flanking region of rat (20) and mouse albumin genes (this study) indicates that homology extends to -460 bp. This strong homology may indicate that regulatory sequences may extend beyond -160 bp and is the basis for conducting further experiments.

For these studies, we constructed a series of transient expression vectors containing as much as 3000 bp of the mouse albumin gene 5”flanking sequences. To generate high levels of tissue-specific expression, we included the Mo-MuLV enhancer in these constructions. These vectors allowed us to determine the effects of deletions on the activity of the mouse albumin promoter with emphasis on determining if there are regulatory elements upstream of -160 and to investigate the differences between albumin promoter deletion studies dis- cussed previously. We also perform DNase I protection studies to determine if there are specific nuclear protein-binding regions upstream of -160.

EXPERIMENTAL PROCEDURES

Plasmid Construction-Plasmids were constructed and amplified using standard protocols (21, 22). All enzymes were purchased from commercial suppliers (Bethesda Research Laboratories, Amersham Corp., International Biotechnologies Inc., Du Pont-New England Nuclear, New England BioLabs, and Worthington). The albumin genomic clone contains approximately 3 kilobase pairs of 5”flanking DNA and exons 1-7. An EcoRI fragment containing all the 5’- flanking sequence and the first five exons was inserted into pBR325 (see Fig. 2, p3-I) . Subsequently, the HindIII fragment containing the albumin promoter and first exon was subcloned into pUC8 (see Fig. 2, pHH3-I ) . The albumin promoter was localized by DNA sequence analysis (23) of the HindIII fragment by a combination of the Ba131 deletion method (21) and directed cloning.

Transient Expression Vectors-To construct a transient expression vector containing the albumin promoter fused to the chloramphenicol acetyltransferase (CAT) gene, the HindIII (-2042) to MspI (+76) fragment was subjected to a controlled Ba131 digestion to remove the protein coding region of the first exon and cloned into the HincII site of pUC8. By sequence analysis, we identified a clone (plasmid HindIII/MspI deleted; see Fig. 2, pHMD) that had a deletion end point of +12. The transient expression vector -1612 Albcat was constructed by removing the simian virus (SV40) promoter and enhancer from pSV2cat (24) with a HindIII/AccI digestion and by inserting the HindIII/AccI (the AccI site is located at -1612) fragment of pHMD (see Fig. 2). Therefore, the albumin promoter/chloramphenicol acetyltransferase construction contains from -1612 to +12 of the albumin gene and 11 bp of the pUC8 multiple cloning site fused to the HindIII site of pSV2cat. Both AccI and SphI (-308) sites are unique to -1612 Albcat. Additional transient expression plasmids were constructed as follows. -308 Albcat was created by digesting -1612 Albcat with AccI and SphI, filling in the ends, and recircularizing. The genomic clone containing the 5”flanking sequence is bordered by a linker-derived EcoRI site. An EcoRI fragment was isolated from p3-1, the ends were filled in, and the fragment was subjected to a partial SphI digestion. The fragment -3000 to -308 was isolated and cloned into the -1612 Albcat that had been linear- ized and blunt ended at the AccI site and subsequently digested with SphI.

Plasmids -3000, -1612, and -308 Albcat-Mo-MuLV were constructed by digesting the three plasmids described previously with BamHI and inserting a BamHI DNA fragment (approximately 280

bp) containing the Mo-MuLV enhancer. Additionally, -623 Albcat- Mo-MuLV was constructed by digesting -1612 Albcat-Mo-MuLV with AccI and AuaI, blunt ending, and recircularizing.

The deletion mutants were constructed by digesting -1612 Albcat- Mo-MuLV with SphI (-308), generating a series of deletions by performing a time course digestion with Ba131, digesting with AccI, and religating after filling in the protruding ends. Deletion end points were determined by sequence analysis (23).

The promoterless plasmid pSVOcat-Mo-MuLV was constructed by removing the albumin 5”flanking sequence from -1612 Albcat-Mo- MuLV with an AccI/HindIII digestion, filling in the ends, and recircularizing. The plasmid pSVlcat-Mo-MuLV was generated by digesting pSV2cat with AccI and SphI, filling in the ends, and recircularizing. The Mo-MuLV enhancer was subsequently cloned into the BamHI site.

SI Nuclease Mapping-The DNA templates used to generate the single-stranded probes for S1 nuclease mapping were constructed as follows. The Hue111 fragment (containing from -294 to +187 bp and including 127 bp of CAT gene coding region) of -308 Albcat was cloned into the SmaI site of M13mp19, and the orientation that produced anti-sense-labeled probe was used (see Fig. 7). The HindIII- XhoII fragment of the 0-galactosidase expression vector, pCHllO (25), was cloned into a HindIII/BamHI-digested M13mp19.

DNase Z Protection-The restriction fragments used for the protection assay were derived from either the -623 or -208 Albcat constructions. The HindIIIIRsaI fragment of -208 Albcat (the RsaI site is located within the pSV2cat vector 540 bp upstream of the HindIII site) was cloned into HindIII/SmaI-digested pUC18. The RsaI fragment of -623 Albcat, located at -375, was cloned into the SmaI site of pUC18. Finally, the RsaI/HindIII fragment (-374 to the polylinker beyond +E) was partially digested with AluI and cloned into the SmaI site of pUC18. The clone that contained from -374 to -164 was used. In all three cases, the inserts are bordered by EcoRI and HindIII sites.

Cell Cultures-The BWTG3 cell line is derived from a mouse hepatoma (BW7756), and its biochemical phenotype has been described in detail (26). The mouse cell line NIH3T3 has been described. All cells were cultured in Dulbecco’s modified Eagle’s medium sup- plemented with 10% bovine calf serum (HyClone Laboratories, Logan UT), 100 units of penicillin, and 0.1 mg of streptomycin/ml. Cultures were maintained in log growth phase at all times.

Transfection Conditions for Chloramphenicol Acetyltransferase As- says-Two to four days before transfection, cells were passed to 50- 70% confluency. One day prior to transfection, cells were seeded at 1 X lo6 (BWTG3) and 0.5 X lo6 (NIH3T3) in a 100-cm dish. A DNA mixture containing 1.5 pmol of transient expression vector and carrier sheared salmon sperm DNA (final DNA concentration, 20 pg/ml) was precipitated by the calcium phosphate method (27). In the experiments including pCH110, 0.75 pmol of DNA was included in the precipitation mix. The cells were refed 2-3 h prior to the addition of DNA. The medium was removed 4 h later, the cells were treated for 2 min with 15% (v/v) glycerol in phosphate-buffered saline, washed twice with HEPES-buffered saline, and refed. Cells were harvested 48 h after the addition of DNA. Chloramphenicol acetyltransferase assays were performed as described by Gorman et al. (24), except 0.1 or 0.2 pCi of [‘4C]chloramphenicol (Du Pont-New England Nuclear) was used. The reaction products were separated by thin layer chromatography (Whatman), visualized by autoradiography (Kodak XAR film), and the spots that correspond to the acetylated forms were cut out and counted. Counting was done to 2% sigma error using a quench correction program (Beckman). Protein concentrations were determined by the method of Bradford (28). Promoter activities are expressed in pmol of chloramphenicol acetylated/mg of cytoplasmic protein/h. In the transfections containing pCH110, one- fifth volume of supernatant was used for either chloramphenicol acetyltransferase or P-galactosidase assay (25, 29). 0-Galactosidase assays were carried out as follows. 20 pl of supernatant was added to 0.6 ml of reaction mix (0.1 M sodium phosphate, pH 7.8, 10 mM KC1, 50 mM P-mercaptoethanol, 1 mM MgC12). This reaction mix was warmed to 37 “C, and 120 pl of ortho-nitro-phenol-P-D-galactopyran- oside (2 mg/ml) was added and incubated at 37 ‘C. The reactions were allowed to proceed until a visible color developed. The reactions were stopped by the addition of 0.3 ml of Na2C03. The reactions were extrapolated to AdZ0/h and promoter activities expressed in pmol of chloramphenicol acetylated/mg of cytoplasmic proteinlhlA420.

Transfection Conditions for mRNA Analysis-Transfections were performed as outlined above except (a ) pCHllO was included in all precipitations; ( b ) the volume of the precipitation was reduced SO

Tissue Specificity of Mouse Albumin Promoter 9173

that the final DNA concentration was 30 pg/ml; and (c) the precipi- tates were left on the BWTG3 and NIH3T3 cells for 8 and 16 h, respectively, prior to the glycerol shock. We determined that the transfection efficiency of the BWTG3 cell line was not affected by increasing the number of cells seeded. BWTG3 cells seeded at 2 X lo6 yielded 100 pg of total RNA per plate. On the other hand, transfection efficiency of NIH3T3 cells was decreased at least 100- fold by increasing the number of seeded cells over 0.7 X lo6. A density of 0.5 X lo6 NIH3T3 cells yielded 75 pg of total RNA per plate.

Transcription Initiation Analysis-Total cytoplasmic RNAs were harvested by the guanidine HCI method (30). The transfected cells were washed twice in Tris-buffered saline. Five milliliters of Tris- buffered saline was added to the tissue culture dishes, and the cells were mechanically dislodged and pelleted for 5 min at 3200 rpm. The Tris-buffered saline was removed, and the cell pellets were lysed by vigorous vortexing after the addition of 2.5 ml of lysis solution (7.5 M guanidine HC1, 50 mM sodium citrate, pH 6.0, and 5 mM p- mercaptoethanol). The lysate was layered over a cushion of 5.6 M cesium chloride and centrifuged, using a Beckman SW 50.1 rotor, for 18 h at 35,000 rpm. The RNA pellets were washed in 70% ethanol and resuspended in TE (10 mM Tris-HC1, 1 mM EDTA, pH 7.0). Sodium acetate, pH 4.8, was added to a final concentration of 0.2 M, and the RNAs were precipitated overnight at -20 "C after the addition of 2.5 volumes of ethanol.

Single-stranded 32P-DNA was synthesized and purified as outlined by Davis et al. (22), except 4 pl of [a-"PIdCTP (3,000 Ci/mM, ICN Biomedicals) was used, and the reaction mixture contained 120 p M dATP, dGTP, dTTP, and 4 p~ dCTP. Total RNA (50 pg) was precipitated from each transfection, partially dried, and resuspended in 30 pl of hybridization mix (40 mM PIPES, pH 6.4, 1 mM EDTA, 0.34 M NaC1, and 80% formamide) containing 100,000 cpm of the Albcat and 20,000 cpm of the p-galactosidase probes. After heating at 75 "C for 15 min, the mixture was allowed to hybridize for 16 h at 46 "C. Ice-cold S1 digestion buffer (300 pl) (0.28 M NaC1, 0.05 sodium acetate, pH 4.8, 4.5 mM ZnSOd, and 24 pg/ml sheared salmon sperm DNA) containing 30 units/ml S1 nuclease was added and incubated for 1 h at 37 "C. The samples were phenol/chloroform (1:l)-extracted, ethanol-precipitated, and resuspended in 10 pl of 50% formamide, 1 mM EDTA, pH 8.0. After boiling for 3 min, 5 pl of the samples were run on a 40-cm 6% polyacrylamide, 7 M urea sequencing gel at 30-mA constant current for 2 h. After drying, the gels were autoradiographed at -90 "C with an intensifying screen (Corning Glass Works).

DNase I Footprinting Reaction-Crude nuclear extracts were pre- pared from mouse BALB/c livers according to the method of Gorski et al. (14) except for the following modifications. Homogenization buffer (buffer A) was 0.25 M sucrose, 60 mM KC1, 15 mM NaCl. All buffers contained 1 pg/ml of leupeptin, chymostatin, antipain, and pepstatin A. After homogenization, 2 volumes of 2.3 M sucrose buffer A was added, mixed, and layered over a 10-ml cushion of 2.0 M sucrose buffer A. Nuclear lysis buffer did not contain glycerol.

Probes were labeled by filling in the 5' overhang with [w3*P]dATP and [cY-~'P]TTP using Klenow fragment of DNA polymerase (21) after linearization of the plasmid with either EcoRI or HindIII.

The DNase I protection (31) was performed in a 20-p1 reaction mixture containing 10 mM HEPES, pH 8.0, 0.5 mM dithiothreitol, 5 mM MgC12,O.l mM EDTA, 30 mM KC1,0.2 mM phenylmethylsulfonyl fluoride, and 12% glycerol. Nuclear proteins were incubated with 4 pg of poly(dI.dC) competitor DNA (Sigma) for 20 min at 4 "C prior to the addition of 100,000 cpm of labeled DNA. After a 1.5-h incu- bation at 4 "C, the samples were allowed to equilibrate to room temperature for 3 min and then digested for 1 min with 0.5-4 pl of digestion mix (6.25 pg/ml DNase I (Worthington), 10 mM HEPES, pH 7.6, 25 mM CaC12) depending on the protein concentration. The reactions were stopped by the addition of 100 pl of stop mix (20 mM EDTA, pH 8.0,3 pg/ml sheared salmon sperm DNA). The DNA was phenol/chloroform-extracted and fractionated using a 6% polyacrylamide, 7 M urea sequencing gel.

RESULTS

Sequence Analysis-The murine albumin promoter was identified by sequence analysis of a DNA fragment containing from -2042 to +334 bp of the albumin gene (Fig. 1). The nucleotide sequence is numbered relative to the most 3' transcription start site which was localized using S1 nuclease protection analysis of mRNA from transfection experiments

A Hind111 k c 1 AvaI SpI a Hind111 - Ne01 Ne01

"-4 4 - 4 "" +

C"" - "c_

c c_ - B -2042 -1982 -1922 -1862 -1802 -1742 -1682 -1622 -1562 -1602 -1442 -1382 -1322 -1262 -1202 -1142 -1082 -1022

-962 -902 -842 -782 -722 -662 -602 -542 -482 -422 -362 -302 -242 -182 -122

-62 - 2 59

119

239 179

299

AASCTTSAAA ACASSACTSC CTTASAAGTA ACTAATAATT ATETSSCTCA TCCTCTASTA TAPASTCTAC ATATASGACS A S T S C C C M C AGTTTCASAA 66AASASTAT AAATTCTTAC TCACCASATS CAAAASTSTT TCTCTTCA6T TSAAATGATC T616ASCACS CACACTCTGT

bCAi1616TT A6TACTSCCT AA66AGASGC TCASTTSTTA TTAATTTATS TCTTAAAAPT ATTPCASCTT CATCACTCAC TAAACCASCT CAACCCCCTA CTTCATTCTS ATGCTTTTCT CTCCAGECTT TSSCTSCATA SATSATTTTT TTTTTTTTTT TTACTTAATT CTCAAACAAA ACTATATATA 61ATACATAS TACATCTCAS ASTSCTAAAA SCCTGASTTA ATAACACAAT ATTATACTTA AACACAATAT TACATATAAC TATATASTAC TTSTEAGTTA ATAATACAAA

ATCTATCTAT TCTCTTATTC TTCTCCTTCA EATSASSACA AASAASAATS CTTCTCCTCC TSSSSTTSAT ATTATTATCC ATTSSASTSC TSAAAACATT SACTTCTSAA TSASACACTS

TATTT66611 CCCTATAATG

TSCCAASSCC ATTTSCTSTT 666TTASA.66 TTTSSCAAAC

&S&TCACCTT TSSTTAATSA

CTCCTCCTCT AA6ASTTTTA TAAAATASTT TATTATTSTC SSTATCTTTS

. . . . . . . . . . . . . . . . . . . . . . . CTATCATCTA CATTTCAGPA TTTTSCCTTS CAASATSASS CTTCTCCTCC TTCCTCTTCT TTSSATSTAG TSTSSTSTTA CTSTTCTAGT CTACTCCASA ASTCCTCTSC TGTSCTTTAT ASACCCACCC SAACAACCTA CACACTSAAA TTACATAACT SSAACASCTC ATSSTATSAT TCTACASTTA TCCTATCAAC TCSTCTCC66 TSTTTTTTCA AAATTTTCCT ATTATTT6CA ATGACAATAA

TCTATCTATC CTCTTTTSAS ACTCCTETSS TCATAASACA TCCTCCTCTT CCTTCTTSTC TCTTSACTSA SASGAASTAT STCTCCTCTA CCCPTTTCAC ACTTGTTTTT 66AASATTCT CTCCCTCATA TSCAATTCAS TCCTCAAATC TTAATSAATS CASATSSCAA TTTSTAATSS TTSGTTAAAS CCCACTACCC

TCTCTPCTTP CTCTSCTTTT

TTASTSCTSA TCTSASAACC T6666ATTTS

. . . . . . . . . . . . . TATCATTCTS TSATAAAACA TTSATTACAG TASATTTCTT

ACTSAATAST CCTCCTCCTC

STTACGAT66 CTCATAASTG

AASCACCCCG ACATASCCTE

AACTTTAAAA TATTGCTCTG

TTCTTASCTT CACATTCTTC

SSASACAAAS SACAAASTCT ACATACSCAA 66TASSAACC AASTATATTA TCTSSCAAAA T C C A 6 6 6 6 T S TATTTTTCTA TTTCTASATT C T T A 6 6 T 6 6 T A M C T T

TTCATCTSTS ACCAAGGAAA AATTTSTACA TCATTSTATA CTCTCTTCCT CCCTSTATAT ASATAACT66

ASAAASASGT CAAASACCAT

A C C 1 1 6 6 6 6 T SCTTCTTCTC ASATSASAAA ATTACCTSTA

AGATTAASCT TTAAATSTSS

TSTSCATSSS 666ATTTAST AATSAAATSC SASCSASTCT TSAASTSSST TSTTTCSCCG 6TAAT66AAG ATTATTACT6 TATATTATTS

ACACCACGAC ESAAGTTTAA TCTTTTCCAC TATTCCATSS TCTATCTATC CTATCTATCT TAGACSCTAC CACTTTTCCC CCTTTCTSAA ACCASATATA CCTTTTCCTT TTAGGAACCA TCTSCTACTA CATCACTSTC TCATGTTAGA SSSEACACAA ASTTCACATT SACTTSSAAE CATAAAASCA CATSCTTCCA CTTATSTAAA 6616666516 CAAACAACTT 6A66TAASTA TTCTSCACAC AACCTTTCTC ASAASCACST

TTSTTSTTST CCTSSTATTT

ATATATTTTT

FIG. 1. The sequence of plasmid pHH3-1. (A) mouse albumin gene exon a is indicated by the black box. Relevant restriction enzyme sites are shown. The arrows indicate the direction and extent to which different templates were sequenced. ( B ) The sequence of pHH3-1 is shown. Numbering is relative to the most 3' transcription initiation site determined by S1 nuclease analysis of mRNA generated from Albcat-Mo-MuLV constructions. The two start sites are underlined.

(see below). Previous studies on the mouse and rat albumin promoters indicate that at least six nuclear proteins interact with the first 160 bp of 5"flanking sequence (see Figs. 8 and 9). Potential regulatory elements found in other liver-specific gene promoters are hepatocyte nuclear factor 1 (HNFI) 5'GTTAATNATTAAC (32) from -60 to -48 and the region from -127 to -135 which is the reverse complement of a site (TGTTTGAC) found in the human hepatitis B viral (33) and liver-specific AFP enhancer and promoter (34).

Albumin Promoter-driven Chloramphenicol Acetyltransfer- me Transient Expression-Three albumin promoter/CAT gene expression vectors containing from +12 to -3000, -1612, and -308 bp, respectively, were constructed in an initial attempt to localize the mouse albumin gene enhancer (Fig. 2). None of these plasmids generated levels of chloramphenicol acetyltransferase activity indicative of an enhancer-driven promoter in either mouse hepatoma (BWTG3) or mouse fibroblast (NIH3T3) cell lines, indicating that the albumin gene enhancer was not contained within this region (Fig. 3). While this work was in progress, a region capable of increasing the transcription rate of the albumin promoter tested in transgenic mice was localized 8-10 kilobase pairs upstream of the initiation site (lo), however this enhancer does not appear to function in hepatoma cell lines.

To elevate the level of transcription, we included the Mo- MuLV enhancer which does not affect tissue-specific expression from the liver-specific a-fetoprotein gene promoter in BWTG3 cells (8). Analysis of these three constructions showed that the levels of chloramphenicol acetyltransferase activity for -3000, -1612, and -308 Albcat-Mo-MuLV were significantly higher in the hepatoma than in the fibroblast

9174 Tissue Specificity of Mouse Albumin Promoter E H A V S a H b C

p3-1 I 1‘ d e E

M ~

pHH3-1 1 Kb

a pUC8

Bal-31 digestion

A1 bcat -1612

A1 bcat -308

1 Hindl I I /AccI dlgest lons of pHHD and pSV2cat

CAT mRNA -A-An

CAT

-3000 A I bcat

-623 Albcat-Mo-MuLV

pSVOcat-Mo-MuLV CAT

pSVlcat-Ho-NuLV 9.. i:),, -.~: e::.=, CAT

FIG. 2. Transient expression vectors used to study the proximal regulatory sequences of the mouse albumin gene. The plasmids and DNA fragments used are shown; the strategy is described under “Experimental Procedures.” Solid black boxes indicate albumin gene exons and are labeled a-e. The albumin gene sequence and 5”flanking region are represented by the double line. SV40 promoter, which includes the enhancer (from Ace1 to second SphI) and promoter (from SphI to HindIII) is indicated by the light stippled box. The CAT gene DNA is enclosed in a large box. The Mo-MuLV enhancer is illustrated by the dark hatched box. The mRNA generated by the correct initiation from the albumin promoter is indicated above the first Albcat construction. The message includes the CAT gene coding sequence, the sV40 small t splice site, small t intron and the SV40 early polyadenylation site. E, EcoRI; H, HindIII; A, AccI; V, AuaI; S, SphI; M, MspI.

U W G 3 NIH3T3

I , L

-3000 -1612 -308 -3000 -1612 -308

Albcat Albcat-YoYuLV

FIG. 3. Transient expression with and without the Mo- MuLV enhancer. Chloramphenicol acetyltransferase (CAT) activities were calculated as pmol of chloramphenicol acetylated/mg of cytoplasmic protein/h, and the different constructions are expressed as percentages of activity produced in parallel by the positive control plasmid pSVlcat-Mo-MuLV. Each horizontal bar represents the mean value of three or more separate experiments. The error bars represent the standard deviation.

cells (Fig. 3). These data indicate that the mouse albumin promoter containing 308 bp of 5”flanking sequence can direct tissue-specific expression of the CAT gene. The inclusion of sequence up to -3000 decreased the level of chloramphenicol acetyltransferase activity in the hepatoma cells, thus the region between -308 and -3000 may contain sequence element(s) capable of down-regulating the albumin promoter. Greatly reduced but significant levels of chloramphenicol acetyltransferase activity were observed in the fibroblast cell line.

Cis-acting Regulatory Regions of the Albumin Promoter- Sequential deletions within -308 bp were generated to determine their effects on the levels of expression in hepatoma and fibroblast cells. The deletion mutant end points were -206, -160, -122, -60, and -1, respectively. Although the level of chloramphenicol acetyltransferase activity measured for each deletion varied between experiments, we always observed the same pattern of relative expression among the deletions within any one experiment (Fig. 4). Deletion from -308 to -206 increased the level of chloramphenicol acetyltransferase activity, the deletion from -206 to -160 decreased chloramphenicol acetyltransferase activity to levels similar to the -308 construction, and the deletions to -122 and beyond decreased chloramphenicol acetyltransferase activity to basal levels. In the fibroblast cells, none of the deletions caused a significant change in expression.

One explanation for the variability in chloramphenicol acetyltransferase activities between experiments could be variations in transfection efficiencies. To correct for this possi- bility, we included the 0-galactosidase expression vector pCHllO as an internal control. pCHllO contains the bacterial P-galactosidase gene under the transcriptional control of the SV40 viral promoter and enhancer (25). Results from these experiments (data not shown) indicate that the level of chloramphenicol acetyltransferase activity generated by the constructions -122, -60, and -1 may gradually decrease rather than have similar values.

The construction containing from -1 to $12 of the albumin promoter generated significant levels of chloramphenicol acetyltransferase activity. To determine if this low level of activity was attributable to the remaining 13 bp of albumin gene promoter or due to non-albumin promoter-specific initiation, we constructed pSVOcat-Mo-MuLV, a promoterless enhancer-containing plasmid (Fig. 2). pSVOcat-Mo-MuLV pro- duces chloramphenicol acetyltransferase activity comparable to the -1 Albcat-Mo-MuLV construction in both cell lines (see Fig. 4). Therefore, nonspecific initiations appear to gen-

a U BWTG3 SS NIH313

-308 -206 -1 60 -122 -60 -1 pSVOcat- Albcat-UoYuLV UOUULV

FIG. 4. Effects of promoter deletions on CAT gene expression. The average of four separate experiments using two different isolates of each plasmid is shown. The results are presented in the same manner as Fig. 3.


erate detectable levels of translatable chloramphenicol acetyltransferase mRNA.

Determination of Transcription Initiation-Although the @- galactosidase vector corrected for transfection efficiency, the levels of non-albumin promoter-specific initiation may not contribute equally to the overall chloramphenicol acetyltransferase activity when the mouse albumin promoter region is present in the vector. To determine directly the amount of albumin promoter-specific initiation, we analyzed RNA from hepatoma and fibroblast cells that were transfected with the Albcat-Mo-MuLV constructions and the @-galactosidase control plasmid. We used an S1 nuclease protection protocol (21, 35) to analyze the mRNAs generated from the two expression vectors. Anti-sense, uniformly labeled, single-stranded DNA probes specific for the albumin/CAT and @-galactosidase genes were used (Fig. 5). The specific as well as nonspecific initiations in the hepatoma and fibroblast cells are shown in Fig. 6.

Differences in promoter activities among the deletions were determined by densitometry scanning of the bands that correspond to promoter-specific initiations. The results were A

-160

A1 bumin promoter

e SS DNA PROBE (519nt)

4 WNA; CORRECT INITIATIONS - $ S1 NUCLEASE

PROTECTED SPECIES:

NOW-SPECIFIC INlTlATlONS

481nt 3000.-1612 ) 1-423, -308 )

I) 395nt (-206) * 347nt (-160)

m 309nt (-122) I) 247nt ( -60)

m 188nt ( -1)

CORRECT lNITIATIONS

187nt 189nt

B

&gal gene

e SS DNA PROBE (162nt)

J WNA

122 bp protection

FIG. 5. Schematic drawing of the procedure and probes used in the S1 nuclease experiments. ( A ) the DNA used to generate the single-stranded probe specific for albumin promoter initiation is shown. The albumin promoter sequence is indicated by the dark box, the CAT gene sequence by the stippled box, and the M13 sequence by the single line. Deletion construction end points and the CCAAT and TATA boxes are indicated for the albumin promoter. The probe is 519 bp in length and contains from -294 to +187 of the albumin/ CAT fusion gene and 38 nucleotides of the M13 multiple cloning site. mRNAs corresponding to correct initiations and protected species corresponding to albumin promoter-specific and nonspecific initiations are shown below the probe. ( B ) the DNA used to generate the single-stranded probe to detect &galactosidase mRNA is shown. The SV40 promoter is indicated by the dark stippled box, the B-galactosidase gene by the light stippled b o w , and the M13 sequence by the single line. The probe, containing 124 bp of the untranslated region of the @-galactosidase vector, and 38 bp of M13, mRNA, and protected species (122 bp) are shown.

A NIH3T3 E W T G 3

P N E 1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 S S 519 - rn

B

PIC. 6. Autoradiograph of S 1 nuclease protection experiment. ( A ) protected DNA products of the SI nuclease digestion were separated on a 6% polyacrylamide, 7 M urea denaturing gel. An autoradiograph of an 8-h exposure is shown. Lane P, probe only; lanes N and B, hybridization using NIH3T3 and BWTG3 total RNA; lanes 1-9, hybridization using 50 pg of total RNA from NIH3T3 and BWTG3 transfected with the Albcat-Mo-MuLV constructions in decreasing order, i.e. -3000, -1612, -623, -308, -206, -160, -122, -60, and -1; lane 10, NIH3T3 transfected with pSVOcat-Mo-MuLV, the protected nonspecific species is 162 nucleotides in length and could not be distinguished from the intact P-galactosidase-specific probe. ( B ) a 24-h exposure of the promoter initiation portion of the autoradiograph is shown.

-60 -160 -308 -1612 -1 22 -206 -623 -3000

FIG. 7. Quantitation of promoter initiation in hepatoma and fibroblast cells. Differences in promoter initiation and transfection efficiency between the constructions were determined by densitometry tracing of the appropriate bands of multiple gel exposures. The areas under the curves were integrated; albumin promoter-specific initiation was normalized to transfection efficiency and expressed as a percent of the -623 Albcat-Mo-MuLV construction.

normalized to the @-galactosidase internal standard and expressed relative to -623 Albcat-Mo-MuLV (Fig. 7). The -623 construction is used as the standard level of expression because it contains the entirety of the sequence homology between rat and mouse albumin 5'-flanking sequences. We were not able to examine -1 Albcat-Mo-MuLV because of inter- ference from the nonspecific protected species.

The -60 Albcat-Mo-MuLV construction retains the ability

9176 Tissue Specificity of Mouse Albumin Promoter

to initiate transcription in the hepatoma cells while it is essentially refractile to transcription in the fibroblast cells. Thus, tissue specificity appears to be established within the first 60 bp of the albumin promoter. The construction containing to -122 increases transcription to 40% in the hepatoma and establishes near maximal levels of transcription in the fibroblast cells. The addition of sequences to -160 increases transcription only in the hepatoma cells (80%), indicating that either tissue-specific factor(s) interact with this region of the promoter or ubiquitous positive transcription factor(s) only function with the transcription complex established in the hepatoma cells. The -206 Albcat-Mo-MuLV construction further increased transcription 2-fold, however the -308 Albcat-Mo-MuLV construction reduced transcriptional activity to the -623 Albcat-Mo-MuLV level. The addition of sequence between -1612 and -3000 decreased transcriptional activity %fold.

The level of expression of the -60 Albcat-Mo-MuLV construction in the hepatoma cells was approximately equal to the level of expression of the constructions containing at least -122 bp in the fibroblasts (see Fig. 7). We believe this direct comparison between cell types is valid because the levels of &galactosidase mRNA (Fig. 6A) and the ratio of chloramphenicol acetyltransferase activity/mg of protein between pSV2cat and pSVlcat-Mo-MuLV (data not shown) are approximately the same in both cell lines. Therefore, the intact mouse albumin promoter appears to function approximately 8-fold better in the hepatoma cell.

Detection of Regions of DNA Which Interact with Nuclear Proteins between -166 and -624-Our data demonstrate that sequences between -308 and -160 affect albumin promoter activity, therefore we wanted to determine if regions upstream of -160 of the albumin gene would specifically bind nuclear proteins. We performed DNase I protection experiments (31) using a nuclear protein extract from BALB/c mouse liver.

In uitro DNase I footprinting experiments using coding and noncoding strand end-labeled fragments covering the regions from -624 to -375 and -374 to -166 of the mouse albumin gene are shown (Fig. 8, A and B ) . In order to test the quality of our nuclear protein preparation, we also footprinted a DNA fragment containing between -166 and +12 (Fig. 8C). This region of the rat and mouse albumin promoters has been shown to interact specifically with a number of different nuclear proteins (14-19,36,37). The relative positions where rat liver nuclear factors A-F bind to the mouse albumin promoter (16) are shown. Using mouse liver nuclear proteins, we observe a similar protection pattern. Our probe also included the promoter region to +12. We also observe protection from the cap site to the end of the fragment. Because protection extends beyond +12 and into the polylinker sequence, we believe that RNA polymerase initiation is responsible for the protection observed.

Footprinting experiments of the regions between -166 and -624 indicate that five regions are protected from DNase I digestions. These are labeled G, H, I, J, and K (Fig. 8), and the sequences are shown in Fig. 9. In addition to these five sites, there are four other regions that may bind nuclear proteins. These sites appear to be protected or partially protected on only one strand (see the two sites between -209 and -169, Fig. 8B). This could be due either to specific but weak interactions or to conformational changes in the DNA structure within these regions when protein binding occurs at sites G, H, I, J, and K which could alter the sensitivity of these regions to DNase I.

DISCUSSION

In these studies, we tested the effects of deletions of the mouse albumin gene 5”flanking sequence on the levels of transcription in mouse hepatoma (BWTG3) and fibroblast (NIH3T3) cell lines. The level of expression of the construction containing the entire upstream sequences available to us, i.e. 3000 bp, generated low levels of activity in either cell type (Fig. 3). Because this level of expression was at the lower levels of sensitivity for the assay, we included the Moloney murine leukemia viral enhancer, which generated high levels of cell-specific expression and allowed us to determine the effects of deletions on promoter function. Because of nonspecific initiation, presumably within the bacterial portion of the expression vector, we determined promoter activity by directly examining correctly initiated chloramphenicol acetyltransferase transcripts (Figs. 6 and 7).

Our results indicate that regions of the albumin gene 5’- flanking sequence capable of altering expression of the promoter are located between -3000 and -1612, and within 308 bp of the start site of initiation. Furthermore, our results using transient expression and DNA protection assays indicate that multiple potential cis-acting elements are located between -585 and -160 bp. We confirm that the near maximal level of tissue-specific expression is generated with 160 bp of the promoter using -623 Albcat-Mo-MuLV as a standard. Similar results have been reported with the mouse promoter using cell-free transcription analysis (14) and the rat promoter using transient expression assays (11, 13). Both DNA protection assays and deletion analyses indicate that this region contains multiple potential cis-acting DNA sequences (see Fig. 9), however the effects of similar promoter deletions differ when assayed by either transient expression or in uitro transcription assays.

Transient expression analysis of rat albumin promoter deletions using enhancerless constructions results in loss of tissue- specific promoter function when 93 bp of the promoter remain (11). In contrast, we observe a gradual decrease in promoter activity as a consequence of progressive deletions similar to results observed by Gorski et al. (14). Most significantly, we observe tissue-specific expression with a deletion of the promoter to within 60 bp of the transcriptional start site. The differences in promoter activity in transient expression assays may be ( a ) a function of the assays used; ( b ) greater sensitivity of S1 analysis afforded when promoter activity is elevated by an enhancer; or ( c ) differences between the hepatoma cell lines used. We do not believe that the Mo-MuLV enhancer is compensating for the promoter deletions because similar deletions of the mouse albumin promoter tested by in uitro transcription assays using rat liver nuclear extracts also result in a gradual loss of promoter activity comparable to our results.

Localized within the first 60 bp of the mouse albumin promoter are at least three potential cis-acting elements. They are the TATA box and two regions, -29 to -23 (site A) and -60 to -48 (site B), which contain similar sequence motifs but may bind different nuclear proteins (16). The -60 to -48 element of the rat promoter has recently been shown to interact specifically with albumin proximal factor (APF, 8). This nuclear protein is probably identical to HNFI which has been shown to bind to a number of different liver-specific promoters (32, 36-40). There is now direct evidence using in uitro transcription experiments that APF, which is only found in hepatocytes, is required for transcriptional activity of the rat albumin promoter (18). Our data corroborate the notion that APF is a cell-specific positive transcription factor because we observe tissue-specific expression with -60 Albcat- MO-MuLV.


4 OOZP 00280 aonP a 0240

.e24 .5?5

"

-"

NC

""

"

3 OOZP oon4

-x+-

!-' I

C a 0 s

NC

.Ym t l 2

FIG. 8. DNase I protection analysis of the albumin Coding (C) and noncoding ( N C ) strands are indicated. The fragments of DNA used in the protection assay are

promoter with mouse liver nuclear extracts.

panel A , a long run was necessary to determine the extent of protection in the upper portion of the gel. The arrows depicted below the autoradiographs in panels A, B, and C. Because of the length of the DNA fragment used in

are positioned at sites that become hypersensitive to DNase I. Regions that show protection on both strands are

weak protection are indicated only with a vertical bar. Regions A-F (panel C ) are the relative positions of the six indicated by a vertical bar and are assigned a letter, while those that either show protection of one strand or show

nuclear protein-binding regions described by Lichtsteiner et al. (16). This result ensured us of the quality of our nuclear protein preparation. Lane G is the chemical cleavage reaction; numbering is relative to the cap site. Lanes 0, 15, 30, and 60 designate the pg of nuclear extract included in the reaction.

Some transcription activators, which bind to sequences adjacent to the TATA box, may alter the binding character- istics of the TATA box factor (41, 42). For instance, the adenoviral major late promoter upstream activator facilitates the binding of the TATA box transcription factor TFIID (43). Since the capacity of these activators to fulfill this function is position-dependent, the proximity of APF and/or the factor that interacts with the mouse albumin promoter site A may function in a similar fashion. Interestingly, in dedifferentiated hepatoma cells and somatic cell hybrids where liver-specific function is lost, APF/HNFI is absent. Instead, a smaller variant protein (vHNFI/vAPF) with a similar binding specificity is present (18, 38). Thus, APF/HNFI may be involved in a crucial step in cell-specific transcription initiation complex formation.

In order to generate near maximal levels of transcription, the albumin promoter requires additional cis-acting elements localized upstream of -60. It has been shown that ubiquitous and liver-specific proteins interact with sequences located within the first 160 bp of the promoter region, however the concentration of these proteins may differ between cell types (15, 16). For instance, the region between -122 and -110 contains half of a recognition sequence for nuclear factor 1 and binds a ubiquitous nuclear factor 1 present in similar concentrations in liver, brain, and spleen and a liver-specific form that is enriched in the liver (16). Also, linker-scanning mutants that selectively abolish factor C, D, or E binding result in a drastic decrease of transcription in uitro (16). Thus, the albumin promoter appears to contain tissue-specific cis- acting elements interspersed within core promoter elements, and the combination of these elements may be important for efficient promoter function.

The 5'-flanking sequences of the mouse and rat albumin genes contain regions of homology to -460 relative to the mouse gene. We observe variations in the level of expression

when 5"flanking sequences are deleted between -623 and -160 (Figs. 6 and 7). Comparison of promoter sequences between divergent species reveals regions of homologies corresponding to sites C, D, and E (40), all of which are important in transcription (14, 18). Similarly, site G (Figs. 8 and 9) is conserved in sequence and relative position between chicken, rat, and human (44). Therefore, it is also likely that this cis- acting element will be important, if not in the promotion of initiation then in the modulation of transcription. The 5'- flanking sequence upstream of site G contains four additional regions clearly protected by nuclear proteins. One of these regions, labeled K (Fig. 8A) , is homologous to a sequence similarly located in the rat 5"flanking sequence. Because these regions are not required for efficient expression in hepatoma cell lines or in in vitro transcription assays, they may have a more central role during development or in response to positive or negative transcriptional signals known to potentiate or attenuate transcription, for instance during the acute phase response.

We also observe light protection or protection of only one strand in four additional regions upstream of -160. In two cases, regions that contain the CACCC box, which binds a ubiquitous nuclear protein and may also be important in imparting hormone inducibility upon promoters (45), are protected on one strand. These are located from -172 to -193 and from -550 to -558 (Figs. 8, A and B, and 9). In both cases, the strand containing GGGTG is less clearly protected than the strand containing the CACCC sequence. Finally, the region from -289 to -276 may be protected on one strand. The lack of strong DNase I-sensitive sites and the small region of potential binding on the other strand make it more difficult to say unequivocally that there is protection. How- ever, this region (5"CACTGAAATGCAA) is homologous to the CCAAT box region at -80 (5'-CAATGAAATGcgA, Figs. 8B and 9). These homologies to known functional cis-acting

9178 Tissue Specificity of Mouse Albumin Promoter A

HOUSE CAGACCCGTTTCAC

RAT CAGttCCaTTTgAt I l l IIIIIII I I

B -460

tg;gacactgtgigctttatg&GATtCTMhTAAAAAGATGAGaMGA?tTGMAG .-

-400

CCiUTaat6AG;CcCaCCCCTcCCTCAtACa~attCTTCAtiaCCTgTACAiAaMgCA

-340 -308 (132%)

TAiTTGGGAtGMCMCCTaTGCAAttcagttCTUGC~AAATGTGgCaTGCTTCCa

-280 - * - TGcCMggcccacACTGMATGCtCAAAT6GGAGACAaAGAGATTMgClCTTATGTAAA - """""""_

-220 -206 (196%) .-""--"- """""

ATllGcTGTTTTACATAACTTTMTGAATgGACAAAGtCTTgtgcaTGGGGGTGGGGGtG "_ -160 (79%)

"""""""

gggttagaggggaacagctccAGATGGC~C~TACGCMGG~TTTAGTc~CMCTT - - - - - - -1 i2 (43%) - 100

TTiGGCMaGATiGTATGATrriGTMTGGGGiAGGMCCMTGAAATGcgAiGTaAGTa -

-60 (12%) -40 * TGGTTMTGATCiACAGTTATTiGTTAaAGMiTATATTAGAiCGAGTcTtT~TGCACAC

- 1 +20

AGAtCACCTTTCCTaTCMCCCCACTagcCTcTGGCAaMTGMGTGGGTMCCTTTCTC *

FIG. 9. Nuclear protein-binding sites of the mouse albumin promoter. Comparison of the promoter region sequences of albumin genes from the mouse and rat reveals several conserved segments. Sequence homologies are indicated by capital letters. The vertical lines in A denote homology of purine-purine or pyrimidine-pyrimidine bases. ( A ) comparison of mouse protein-binding site K (-585 to -572) with the analogous region in the rat albumin promoter. The binding site exhibits a strong homology between mouse and rat albumin promoters. Since there is a breakdown in homology in the regions 5' and 3' to binding site K, it appears that this site has been conserved and may play in important role in the structure and/or function of the albumin gene. ( B ) sequences of interest within nuclear protein-binding regions between -160 and -40 (see Refs. 15-19) and regions protected between -160 and -308 are underlined. Nuclear factor-binding sites are: albumin proximal factor (APF), -62 to -48 (see Ref. 18); albumin CCAAT factor/nuclear factor (ACF/NFY), -86 to -74 (see Ref. 19); CCAAT-binding protein/enhancer-binding protein (C/EBP), -103 to -95 (see Ref. 39); nuclear factor l/pseu- donuclear factor 1 (NFl/pseudo-NFl), -122 to -110 (see Ref. 16). An 8-bp reiterated sequence is indicated by brackets above the sequences at -280 and -80.

elements suggest that these regions may also be involved in the modulation of promoter function.

The sequences between -1612 and -3000 decrease albumin promoter-specific initiation by approximately 50%. This region of the 5'-flanking sequence contains one copy of a B1 repeat, which is a short interspersed repetitive sequence found in a transcriptional orientation opposite the albumin gene promoter (data not shown). A long interspersed repetitive sequence element located upstream of the rat insulin 1 gene reduces transcription by the insulin promoter/SV40 enhancer construction in transient expression assays (46). The B1 repeat present in the -3000 Albcat-Mo-MuLV construction may act similarly. However, this is inconsistent with data from transgenic mice studies where introduction of vectors containing an increased amount of the 5'-flanking DNA of

the mouse albumin gene increases the level of expression of the introduced gene (10). Interestingly, preliminary studies indicate that the -3000 Albcat-Mo-MuLV construction, when tested in primary mouse hepatocytes, does not exhibit a decrease in activity but rather increases chloramphenicol acetyltransferase activity relative to the -1612 Albcat-Mo- MuLV construction (data not shown). Therefore, this decrease in promoter activity in the hepatoma cell line may be a characteristic of transformed cells.

The lack of expression of the -60 bp promoter construction in the fibroblast cell line may be due to the absence of APF in this cell type. Alternatively, the factor that binds to the region adjacent to the TATA box may not be present in fibroblasts, or its interaction with the promoter may require APF. Since additional upstream sequences starting at -122 bp generate low but significant levels of transcription in the fibroblasts, differences in factor binding to the proximal promoter region do not appear to repress promoter initiation irreversibly. I t seems likely that the initiation complexes generated in either cell type probably differ with respect to their complement of transcription factors which in turn dic- tate the levels of transcriptional activity. The simplest hy- pothesis is that the fibroblasts lack necessary positive factors required for high levels of albumin promoter initiation.

Usually, the enhancer elements of liver-specific genes contribute to tissue-specific expression of their respective promoters. Low levels of promoter activity in the fibroblast may be stimulated by the Mo-MuLV enhancer which functions in both cell types. Previous studies to define regulatory regions of the mouse a-fetoprotein (AFP) gene indicate that transient expression vectors containing both promoter and enhancer of the gene were more effectively repressed in fibroblasts than AFP promoter/Mo-MuLV enhancer or AFP enhancerlSV40 promoter constructions (8). Therefore, the homologous promoter/enhancer combination may be required for the most effective and most tightly coupled tissue-specific regulation. We propose that this may be due to cooperative activity between these cis-acting elements. We are currently determining the stringency of expression of AFP and albumin gene enhancer/promoter constructions in albumin expressing and nonexpressing cell types.

Acknowledgments-We are grateful to Drs. Dong-er Zhang and Peter Hoyt for suggestions concerning the preparation of nuclear proteins and DNase I protection assays. We would also like to thank Dr. Steven G. Widen for his valuable discussions during the initial stages of this work and Dr. Jeffrey P. Rabek for thoughtful discussions and for his critical reading of this manuscript.

REFERENCES 1.

2. 3.

4.

5.

6.

7.

8.

9.

10.

11.

Edlund, T., Walker, M. D., Barr, P. J., and Rutter, W. J. (1985)

Ohlsson, H., and Edlund, T. (1986) Cell 45,35-44 De Simone, V., Ciliberto, G., Hardon, E., Paonessa, G., Palla, F.,

Lundberg, L., and Cortese, R. (1987) EMBO J. 6, 2759-2766 Grayson, D. R., Costa, R. H., Xanthopoulos, K. G., and Darnell,

J. E. Jr. (1988) Mol. Cell. Bwl. 8, 1055-1066 Wu, L., Morley, B. J., and Campbell, R. D. (1987) Cell 48,331-

Science 230,912-916

342

Cell 33.717-728 Gillies, S. D., Morrison, S. L., Oi, V. T., and Tonegawa, S. (1983)

Godbout,.R., Ingram, R., and Tilghman, S. M. (1986) Mol. Cell.

Widen, S. G., and Papaconstantinou, J. (1986) Proc. Nutl. A d .

Costa, R. H., Lai, E., and Darnell, J. E., Jr. (1986) Mol. Cell. Biol.

Pinkert, C. A,, Ornitz, D. M., Brinster, R. L., and Palmiter, R.

Heard, J., Herbomel, P., Ott, M., Mottura-Rollier, A., Weiss, M.,

Biol. 6,477-487

Sei. U. S. A . 83,8196-8200

6,4697-4708

D. (1987) Genes Deuel. 1, 268-276


12. Muglia, L., and Rothman-Denes, L. B. (1986) Proc. Natl. Acad. 29. Miller, J. F. (1972) Experiments in Molecular Genetics, Cold

13. Babiss, L. E., Friedman, J. M., and Darnell, J. E., Jr. (1986) 30. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,

14. Gorski, K., Carneiro, M., and Schibler, U. (1986) Cell 47, 767- 31. Galas, D. J., and Schmitz, A. (1978) Nuckic Acids Res. 5, 3157-

15. Babiss, L. E., Herbst, R. S., Bennett, A. L., and Darnell, J. E., 32. CoUfiOiS, G., Morgan, J. G.9 Campbell, L. A.2 Fourel, G., and

16. Lichtsteiner, S., Wuarin, J., and Schibler, U. (1987) Cell 51,963- 33. Shaul, y., and Ben-LeV, R. (1987) E M B o J . 6 9 1913-1920

17. Cereghini, S., Raymondjean, M., Carranca, A. G., Herbomel, P., Biol. 8,1169-1178

18. Cereghini, S., Blumenfeld, M., and Yaniv, M. (1988) Genes Deuel.

19. Raymondjean, M., Cereghini, S., and Yaniv, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 8 5 , 757-784 37. Kugler, W., Wagner, U., and Ryffel, G. U. (1988) Nucleic Acids

20. Sargent, T. D. (1981) Ph.D. dissertation, California Institute of Technology 38. Baumhueter, S., Courtois, G., and Crabtree, G. R. (1988) EMBO

21. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Cold Spring Harbor Laboratory, 39. Costa, R. H., Grayson, D. R., Xanthopoulos, K. G., and Darnell, Cold Spring Harbor, NY

22. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic 40. Courtois, G., Baumhueter, S., and Crabtree, G. R. (1988) Proc. Methods in Molecular Biology, Elsevier Science Publishing Co., New York 41. Horikoshi, M., Carey, M. F., Kakidani, H., and Roeder, R. G.

23. Sanger, R., Nicklen, S., and Coulson, A. (1977) Proc. Natl. A c d . 42. ~ i ~ , y., carey, M. F,, ptashne, M., and G ~ ~ ~ ~ , M. R. (1988) cell Sci. U. S. A. 74,5463-5467

24. German, c. M., Moffat, L. F., and Howard, B. H. (1982) Mol. 43. Sawadogo, M., and Roeder, R. G. (1985) Cell 43, 165-175 Cell. Biol. 2, 1044-1051 44. Urano, Y., Watanabe, K., Sakai, M., and Tamaoki, T. (1986) J.

25. Hall, C. V., Jacob, P. E., Ringold, G. M., and Lee, F. (1983) J. Biol. Chem. 261,3244-3251 Mol. Biol. 2, 101-109 45. Schule, R., Muller, M., Otsuka-Murakami, H., and Renkawitz,

26. Szpirer, C., and Szpirer, J . (1975) Differentiation 4, 85-91 R. (1988) Nature 332.87-90 27. Graham, F. L., and Van Der Eb, A. J. (1973) Virology 52, 456- 46. Laimins, L., Hulmgren-Konig, M., and Khoury, G. (1986) Proc.

and Yaniv, M. (1987) Mol. Cell. Biol. 7 , 2425-2434 28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254

Sci. U. S. A. 83, 7653-7657 Spring Harbor Laboratory, Cold Spring Harbor, NY

Biologist 6, 3798-3806 W. J. (1979) Biochemistry 18,5294-5299

776 3170

Jr. (1987) Genes Deuel. 1, 256-267 Crabtree, G. R. (1987) Science 328,688-692

973 34. Godbout, R., Ingram, R. S., and Tilghman, S. M. (1988) Mol. Cell.

2,957-974 36. Hardon, E. M., Frain, M., Paonessa, G., and Cortese, R. (1988)

and Yaniv, M. (1987) Cell 50, 627-638 35. Berk, A. J., and Sharp, P. A. (1978) Proc. Natl. Acud. Sci. U. S. A. 75, 1274-1278

EMBO J. 7,1711-1719

Res. 16,3165-3174

J. 7,2485-2493

J. E. Jr. (1988) Proc. Natl. Acud. Sci. U. S. A. 85, 3840-3844

Natl. Acad. Sci. U. S. A. 8 5 , 7937-7941

(1988) Cell 54,665-669

54,659-664

467 Natl. Acud. Sci. U. S. A. 83, 3151-3155

cell-specific expression of mouse albumin promoter · 9172 tissue specificity of mouse albumin...

Documents