determination of tissue specificity of the enhancer by

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1904 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 2, of January 14, pp. 1323-1331,1994 Printed in U.S.A.

Determination of Tissue Specificity of the Enhancer by Combinatorial Operation of Tissue-enriched Transcription Factors BOTH HNF-4 AND CIEBPB ARE REQUIRED FOR LIVER-SPECIFIC ACTIVITY OF THE ORNITHINE TRANSCARBAMYLASE ENHANCER*

(Received for publication, August 2, 1993)

Atsushi NishiyoriSQ, Hidetoshi TashiroS, Akira KimuraS, Kiwamu AkagiST, Ken-ichi Yamamurav, Masataka MoriS, and Masaki TakiguchiSII From the $Department of Molecular Genetics and lllnstitute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan

The enhancer of the rat ornithine transcarbamylase gene is located 11 kilobases upstream from the transcription start site and has been shown to be hepatoma cell-specific. Using transgenic mice, we showed that this enhancer is capable of activating transcription in a liver-specific manner, inverting the tissue specificity of the homologous promoter that is by itself more active in the small intestine than in the liver. Transient transfection analysis with cultured hepatoma cells indicated that the enhancer activity resides in the -110-base pair region containing four protein-binding sites, two for hepatocyte nuclear factor-4 (HNF-4) and two for CCAAT/enhancer binding protein (C/EBP), both of which are liver-selective transcription factors. Con- catemerization of a region containing one HNF-4 and one C/EBP site led to reconstitution of the hepatoma cell-specific enhancer, and intactness of these two sites was strictly required for the enhancer activity. Fur- thermore, cotransfection experiments showed that both HNF-4 and C/EBPB are necessary, and neither alone sufficient, for activation of the reconstituted enhancer in nonhepatic cells. Requirement of combinatorial operation of at least two liver-enriched transcription factors for transcriptional activation successfully explains why these liver-selective but not strictly liver-specific factors can confer more restricted liver specificity on transcription of their target genes.

Studies on the transcriptional regulation of tissue- or cell- specific genes have led to identification of a number of transcription factors that are enriched in restricted cells and are involved in the tissue-specific transcription of their target genes (Weintraub et al., 1991; Bodner et al., 1988; Ingraham et al., 1988, Muller et al., 1988b; Scheidereit et al., 1988; Clerc et al., 1988, Tsai et al., 1989; Evans and Felsenfeld, 1989; Friedman et al., 1989; Frain et al., 1989). While some of these

*This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan ( t o M. M., M. T., and K. Y.), and a grant from the Ministry of Health and Welfare of Japan (to M. M.). The costa of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 On leave from the Dept. of Pediatrics, Kurume University School of Medicine, Kurume 830, Japan.

I( To whom correspondence should be addressed: Dept. of Molec- ular Genetics, Kumamoto University School of Medicine, Kuhonji 4- 24-1, Kumamoto 862, Japan. Tel: 0-96-344-2111 (ext. 6772); Fax: 0- 96-364-3554.

factors were verified to be indispensable for efficient transcription of their target genes (Li et al., 1990; Pevny et al., 1991), it is not certain whether only one tissue-enriched factor in combination with general transcription factors is sufficient to conduct the tissue-specific transcription. In some cases, several lines of evidence argue against it. First, although these transcription factors are enriched in specialized tissues, they are usually not strictly tissue- or cell-specific (Simmons et al., 1990; Martin et al., 1990; Xanthopoulos et al., 1991). Second, when introduced to and forcedly expressed in cells, they activate transcription of cotransfected or endogenous target genes in some cell types but not in others (Friedman et al., 1989; Schiifer et al., 1990). Conversely, the observation that transcriptional regulatory regions such as promoters and enhancers usually bind multiple tissue-enriched protein factors suggests that tissue-specific transcription may be conducted by the cooperation of more than one tissue-enriched factor. It waits to be clarified what kind of combination of protein factors actually enables such cooperative activation of transcription.

We have investigated the regulatory mechanisms of liver- selective transcription of the gene for ornithine transcarbamylase (OTC),’ an ornithine cycle enzyme. The mammalian OTC gene is mainly expressed in the liver and to a lesser extent in the intestine (Ryall et aZ., 1986; Wraight et al., 1985). Transient transfection analysis using a hepatoma cell line HepG2 revealed that in the promoter region of the rat OTC gene there are two &activating elements (Murakami et al., 1990), both of which are recognized by at least two orphan members of the steroid receptor superfamily, hepatocyte nuclear factor-4 (HNF-4; Sladek et al. (1990)) and chicken ovalbumin upstream promoter-transcription factor (COUP- TF; Wang et al. (1989)) (Kimura et al., 1993). HNF-4, a liver- enriched factor, activates the transcription from the cotransfected OTC promoter construct, while COUP-TF, a ubiqui- tous factor, represses the transcription (Kimura et al., 1993). In transgenic mice, the OTC promoter was capable of con- ducting liver- and small intestine-specific transcription, although the expression level of the introduced gene in the liver was lower than in the small intestine (Murakami et al., 1989; Jones et al., 1990). This expression pattern of the transgene was contrary to that of the endogenous OTC gene, suggesting the presence of a remote enhancer-like element(s) that up-

’ The abbreviations used are: OTC, ornithine transcarbamylase; HNF-4, hepatocyte nuclear factor-4; C/EBP, CCAATIenhancer binding protein; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; CHO, Chinese hamster ovary; EF-la, elongation factor-la.

1323

1324 Determination of Tissue Specificity of Enhancer

regulates the expression of the OTC gene in the liver. Subse- quent search for such an element with the transient transfection system revealed the presence of a hepatoma cell-specific enhancer located 11 kilobases (kb) upstream of the gene (Murakami et aL, 1990). In this enhancer region, we detected four protein-binding sites clustered in the -110-base pair (bp) area (Murakami et aL, 1990; Kimura et al., 1993); two are recognized by HNF-4 and the other two by a factor(s) related to CCAAT/enhancer binding protein (C/EBP; Landschulz et al. (1988)). C/EBP is a transcription factor of the basic domain-leucine zipper type and is also enriched in the liver.

Here we report that the OTC enhancer augments transcription from the homologous promoter in the liver of transgenic mice, inverting the tissue specificity of this promoter. In transient transfection analysis using cultured hepatoma cells, the enhancer activity was delimited to the -110-bp region exactly containing two HNF-4 and two C/EBP sites. Both HNF-4 and C/EBP sites were strictly required for reconstitution of the enhancer activity by combinatorial catenation of the protein-binding sites. Furthermore, it was shown that, in cotransfection experiments using nonhepatic cells, both HNF-4- and CIEBPB-expressing plasmids were necessary, and neither alone sufficient, for activation of the reconstituted enhancer. Therefore, cooperation of at least two liver-enriched transcription factors, HNF-4 and CIEBPB, is prerequisite for the liver-specific activity of the OTC enhancer.

EXPERIMENTAL PROCEDURES

Transgenic Mice-The plasmid pOCGl harboring the rat OTC promoter/cDNA was described previously (Murakami et al., 1989). Into the EcoRI site located in front of the OTC promoter segment of pOCG1, the 2.7-kb EcoRI fragment spanning from -13.8 to -11.1 kb of the OTC gene and containing the enhancer region situated around -11.2 kb was inserted, and a recombinant bearing the enhancer segment in the same direction relative to the promoter was selected by restriction mapping. From the resultant plasmid named pOCGlE, the 6.7-kb SphI fragment bearing the OTC enhancer/promoter/ cDNA (the enhancer region resides in the 1.6-kb segment spanning from -12.7 to -11.1 kb) was excised and injected into fertilized eggs from (C57BL/6 X DBA/2) Fl parents. Integration of the transgene in progeny was screened by polymerase chain reaction (PCR) analysis of ear piece DNA as described (Qi et al., 1991). The primers used were 5’-GGTTATGAGCCAGATCCTAA-3’ and 5”GTAACCTGG TAACCTTGGAA-3’ corresponding to sequences in exons 7 and 8, respectively, of the OTC gene and identical in rat and mouse (Taki- guchi et al., 1987; Scherer et al., 1988). Expected sizes of PCR products for the rat cDNA recombinant transgene and the mouse endogenous gene were 197 and 285 bp, respectively. The copy number of the transgene per cell was estimated by Southern blot analysis of tail DNA on the assumption that mouse 112, which gave PCR reaction products of similar intensities for both the endogenous and introduced OTC genes, has one copy of the transgene per cell (the OTC gene is X-linked and mouse 112 was male). Founder transgenic mice were mated with C57BL/6 mice, and their offspring were examined for expression of the transgene.

RNA Blot Analysis-Total RNA was isolated from various tissues by acid guanidinium thiocyanate-phenol/chloroform extraction (Chomczynski and Sacchi, 1987). After electrophoresis in formalde- hyde-containing agarose gels, RNAs were transferred onto nylon membranes. Hybridization was performed using as probes the random primer-labeled rat OTC cDNA (the mixture of the 417-bp X h I - Hind111 fragment and the 389-bp HindIII-HindIII fragment excised from pOCG1) or rat growth hormone gene (the -700-bp HindIII-PstI fragment). Autoradiographs were quantified with Bio-Image Analyzer BAS2000 (Fuji Photo Film Co., Tokyo).

Reporter Plasmids-The basic chloramphenicol acetyltransferase (CAT) plasmid pUC/OTC/CAT was constructed as follows. The PstI- BarnHI fragment containing the OTC gene 5’ region (-847 to +53 bp), the CAT gene, and the SV40 early gene 3’ region was excised from the plasmid pOC1.3kCAT described previously (Murakami et al., 1990) and inserted into the PstIIBarnHI site of pUC18. The resulting plasmid was cut a t the SmaI site just downstream of the BamHI site, and the double-stranded oligonucleotide 5’-

AGGGCCCAGATCTCGGGAGATCT-3’ was ligated in the opposite orientation relative to the CAT transcription unit. This oligonucleotide contains non-palindromic AuaI site 5”CTCGGG-3’ which allowed the unidirectional insertion of test oligonucleotides (Fromental et al., 1988). Restriction fragments examined for enhancer activity were excised from the 232-bp enhancer region subcloned in pUC19, and inserted into the BarnHI site of pUC/OTC/CAT after attachment of the BamHI linker. Synthetic oligonucleotides bearing AuaI ends on both sides were inserted into the AuaI site. Concatemerized oligonucleotides were separated by polyacrylamide gel electrophoresis, excised from the gel, and also inserted into the AuaI site. The plasmid for lune 19 of Fig. 2C was constructed by inserting size-selected ligation products of oligonucleotides 1-11 and 111-IV, and verified by sequencing the insert. CAT plasmids bearing the SV40 early promoter were constructed as follows. The SphI-HpaI fragment containing the SV40 early promoter, the CAT gene, and the SV40 early gene 3’ region was excised from pSV2CAT (Gorman, 1985). The HpaI-SphI fragment containing the test enhancer segment and pUC18 was excised from each of the pUC/OTC/CAT derivatives appearing in Fig. 2. These two fragments were ligated, producing the enhancer- less construct pUC/SV/CAT and its derivatives containing test enhancer segments. The plasmid for lune 10 of Fig. 5B was constructed by inserting the FokI-PuuII (positions 98-272 of SV40) fragment into the BarnHI site of the pUC/SV/CAT.

Effector Plasmids-Construction of the HNF-4-expressing plasmid pEF-HNF-4 under the control of the human elongation factor-la (EF-la) promoter was described previously (Kimura et al., 1993). Plasmids expressing C/EBP family members were constructed after isolation of mouse genomic clones for these factors as follows. A phage library constructed from Balb/c mouse liver DNA in the EMBL-3 SP6/T7 vector (purchased from Clontech) was screened for C/EBP family members. The probe used was the 80-mer synthetic oligonucleotide corresponding to the basic domain well conserved between the family members (nucleotide positions 994-1073 in the sequence of Landschulz et al. (1988)). It was labeled with the Klenow enzyme and [ c Y - ~ ~ P I ~ C T P , priming from the 5’-most 17 mer against the antisense strand template. Under the washing condition of 1 X

identical with those for C/EBPa, C/EBP@/CRP2, C/EBPG/CRP3, SSC at 68 “C, four independent genes were isolated and found to be

and CRPl (Cao et al., 1991; Williams et al., 1991) by sequence determination after subcloning appropriate fragments into pUC19. From C/EBPa, -@, and -6 subclones, DNA fragments containing protein-coding regions just from nucleotide position -1 relative to the initiation methionine codon were excised for C/EBPa, the native NcoI site containing the initiation codon was utilized by partial NcoI digestion; for CBBPj3, the sequence around the initiation codon was converted to the NdeI site, replacing the NdeI (in pUClS)-SphI (position +32 relative to the initiation codon of C/EBP@) segment with the synthetic oligonucleotide corresponding to -1 to +32; for C/ EBP6, the initiation codon-containing sequence was converted to the NdeI site by site-directed mutagenesis with PCR employing primers 5’-GCCMATGAGCGCCGCGCTTTTCAG-3’ (-6 to +20, under- lined nucleotides were substituted) and 5”TGCTGTTGAA- GAGGTCGGCG-3’ (+265 to +246). The NdeI (-1)-NcoI (+200) fragment excised from the resultant PCR products was substituted for the NdeI (in pUC19)-NcoI (+200) fragment of a C/EBPC subclone. Restriction sites for 3’ ends of excised fragments were Hind111 (-+1.9 kb), EcoRI (-+1.6 kb), and SphI (-+1.1 kb) sites for C/EBPa, -8, and -6, respectively. After attachment of the BstXI adaptor containing the consensus sequence of vertebrate translation start sites (-9 to -2 relative to the initiation codon; Kozak (198711, each fragment was inserted into the BstXI sites of the mammalian expression plasmid pEF-BOS that utilizes the EF-la promoter (Mizushima and Nagata, 1990).

CAT Assay-HepG2 cells were grown in Dulbecco’s modified Ea- gle’s medium, and Chinese hamster ovary (CHO) cells in F-12 medium supplemented with 10% fetal calf serum. CAT assay was performed as described (Gorman, 1985; Murakami et al., 1990), transfecting plasmids transiently into cultured cells (Gorman, 1985; Chen and Okayama, 1987). DNA mixtures containing 10 pg of a reporter CAT plasmid, 5 pg of an internal control @-galactosidase plasmid pSAc- lacZ (Miyazaki et al., 1989), and 1 pg of an effector plasmid were applied. CAT activity was normalized for transfection efficiency to @-galactosidase activity. In most cases, the results represent the mean of at least three independent experiments. Since, in cotransfection experiments, the internal control @-galactosidase activities were affected by effector plasmids, the normalization was omitted and independent transfections were done at least four times.

Determination of Tissue Specificity of Enhancer 1325

RESULTS

The OTC Enhancer Activates the Transcription from the Homologous Promoter in a Liver-specific Manner in Trans- genic Mice-We (Murakami et al., 1989) and Jones et al. (1990) showed that the OTC promoter is capable of directing liver- and small intestine-specific transcription in transgenic mice, but the expression level of the introduced gene was higher in the small intestine than in the liver, contrary to the expression pattern of the endogenous OTC gene. Here we examined whether the OTC enhancer located 11 kb upstream of the gene can modulate the tissue-specificity of the promoter and bring about a higher level of transcription in the liver. The structure of the introduced genes is shown in Fig. lA. The OTC promoter/cDNA construct used in our previous study (Murakami et al., 1989) bears the rat OTC promoter 1.3-kb region that drives the transcription of the OTC cDNA. The OTC enhancer/promoter/cDNA construct used in the present study has the 1.6-kb enhancer segment placed in front of the promoter/cDNA construct. With this enhancer/promoter/cDNA construct, we produced eight lines of transgenic mice, five of which expressed the introduced gene.

The expression of transgenes in various organs was examined by RNA blot analysis (Fig. 1B). The endogenous mouse OTC mRNA was detected at a high level in the liver, moder- ately (about 30% of the liver level) in the small intestine, and at a lower level (about 10%) in the large intestine. As we reported (Murakami et al., 1989) and reproduced here for a representative case, the mRNA level of the promoter/cDNA transgene was higher in the small intestine than in the liver. In contrast, the mRNA level of the enhancer/promoter/cDNA transgene was higher in the liver than in the small intestine in four lines (30, 35, 36, and 112), while the level was much the same in both organs in one line (71). On average, the mRNA level was 3-fold higher in the liver than in the small intestine (Fig. IC). This ratio resembled that of the endogenous OTC mRNA levels. Thus, the OTC enhancer can bring about a higher level of transcription in the liver, inverting the tissue specificity of the homologous promoter. The maximum mRNA level of the transgenes in the liver was estimated to be only 15% (line 36) of that of the endogenous gene even in the presence of the enhancer. This low level expression may be attributable to a cause(s) such as the presence of an unidentified enhancer region(s) and/or unfitness of the transgene construction such as a lack of an appropriately placed intron (Palmiter et al., 1991).

Contribution of HNF-4- and C/EBP-binding Sites to En- hancer Activity-We previously delimited the OTC enhancer to a 232-bp region (Murakami et al., 1990) by using a transient expression system with a hepatoma cell line HepG2. This enhancer region contains four prominent footprint regions I- IV (Fig. 2 A ; Murakami et al. (1990)). Regions I and IV are recognized by a liver-enriched transcription factor HNF-4 (Kimura et al., 1993). On the other hand, regions I1 and I11 are recognized by a factor(s) related to C B B P (Murakami et al., 1990), another liver-enriched transcription factor.

To examine the contribution of these protein-binding sites to enhancer activity, the 232-bp enhancer region was further divided into portions, which were then inserted into the plasmid carrying Escherichia coli CAT gene under the control of the OTC promoter (Fig. 2B). The resulting plasmids were introduced into human hepatoma HepG2 cells, and CAT activities were measured (Fig. 2C). The 114-bp DdeI fragment exactly covering all footprint regions I-IV (lanes 6 and 7) exhibited enhancer activity comparable to that of the original 232-bp region (lanes 2 and 3 ) , whereas upstream (lanes 4 and 5) and downstream (lanes 8 and 9) regions flanking the

footprint regions showed no significant enhancer activity. Truncation of the segment I-IV by deleting either region I (lane 11) or IV (lane 10) led to loss of the enhancer activity. Further truncation (lanes 12-18) did not lead to its recovery. Catenation of oligonucleotides 1-11 and 111-IV caused recovery of the activity (lane 19). Therefore, the OTC enhancer was delimited to the -110-bp region exactly containing four protein-binding sites. We speculate that all protein-binding sites I to IV are required for the enhancer activity, although strict examination of the indispensability of regions I1 and I11 remains to be performed.

Both HNF-4- and CIEBP-binding Sites Are Required for Reconstitution of Enhancer Activity-To analyze the contribution of each protein-binding site to the enhancer activity in a more simplified manner, we concatemerized oligonucleotides containing one or two protein-binding sites, and examined the enhancer activity of the resultant multimers (Fig. 3) High enhancer activities were detected with concatemers of an oligonucleotide containing both regions I and I1 (lanes 3 6 ) , whereas concatemers of the 11-111 or 111-IV oligonucleotide showed no obvious activity (lanes 6-8). Since multimer- ization of region I or I1 alone did not lead to any enhancer activity (lanes 9-12), the enhancer activity of 1-11 concatemers appears to depend on the presence of both region I, the HNF- 4-binding site, and region 11, the C/EBP-binding site. Al- though the oligonucleotide 111-IV also contains both HNF-4- and C/EBP-binding sites, its concatemers failed to exhibit enhancer activity. This might be due to the more distant separation of I11 and IV sites than I and I1 sites (Fig. 2 A ; Murakami et al. (1990)), to a higher affinity for HNF-4 in region I than in region IV (Kimura et al., 1993), and/or to possible differences between the population of C/EBP-related proteins binding to region I1 and that binding to region I11 (Kimura et al., 1993).

The indispensability of regions I and I1 for the enhancer activity was further confirmed by examining the effects of introducing clustered point mutations into regions I and/or I1 (Fig. 4). Oligonucleotides harboring base changes in either region I or 11, or both, failed to exhibit enhancer activity even after concatemerization. Therefore, intactness of these two sites was strictly required for the enhancer activity. The loss of protein-binding activities of mutated sites and the intactness of non-mutated sites were confirmed by gel mobility retardation analysis using rat liver nuclear extracts containing HNF-4 and C/EBP family members (data not shown).

Hepatoma Cell Specificity of the Reconstituted Enhamer- Cell specificity of the enhancer activity of 1-11 concatemers was examined by introducing plasmids into nonhepatic cells (Fig. 5). In this case, the SV40 early promoter was used as a CAT transcription unit promoter (Fig. 5A) instead of the OTC promoter that is by itself cell type-selective. As we showed previously (Murakami et al., 1990) and reproduced here (Fig. 5B, lanes 9 and IO), the 232-bp OTC enhancer is active in HepG2 cells, but it is apparently inactive in CHO cells. On the other hand, the SV40 enhancer is functional in both cells. Among concatemers of protein-binding sites of the OTC enhancer region (lanes 2-8), only the 1-11 concatemer exhibited a marked enhancer activity in HepG2 cells in the context of the SV40 early promoter (lane 6), and this enhancer activity was clearly cell-specific.

Cotransfection of Both HNF-4- and C/EBPp-expressing Plasmids Is Required for Activation of the Reconstituted En- hancer in Nonhepatic Cells-Inactivity of the 1-11 concatemer enhancer in CHO cells may be attributable to a lack or low levels of HNF-4 and/or C/EBP family members in this cell line. We tested whether cotransfection of HNF-4- and/or C/


A 10 kb OTC Gene

PromoterkDNA Construct

- cDNA Construct I - & I Enhancer/Promoter/

1 kb - .. OTC GH Probe Probe

25s 18s

Enhancer/Promoter/cDNA Transgene

C 0 1 2 3 4

Relative mRNA Level

Transgene Promoter/cDNA Average erlnt. cDNA Transgene EnhanceriPromoterl Average Liver

Sm. Int.

No. 30 ( 1 ) gzrlnt. No. 35 ( 3 ) :herlnt.

No. 36 (15) ~ ~ r l n t .

N0.71 (15) zrlnt. No. 112 ( 1 ) zr,nt.

FIG. 1. Organ specificity of the OTC enhancer activity in transgenic mice. Panel A, structure of the introduced gene. The first line shows the organization of the OTC gene in the rat genome (Takiguchi et al., 1987). Exons are drawn as boxes. Brackets indicate positions of promoter and 11-kb upstream enhancer segments used for construction of the transgene. The OTC promoter/cDNA construct (the 5.1-kb EcoRI-SphI fragment from the plasmid pOCG1) was described in the previous study (Murakami et al., 1989). This construct contains the 1.3-kb promoter region ligated to the rat OTC cDNA. The 3' portion of the construct derives from the rat growth hormone (GH) gene segment ranging from the midst of exon 4 through the 3"flanking region. The OTC enhancer/promoter/cDNA construct (the 6.7-kb SphI- SphI fragment from the plasmid pOCGlE, see "Experimental Procedures") bears the enhancer-containing 1.6-kb segment placed in front of the promoter region. The small solid box indicates the -110-bp minimal enhancer region (see below). Positions of hybridization probes specific to OTC and growth hormone genes are shown by spans. Panel B, RNA blot analysis for expression of the transgene in various tissues. Total RNAs (5 pg) isolated from various tissues of a control littermate mouse, a transgenic mouse of the promoter/cDNA construct (mouse line 94 as a representative of five lines in the previous study (Murakami et al., 1989)), and transgenic lines of the enhancer/promoter/cDNA construct (lines 30,35,36, 71, and 112 in the F1 generation) were analyzed as described uner "Experimental Procedures." The positions of 28 S and 18 S rRNAs are indicated by bars on the left. A portion of the OTC cDNA was used as a probe to detect the endogenous OTC mRNA in the control mouse, and a portion of the growth hormone gene to detect the transgene mRNA. Integrity of the RNAs and equal loading were verified by comparison of 28 S and 18 S rRNA band intensities following the ethidium bromide staining after gel electrophoresis (data not shown). Panel C, quantification of the transgene expression. The mRNA levels in the liver and small intestine were measured by counting the radioactivity of hybridized bands, and represented relative to the average mRNA level of the promoter/cDNA transgene in the livers of five strains described in the previous study (Murakami et al., 1989). This average value was estimated to be about 4% of the endogenous OTC


A 1 50 100 1 50 200 282 (bp) I I I I

FIG. 2. Delimitation of the OTC enhancer. Panel A, protein-binding sites of the OTC enhancer and location of oligonucleotides used in this study. On the top, the 232-bp enhancer region delimited in the foregoing study (Murak- ami et al., 1990) and its binding proteins are represented schematically. Below, the sequence of protein-binding regions is enlarged. Boxes marked I-IV show footprint regions detected with rat liver nuclear extracts (Murakami et al., 1990). DdeI restriction sites allowed excision of almost the whole range of these footprint regions. Locations of synthetic oligonucleotides are indicated by spans. Panel E, structure of the CAT plasmid. The basic plasmid pUC/OTC/CAT bears the 5’ region from -847 (PstI site) to +53 bp (XbaI site, HindIII linker-attached) of the rat OTC gene (Takiguchi et al., 1987) as a promoter that drives the CAT gene. Restriction fragments and oligonucleotides to be tested for enhancer activity were inserted into the BamHI and AuaI sites, respectively. Panel C, delimitation of the OTC enhancer activity to the region exactly covering the four footprint sites. On the left, the ranges and orientations of the DNA inserts are shown by arrows with or without ovals representing the footprint sites. Right- ward and leftward arrows indicate, respectively, the same and opposite directions relative to the CAT transcription unit. Plasmids were transfected into HepG2 cells. After 48 h of culture, CAT activities were measured, normalized for transfection efficiencies, and represented as fold enhancement relative to the activity obtained with the enhancer- less plasmid pUC/OTC/CAT (lane 1 ).

I I 1-11-111

k I 11-1x1-IV 1-11 I

I 111-IV 11-111

1 - I

6 I I1

I I I I I11 IV

Avail \&MI Oligonudeotides Restriction Fragments

‘ y ’ y 2oo 232(bp) Fold Enhancement of CATActivity I ,

- 0 1 2 3 4 5 6 7 8 9 1 0 1 ( 4

3- 2

4- 111 11 1

6 7 8 9 10 1 1 12 13 14 15 16 17 18 19

5-

EBP family member-expressing plasmids results in activation of the reconstituted enhancer in CHO cells (Fig. 6). Plasmids expressing rat HNF-4, and those expressing mouse C/EBPa, -8, and -6 were used as effector plasmids (see “Experimental Procedures”). Expression from the basal reporter construct under the control of the SV40 early promoter and without the enhancer was not significantly activated by cotransfection of any of these effector plasmids and their combinations (lanes 1-8). When the reporter plasmid harboring the 1-11 concatemer enhancer was used, a marked activation of the reporter gene expression was caused by the cotransfection of both HNF-4- and C/EBPB-expressing plasmids ( l a n e 15). On the other hand, the cotransfection of neither effector plasmid alone (lanes 9 and 12) caused any remarkable activation above the marginal level. Since a combination of HNF-4 plus C/

EBPa ( l a n e 14) or C/EBPG ( l a n e 16) was far less effective than that of HNF-4 and C/EBPB, the effect of C/EBPB seems to be specific at least under the present experimental condi- tions. On the other hand, our preliminary results showed that expression from the reporter construct containing the OTC promoter alone was activated by the cotransfection of any of the C/EBPa-, C/EBPB-, and C/EBP6-expressing plasmids in combination with the HNF-4-expressing plasmid.’ In conclu- sion, an apparently specific combination of HNF-4 and C/ EBPB is required, and neither alone is sufficient, for activation of the reconstituted hepatoma cell-specific enhancer in the nonhepatic cell line CHO.

* H. Tashiro, A. Kimura, M. Mori, and M. Takiguchi, unpublished observations.

mRNA level in the liver by normalizing the difference of OTC and growth hormone probes experimentally (not shown), and by assuming that the endogenous mouse OTC mRNA and the rat transgene mRNA hybridize with the rat OTC cDNA probe at the same efficiency. In parentheses are the estimated copy numbers of the transgene (see “Experimental Procedures”).


DISCUSSION

OTC Enhancer and Promoter That Are Preferentially Active in the Liver and Small Intestine, Respectively-One of the characteristic features of the OTC enhancer is that it can invert the tissue specificity of transcription from the homologous promoter, as was demonstrated by the study using transgenic mice. This property of the OTC enhancer is unique compared to other enhancers previously analyzed with transgenic mice. The enhancer of the immunoglobulin heavy chain gene (Gerlinger et al., 1986) activates transcription from the homologous B cell-specific promoter, apparently in a B cell- specific manner. The enhancers of the a-fetoprotein (Hammer et al., 1987a) and elastase I (Hammer et al., 198713) genes tissue-specifically stimulate transcription from the homologous promoters whose activities by themselves are undetect- able in almost all tissues examined. On the other hand, the OTC promoter and enhancer operate as discrete “cassette” regulatory units, each exhibiting different tissue specificity. The presence of differentially tissue-selective cassette units serves as a paradigm for the mechanisms that bring about the transcription specific for several restricted tissues. Availabil- ity of several discrete cassette units in transcriptional regu-

Fold Enhancament of CAT k w i i 0 1 2 3 4 5 6 7 8 Q 1 0 1 1 1 2 1 3

1 (-1 2 I I1 111 Iv 3 4F[:: 5 x 4 6 ”@ x 4 7 8 @-@[:t

1: a [: d 13 14

FIG. 3. Enhancer activity of concatemerized protein-binding sites. Indicated copies ( X 2 to 6 ) of oligonucleotides covering one or two protein-binding sites shown by ouak were linked in head-to- tail form, and inserted into the AuaI site of pUC/OTC/CAT in opposite orientation relative to the CAT transcription unit. The resulting plasmids were transfected into HepGZ cells, and CAT activities were measured as in Fig. 2C. Lane 2, the CAT construct bearing the 232-bp OTC enhancer segment.

I I1

lation processes seems to be especially suited for cases where a gene should be differentially regulated in separate tissues.

In this context, it is noteworthy that the regulatory profile of OTC gene expression in the liver is different from that in the small intestine. OTC in the liver participates in ammonia detoxification as a member of the ornithine cycle through which ammonia is converted into urea, whereas the enzyme in the intestine is believed to be involved in the arginine- biosynthetic pathway. In the rodent development, the OTC mRNA levels in the liver increase steadily late in gestation through the neonatal period, while those in the small intestine rapidly increase and then decrease during the perinatal period (Ryall et al., 1986). The OTC enzyme levels in the adult liver and small intestine change in response to the increase of dietary protein in different directions, i.e. upward and down- ward, respectively (Wraight et al., 1985). In addition, the OTC mRNA levels in the liver are increased by glucagon injection and decreased by dexamethasone treatment, but they are not significantly affected in the small intestine by either reagent (Ryall et al., 1986). Finally, during the evolution, the expression pattern of the ornithine cycle enzyme genes including that of OTC has undergone dramatic changes especially in the liver (Takiguchi et al., 1989). It is interesting to know to what extent these differential regulations of the OTC gene in the liver and in the small intestine in developmental, environ- mental, and evolutional aspects are attributable to the differential involvement of the OTC enhancer and promoter in each transcriptional regulation event.

Judging from the average mRNA level of individual mice in each tissue for each transgene construct (Fig. IC), activation of the promoter by the enhancer in the liver and/or repression in the small intestine may cause the inversion of tissue specificity of transcription from the OTC promoter by the enhancer. However, an absolute comparison of the transgene mRNA levels in one organ from different mouse lines should be carefully done, since the transgene expression levels fluctuate broadly from one mouse line to another. While the OTC enhancer was shown to activate the homologous promoter in cultured hepatoma cells (Murakami et al., 1990), it is not known if this enhancer can repress the promoter in intestinal cell lines. It is also unclear what determines the differential activity of the enhancer between the two tissues. One of the most probable explanations is that qualitative and/ or quantitative differences between the liver and small intes-

Fdd Enhancement of CAT Actiiity 0 1 2 3 4 5 6 7 0 9

(-1

FIG. 4. Requirement of intactness of both regions I and I1 for the enhancer activity. Sequences of oligonucleotides tested for enhancer activity are shown on the left. Bores marked Z and IZ in the wild-type oligonucleotide shown on the top represent footprint areas. The armus in box I indicate the sequence elements that concord with the heptamer direct repeats proposed as the consensus sequence for the HNF-4-binding sites (Kimura et al., 1993). The span in box I1 shows the sequence element that coincides with other C/EBP sites (Murakami et al., 1990). Base substitutions indicated by lower case letters were introduced into regions I and/or 11. A single copy ( X I ) or four copies ( X 4 ) of oligonucleotides were inserted into the AuaI site of pUC/OTC/CAT in opposite orientation relative to the CAT transcription unit. The resulting plasmids were transfected into HepG2 cells, and CAT activities were measured as in Fig. 2C.


A SV40 Early

FIG. 5. Cell specificity of the enhancer activity of the 1-11 concatemer. Panel A, structure of the plasmid used. As a promoter of the CAT transcription unit, the SV40 early promoter was employed instead of the OTC promoter that is by itself cell-specific. Re- striction fragments and oligonucleotides to be tested for enhancer activity were inserted into the BanHI and AuaI sites, respectively. Panel B, CAT assay was performed using HepG2 (hatched col- umns) and CHO (open columm) cells. CAT activities are expressed as fold enhancement relative to the activity obtained with the enhancer-less construct ( l o n e 1 ) for each cell type. Lanes 2-9, the sequences and orientations of the test inserts represented by ovals and lines on the left correspond with those in Fig. 2. Lane 10, the CAT construct bearing the SV40 enhancer segment.

Reporter

B 1

2

3

4

5

6 7

8

9

10

pUC/SV/CAT

Oligonucleotides Restriction Fragments

Fold Enhancement of CAT Activity

I

-

HNF-4

HNF-4 HNF-4

Effector o 1 2 3 4 5 6 7 6 Fold Enhancement of CAT Activity

I i

( 4 HNF-4 CIEBPa CIEBPP

+ CIEBPa CIEBPG

+ CIEBPP + CIEBPG

HNF-4 t HNF-4 +

HNF-4 +

(-1 HNF-4 CIEBPu CIEBPP

. CIEBPa CIEBPG

CIEBPP . CIEBPS

FIG. 6. Activation of the reconstituted enhancer by cooperation of HNF-4 and C/EBPB in a nonhepatic cell line CHO. Reporter CAT constructs under the control of the SV40 early promoter without the enhancer (lanes 1-8) or with the 1-11 concatemer enhancer (lanes 9-16) schematically represented on the left were transfected into CHO cells with effector plasmids expressing HNF-4 or the C/EBP family members indicated. The parental plasmid of the effector plasmids, namely pEF-BOS, employs the EF-10 promoter to drive the expression of inserted transcription factor genes. CAT activities are expressed as fold enhancement relative to the activity obtained with the reporter plasmid harboring no enhancer in the presence of the pEF-BOS vector ( l a n e 1 ).

tine are present in OTC enhancer-recognizing proteins in- Requirement of Cooperation of ut Least Two Liver-selective cluding HNF-4- and C/EBP-related factors, which were Transcription Factors for the Liver-specific Enhancer Actiu- shown to play crucial roles in the mediation of OTC enhancer ity-It is generally accepted that promoters and enhancers of activity in the present study. eukaryotic genes are composed of multiple cis elements that


interact with distinct protein factors. Nevertheless, the reason why such multiple factors are required for the enhancer activity has not sufficiently been clarified. In several cases, it was suggested that enhancers have redundancy in a repertory of binding factors (Lenardo et al., 1987), and that multimeri- zation of a single protein-binding sequence can result in the construction of a strong enhancer (Miiller et al., 1988a). In the present study, however, both HNF-4- and C/EBP-binding sites were indispensable for the OTC enhancer activity. This is still speculative in the case of the native OTC enhancer, but it is evident in the case of the enhancer reconstituted by the concatemerization of regions I and 11. In addition, cotransfection of both HNF-4- and C/EBPB-expressing plasmids was required for the reconstituted enhancer to exhibit its activity in a nonhepatic cell line.

Both HNF-4 and C/EBPB are liver-enriched transcription factors. HNF-4 has been described as a factor crucial for the liver-selective transcription of a number of genes including those for transthyretin (Costa et al., 1989), apolipoprotein CIII (Mietus-Snyder et al., 1992), and another liver-selective transcription factor HNF-1 (Tian and Schibler, 1991; Kuo et al., 1992). Molecular cloning revealed that HNF-4 is an orphan member of the steroid receptor superfamily (Sladek et al., 1990). HNF-4 binds to DNA sequences comprised of direct repeats of the heptamer motif TGA(C/A)C(T/C)(T/C) (Mie- tus-Snyder et al., 1992; Kimura et al., 1993) as a homodimer (Sladek et al., 1990; Mietus-Snyder et al., 1992). The HNF-4 mRNA is present in the liver, intestine, and kidney in a tissue-specific manner (Sladek et al., 1990). C/EBPB is a member of a basic domain-leucine zipper protein family, the C/EBP family. The prototypic member of this family, C/ EBPa (Landschulz et al., 1988), is also a liver-enriched transcription factor (Birkenmeier et al., 1989). C/ESPB (Cao et al., 1991) has also been named LAP (Descombes et al., 1990), NF-IL6 (Akira et al., 1990), IL-6DBP (Poli et aZ., 1990), AGP/ EBP (Chang et al., 1990), and CRP2 (Williams et al., 1991). C/EBPB seems to be involved in many aspects of transcriptional regulation including the acute-phase gene induction in the liver (Akira et al., 1990; Poli et al., 1990; Chang et al., 1990), interleukin-1-induced interleukin-6 gene expression in various cells (Akira et al., 1990), adipocyte differentiation (Cao et al., 1991), and transcriptional stimulation by CAMP (Metz and Ziff, 1991) and calcium (Wegner et al., 1992) in neural cells, in addition to liver-selective transcription (Des- combes et al., 1990; van Ooij et al., 1992). The C/EBPB/LAP mRNA is also enriched in other organs such as the lung, but its protein concentration in the nuclei reaches a much higher level in the liver than in other tissues, apparently under post- transcriptional regulation (Descombes et al., 1990). C/EBP- binding sites in regulatory regions of a number of liver- selective genes yield multiple DNA-protein complexes in gel shift analyses using liver nuclear extracts (Costa et al., 1988; Descombes et aZ., 1990; Howell et al., 1989; Takiguchi and Mori, 1991), possibly reflecting the presence of multiple forms of homodimers and heterodimers of C/EBPa, -8, and other related factors in the liver.

The coexistence of discrete HNF-4- and C/EBP-binding sites has also been reported in regulatory regions of other liver-selective genes including mouse transthyretin and al- antitrypsin genes (Xanthopoulos et al., 1991), and the chicken very low density apolipoprotein I1 gene (Beekman et al., 1991). Potentially, through these regulatory regions, the cooperative activation of transcription in a manner similar to that of the OTC enhancer can occur. The molecular mechanism under- lying the cooperation between HNF-4 and C/EBP was not investigated in the present study. Cooperative activation of

transcription by a steroid receptor superfamily member and a basic domain-leucine zipper protein has been reported for different combinations including that of the estrogen receptor and Fos/Jun (Gaub et al., 1990). In this case, the coactivation appears to be caused through direct binding of only Fos/Jun to a relatively short seven-bp sequence; the DNA-binding of the estrogen receptor was not required for the activation. Therefore, the molecular mechanism operating in the estrogen receptor-Fos/Jun coactivation seems to differ from that operating in the HNF-4-CIEBPB cooperation in the present study.

Stimulation of transcription by the combined interaction of two liver-enriched transcription factors, HNF-4 and C/ EBPB, successfully explains why these liver-selective transcriptional activators, in spite of being not strictly liver- specific, can confer more restricted liver-specificity on transcription of the OTC gene. For example, the absence of OTC gene expression in the kidney, where the HNF-4 mRNA and HNF-4-related DNA-binding activities are at a level comparable to those in the liver (Sladek et al., 1990; Xanthopoulos et al., 1991), can be attributed to a lack of C/EBPB/CRP2 protein at a detectable level in the kidney (Williams et al., 1991). Our preliminary results show that cotransfection of both HNF-4- and C/EBP family member-expressing plasmids is also required for activation of transcription from the OTC promoter by itself in nonhepatic cells.' A similar mechanism in which cooperation of several protein factors is prerequisite for transcriptional activation may be applicable to other promoter and enhancer systems, and to other combinations of transcription factors. This mechanism potentially accounts for the tissue-specific transcription of many other genes under the control of a number of transcription factors that exhibit less strict tissue specificities.

Acknowledgments-We thank S. Hata, T. Tsukamoto, and T. Osumi for the HNF-4-expressing plasmid; H. Nomiyama for valuable suggestions on the construction of plasmids, A. Rosen for comments on the manuscript; M. Yoshino, F. Yamashita, and H. Kata for encouragement throughout this work; and F. Tashiro, J. Miyazaki, T. Murakami, and colleagues for suggestions, discussions, and assist- ance in transgenic mouse experiments.

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