- Email: [email protected]
An Upstream Repressor Element That Contributes to Hepatocyte-Specific Expression of the Rat Serum Amyloid A1 Gene
Biochemical and Biophysical Research Communications 264, 395– 403 (1999) Article ID bbrc.1999.1527, available online at http://www.idealibrary.com on
An Upstream Repressor Element That Contributes to Hepatocyte-Specific Expression of the Rat Serum Amyloid A1 Gene Lei Li and Warren S.-L. Liao 1 Department of Biochemistry and Molecular Biology, Box 117, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030
Received September 14, 1999
Serum amyloid A (SAA) is a major acute-phase protein whose expression can be dramatically induced in response to tissue damage, infection, and inflammation. Its expression is highly tissue-specific, restricted almost exclusively to liver hepatocytes. We have shown that a 320-bp fragment of the rat SAA1 promoter could confer liver-cell-specific expression on a reporter gene when transfected into cultured cells. Here we report the identification of a 29-bp regulatory element that possesses inhibitory activities on SAA1 promoter in HeLa cells but has no such effects in liver cells. Moreover, this regulatory element has properties of a transcriptional repressor; in that, it can function with a heterologous promoter and is independent of orientation and distance from the transcription initiation site. Protein binding studies showed that this regulatory element can form specific protein–DNA complexes with nuclear proteins from several nonliver cell lines (HeLa, 10T 1/2 , and C2) and placenta. However, the same DNA–protein complex was not detected in extracts from liver or liver-derived cell lines (HepG2 and Hep3B). Taken together, our results demonstrate the presence of a DNA-binding protein, termed tissuespecific repressor, found only in nonhepatocytes which may function to repress SAA1 gene expression by interacting with a repressor element. Thus, liverspecific expression of the SAA1 gene is apparently regulated by both positive and negative regulatory elements and their interacting transcription factors to ensure that it is expressed only in suitable cell types. © 1999 Academic Press
In a multicellular organism, the expression of cellular genes is highly regulated in specific spatial and temporal patterns. A major control of cell type-specific gene expres1
To whom correspondence should be addressed. Fax: (713) 7919478. E-mail: [email protected].
sion is at the level of gene transcription. Studies of cell type-specific gene regulation have led to the identification of both positive and negative regulatory elements. Among the positive regulatory sequences, some interact with widely distributed cellular transcription factors while others interact with cell-specific factors (1–5). Therefore, a gene that employs multiple cellular transcription factor-binding sites in the promoter region could display distinct transcription rate, inducibility, and tissue specificity by the combinatorial use of these regulatory elements and their binding factors. Additionally, the gene expression patterns can also be controlled by negative regulatory elements and their DNA-binding transcription repressors. For example, in the mouse albumin gene far-upstream enhancer, a negative regulatory element has been found that is capable of negating the effects of both homologous and heterologous enhancers (6, 7). Thus, this repressor element modulates the expression levels of albumin by suppressing its promoter activities in nonhepatic cells (7). Negative regulatory elements have also been shown to control the developmental appearance or disappearance of gene expression in a stagespecific manner (8–12). Vacher et al. (13, 14) have identified a dominant negative element that acts as a repressor in a position-dependent manner to extinguish mouse a-fetoprotein gene transcription in adult liver. Similarly, a negative regulatory element in MHC class I promoter was found to be active only in undifferentiated F9 embryonal cells (8). Moreover, tissue-specific genes with their distinct tissue specificity, such as liver-specific retinol binding protein (15, 16), a 1-antitrypsin (17), glutathione transferase P (18), acetylcholinesterase (19), transthyretin (20), and B cell-specific immunoglobin (21) genes, all contain either silencer that allows only the expression of the genes in the correct cell type or repressors that contribute to the tissue specificity of gene transcription. In some cases, specific DNA sequences and interaction with their cognate DNA-binding proteins have been delineated (10, 22, 23).
395
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Serum amyloid A (SAA), one of the major acutephase proteins, is synthesized and secreted primarily by the liver (24 –27). During normal conditions, the plasma concentration of SAA is very low or undetectable. However, in response to tissue damage, infection, or inflammation, its plasma concentration rises several hundredfold as a consequence of increased production of SAA mRNA at the transcriptional level (28). In cultures of primary hepatocytes and hepatoma cells, SAA gene expression can be induced by inflammatory cytokines, interleukin-1 and tumor necrosis factor, and by the phorbol ester, 12-O-tetradecanoyl-phorbol-13acetate (TPA) (25, 29, 30). In mice, the SAA gene family consists of three acutephase genes (SAA1, 2, and 3), one constitutively expressed gene (SAA5) and a pseudogene (31–33). Among the three acute-phase SAA genes, SAA1 and 2, are highly homologous to each other having over 95% sequence homology over a region that includes four exons, introns, and 59 and 39-flanking DNA sequences. The SAA3 DNA sequence, however, shows significant homologies only to the protein coding exons of SAA1 and 2; otherwise, this sequence has diverge considerably (33). Consistent with their DNA sequence divergence, the expression patterns of these three acutephase genes also differed. At the peak of inflammatory response, all three SAA genes are expressed in the liver with each gene accounting for approximately onethird of total SAA mRNA (28). However, while SAA1 and 2 genes are expressed almost exclusively in the liver, expression of SAA3 gene is less restricted where high levels of expression are also found in the macrophages, and at lower levels in the kidney and intestine (34 –36). Thus, the expression of SAA1 and 2 are tightly regulated in a liver cell-specific manner and provide good experimental models to study the control of tissue-specific gene expression. Our previous studies on the molecular mechanisms of rat SAA1 gene regulation have identified several regulatory regions that are essential for its dramatic response to inflammatory cytokines (30, 37, 38). Transient transfection analysis of the promoter demonstrated that a 66-bp fragment (positions 2138 to 273) could confer cytokine responsiveness onto a heterologous promoter in liver and nonliver cells (37). This fragment thus functions as a non-cell-specific cytokine response unit (CRU). Within the CRU resided binding sites for two positive transcription factors C/EBP and NFkB that function synergistically to transactivate the target promoter (37). A third transcription factor YY1 also binds to the CRU and competes with NFkB for overlapping binding sites. Thus, in the rat SAA1 promoter, YY1 functions as a transcriptional repressor, not only contribute to SAA1’s low basal expression, but also perhaps play a role in the transiency of its expression in response to acute inflammation (38).
In this report, we provide evidence for a second negative regulatory element in the rat SAA1 promoter. This regulatory element, however, differs from YY1 in that, while YY1 participates in modulating the cytokine responsiveness of the SAA1 promoter, this negative regulatory element contributes to its liver-specific expression. We show that a 29-bp regulatory element located upstream of the CRU could repress the expression of SAA1/CAT reporter gene in HeLa cells, but has no inhibitory effect in liver cells. Furthermore, it can function with a heterologous promoter and is independent of orientation and distance from the transcription start site. Protein binding studies revealed DNA sequence-specific binding activities in several nonliverderived cell lines; however, this binding activity is completely absent in liver or liver-derived cell lines. Thus, this DNA-binding protein, termed tissue-specific repressor (TSR), may function to repress SAA1 gene expression in nonliver cells, contributing to SAA1’s liverspecific expression pattern. MATERIALS AND METHODS Plasmid construction and oligonucleotides. Two restriction enzyme fragments KpnI to AvaII (2289/273) and HinfI to AvaII (2138/ 273) from the rat SAA1 gene promoter were isolated from pSAA1/ CAT(2304) (30) and inserted as blunt-ended fragments into the SmaI site of pBLCAT81 to generate pTK/SAA1(2289/273) and pTK/SAA1(2138/273), respectively. A PCR fragment from bp 2226 to 273 was amplified and inserted similarly to yield pTK/ SAA1(2226/273) construct. The double-stranded wild type TSR-binding site oligonucleotides (59-CTTTCACTCTATACCTCAGGCAGCTAAGG-39) corresponding to bp 2285 to 2257 and the mutated TSR-binding site oligonucleotides (59-CTTTCACTCTATAggaCAGGCAGCTAAGG-39) were synthesized and used as radiolabeled probes or as competitors in gel shift assays. Plasmids p-138/273(TSR)1x and p-138/273(TSR)3x, containing one and three copies of wild type TSR-binding site oligonucleotides, respectively, were constructed by inserting annealed oligonucleotides into the HindIII site of pTK/SAA1(2138/273). p-138/273(mTSR)1x was constructed by inserting mutated TSRbinding site oligonucleotides into pTK/SAA1(2138/273). Cell culture and transient transfection assay. Human hepatoma HepG2 cells and HeLa cells were cultured as monolayers in modified Eagle’s medium and Waymouth MAB (3:1, vol/vol) plus 10% fetal calf serum (39) and passaged at confluence by trypsinization approximately once a week. DNA transfection was performed by the Polybrene procedure (40). Approximately 18 h after transfection, cells were stimulated with basal medium, 50% conditioned medium (CM), or 100 ng/ml TPA (Sigma) (30, 41). Cell extracts were assayed for protein content by the Bradford assay (42), and chloramphenicol acetyltransferase (CAT) activity was determined by modifications (43) of procedures described by Gorman et al. (44). To determine CAT activities, [ 14C]chloramphenicol spots corresponding to acetylated and nonacetylated forms were quantitated using a PhosphorImager (Molecular Dynamic). CM was prepared from mixed human lymphocyte cultures stimulated with 0.75% phytohemagglutinin as described previously (30, 41) and was used as a mixture with an equal volume of basal medium. Nuclear extracts. Nuclear extracts from cultured cells were prepared as described (45). Briefly, isolated nuclei were resuspended at a concentration of 10 9 nuclei in 6 ml of final nuclear resuspension
396
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
buffer (9 vol of nuclear resuspension buffer and 1 vol of saturated ammonium sulfate; nuclear resuspension buffer consists of 20 mM N-2-hydroxyethylpiperazine-N9-2ethane-sulfonic acid (Hepes), pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 0.2 mM EGTA, 2 mM dithiothreitol (DTT), and 25% glycerol. The resulting suspension was mixed by gentle rocking at 4°C for 30 min. The chromatin was subsequently removed by sedimentation at 150,000g for 90 min at 2°C. The supernatant was collected and the protein precipitated by the addition of powdered ammonium sulfate (0.33 g/ml), and collected by centrifugation at 85,000g in SW28 rotor for 20 min at 4°C. Protein pellets were dissolved in 1 ml of nuclear dialysis buffer (20 mM Hepes, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, and 2 mM DTT) per 10 9 nuclei and dialyzed for 4 h at 4°C against 300 ml of nuclear dialysis buffer, with one change of buffer. The dialyzed nuclear extract was then clarified by centrifugation at 10,000g for 10 min, and frozen in small aliquots in dry ice and stored at 280°C. Nuclear extracts from human placenta were prepared by a modification of the method described by Dignam et al. (46). Electrophoretic mobility shift assay (EMSA). Reactions for EMSA were performed in a 20-ml reaction mixture consisting of 12 mM Hepes (pH 7.9), 60 mM KCl, 1.2 mM DTT, 0.12 mM EDTA, 12% glycerol, 4 mg poly(dI:dC), nuclear protein extracts, and 2 3 10 4 cpm (;1 ng) of 32P-labeled DNA fragment. The reaction mixture was preincubated for 5 min at room temperature before the radioactive probe was added. The reaction mixture was then incubated for 20 min at room temperature. Samples were loaded onto a low-ionic strength, 6% acrylamide gel (19:1 crosslinking ratio) containing 0.253 TBE (13 TBE: 89 mM Tris, pH 7.8, 89 mM boric acid, 1 mM EDTA) and subjected to electrophoresis at 200 V for 2 h. The gel was then dried and autoradiographed.
RESULTS Rat SAA1 promoter contains a regulatory element that inhibits its cytokine responsiveness in HeLa cells. We have previously reported that the rat SAA1 promoter is highly responsive to cytokine induction when transiently transfected into hepatoma HepG2 and Hep3B cells (30, 37, 38). This dramatic induction by cytokines is mediated primarily by the activation and cooperative interactions of transcription factors NFkB and C/EBP that bind at adjacent sites within a 66-bp CRU in the proximal region of the SAA1 promoter (37, 38). Interestingly, while the SAA1 promoter can direct accurate transcription in liver or liver-derived cells in response to stimulation, it is totally nonresponsive in HeLa cells (37). To examine the cis regulatory elements responsible for this differential expression in HepG2 and HeLa cells, we inserted the 217-bp KpnI–AvaII fragment from the SAA1 promoter into the unique SmaI site of plasmid pBLCAT81 to generate the CAT reporter construct pTK/SAA1(2289/273). This reporter gene construct which contains the CRU showed pronounced response upon stimulation by CM or TPA when transfected into HepG2 cells (Fig. 1). However, there was no increase in CAT activity when the same construct was introduced into HeLa cells. Thus, despite the presence of transcription factors NFkB and C/EBP in HeLa cells, these two transcription factors are nevertheless unable to transactivate the pTK/ SAA1(2289/273) reporter construct. This result indicates that the regulatory region between bp 2289 and
FIG. 1. Identification of a cell-specific regulatory element in the rat SAA1 promoter. (Top) Schematic diagrams of the proximal region of the rat SAA1 promoter and the 59 deletion constructs used for transfection. Deletion constructs containing various promoter regions of SAA1 gene were generated as described under Materials and Methods. The SAA1 promoter fragments were inserted into the pBLCAT81 vector with the minimum thymidine kinase (TK) promoter. The arrow denotes the transcription initiation site. (Middle and bottom) CAT assay analysis. Cultured HepG2 and HeLa cells were transfected with 10 mg of pTK/ SAA1(2289/273), pTK/SAA1(2226/273), and pTK/SAA1(2138/273) DNAs. As a negative control, pBLCAT81 vector (TK) was also included. Transfected cells were not stimulated (2), or stimulated with 50% CM (CM) or 100 ng/ml TPA (TPA). After treatment, cell extracts were prepared and the protein content and the CAT activities determined. Consistent results were obtained from three independent experiments, one of which is shown.
273 in the rat SAA1 promoter contains sequences that contributes to its hepatocyte-specific expression. To further delineate sequences within this regulatory region that inhibits SAA1 promoter function in
397
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HeLa cells, we examined two 59 deletion mutants, pTK/ SAA1(2226/273) and pTK/SAA1(2138/273), with deletions of 63 and 151 bp, respectively, from the KpnI site (Fig. 1). These two constructs, along with the parental plasmid pTK/SAA1(2289/273) and the promoterless pBLCAT81 vector, were tested for CM- and TPA-induced expression in HepG2 and HeLa cells. As shown in Fig. 1, when compared with the parental pTK/SAA1(2289/273) construct, both deletion mutants showed comparable levels of CAT expression in HepG2 cells in response to induction by CM and TPA. Interestingly, these two deletion constructs also showed dramatic response to CM- and TPA-mediated induction in HeLa cells. This is in sharp contrast to the parental pTK/SAA1(2289/273) construct in which there was no response to cytokine induction. As expected, the promoterless vector construct had no CAT activity with or without stimulation. Taken together, these results indicate that while the regulatory region from bp 2138 to 273 in the SAA1 promoter is necessary and sufficient to confer cytokine responsiveness onto a heterologous thymidine kinase promoter, the region from 2289 to 2226 may be crucial for SAA1’s tissue-specific expression by restricting its promoter function in HeLa cells. Binding of a HeLa nuclear factor to the TSR element. Deletion of bp 2289 to bp 2226 in the rat SAA1 promoter resulted in dramatic increase in CM- and TPAinduced transcription of the CAT reporter gene in HeLa cells. This might be taken to represent deletion of a binding region for a transacting factor capable of suppressing the positive effects of NFkB and C/EBP. To determine whether any transcription factor could bind to this region of the SAA1 promoter, two DNA fragments, a 29-bp (2285/2257) fragment and a 31-bp fragment (2256/2226) were generated and used as probes in EMSA with nuclear extracts from HeLa and HepG2 cells (Fig. 2A). When the 31-bp 2256/2226 fragment was used as a probe, no protein-DNA complexes were formed with either HeLa or HepG2 nuclear extracts (data not shown). In contrast, an intense protein-DNA complex was detected with the 29-bp (2285/2257) fragment (Fig. 2B). Interestingly, this protein-DNA complex was only detected with HeLa nuclear extracts while the same complex was not found with HepG2 nuclear extracts. This HeLa nuclear protein that possesses the DNA-binding activity was termed the tissue-specific repressor (TSR) and the 29-bp DNA regulatory element that it interacts with was denoted the TSR element. To test whether the lack of complex formation with HepG2 nuclear extracts might be due to the presence of inhibitory components that interfere with protein binding to the probe, we mixed equal amounts of HepG2 and HeLa nuclear extracts and incubated this mixture with the DNA probe in EMSA. As shown in Fig. 2B, a
FIG. 2. Tissue-specific repressor binding activity in HeLa but not in HepG2 cells. (A) Location of tissue specific repressor element in the rat SAA1 promoter. Nucleotide sequences (bp 2285/2257) corresponding to wild type (TSR) or mutant (mTSR) TSR-binding sites are indicated. (B) End-labeled TSR element was incubated with 5 mg of nuclear extracts from HeLa and HepG2 cells. For the mixing experiment, equal amounts (2.5 mg 1 2.5 mg) of nuclear extracts from each cell line were mixed before addition of the 32P-labeled TSR probe. (C) Specific competition of TSR-binding activities. End-labeled TSR probe was incubated with HeLa nuclear extracts (2 mg) in the presence of a 25- or 100-fold molar excess of unlabeled oligonucleotides corresponding to wild-type (TSR) and mutated (mTSR) TSRbinding sequence, and the binding sequence for NFkB (NFkB). Position of specific DNA-protein complex formed is indicated by the solid arrow and the open arrow denotes the position of the free probe. (D) Heat stability of TSR-binding activity. HeLa nuclear extracts were not heated or heated at 42, 65, and 90°C for 10 min before incubated with radiolabeled probe. Position of specific complex formed is indicated by the solid arrow. The open arrow denotes the position of the free probe.
protein-DNA complex that is indistinguishable from that with HeLa nuclear extract alone was formed. This result strongly indicates that the TSR-binding activity is present only in HeLa cells and that the lack of binding in HepG2 nuclear extracts is due to the absence of TSR in HepG2 cells rather than the presence of an inhibitory factor. To examine the sequence specificity of TSR binding, wild type and mutant TSRbinding site oligonucleotides were used in competition experiments. In addition, an oligonucleotide corresponding to NFkB-binding site was also included as a
398
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
nonspecific competitor. As shown in Fig. 2C, the TSRDNA complex was specifically competed by an excess of unlabeled wild type TSR element but not by mutated TSR element or by the unrelated NFkB competitors. To examine the heat stability of TSR-binding activity, aliquots of HeLa nuclear extracts were heated at 42, 65, or 90°C for 10 min before incubation with labeled DNA probe. No significant change was observed in TSR–DNA complex when HeLa nuclear extract was heated at 42°C. However, at temperatures higher than 65°C, the TSR-binding activities were completely inactivated (Fig. 2D). The TSR element confers inhibition on cytokine responsiveness in HeLa but not in HepG2 cells. Our earlier transient transfection experiments have shown that a 63-bp deletion, that includes the TSR element, from the 59 region of the parental pTK/SAA1(2289/ 273) construct resulted in loss of its liver-specific expression pattern (Fig. 1). To demonstrate that it is the TSR element that is responsible for the repressed cytokine responsiveness in HeLa cells, we inserted one or three copies of wild type TSR element into the HindIII site of the pTK/SAA1(2138/273) construct, yielding p-138/273(TSR)1x and p-138/273(TSR)3x constructs, respectively (Fig. 3A). A p-138/273(mTSR)1x construct that contained one copy of the mutated TSR element was also generated for use as a negative control and to examine the correlation between TSR’s DNA binding activity and its repression function in HeLa cells. These reporter constructs were transfected into HeLa and HepG2 cells and CAT activities determined following stimulation with CM or TPA. While insertion of either wild type or mutant TSR element did not affect the promoter’s cytokine responsiveness in HepG2 cells, they had profound effects when transfected into HeLa cells. In HepG2 cells, all three constructs, p-138/273(TSR)1x, p-138/273(TSR)3x, and p-138/ 273(mTSR)1x, showed essentially identical responsiveness to CM and TPA induction and were indistinguishable from that of the parental pTK/SAA1(2138/ 273) construct (Figs. 3A and 3B). In HeLa cells, however, constructs containing the wild type TSR element showed reduced CM- and TPA-mediated induction, regardless of its orientation and copy number. In contrast, insertion of a mutated TSR element did not have such inhibitory effects (Figs. 3A and 3B). These data demonstrated that one copy of the TSR element is sufficient to confer repression on a heterologous promoter in HeLa cells and that the repression may be independent of its orientation and distance from the transcription initiation site. Results from these functional studies further support the notion that the liverspecific expression of SAA1 gene is regulated, at least in part, by the inhibitory activities of the negative regulatory TSR element. The fact that TSR-binding activity was found in HeLa cells but not in HepG2 cells
is consistent with its role in conferring SAA1’s differential expression patterns in these two cell types. Tissue distribution of TSR-binding activities. Our results showed that the presence of TSR-binding activity in HeLa cells is correlated with the absence of SAA1 gene transcription, suggesting that TSR may play a role in repressing SAA1 expression in nonliver cells and contribute to its cell-type-specific expression. To examine whether the correlation between TSR-binding and absence of SAA1 expression may be extended to other cell types, nuclear extracts were prepared from several nonliver and liver cell lines and tissues and the TSR-binding activities determined by EMSA. As shown in Fig. 4, when compared with HeLa nuclear extracts comparable levels of TSR-binding activities were detected in human placenta nuclear extracts. TSR-binding activities were also detected in two nonliver-derived cell nuclear extracts, mouse myoblast C2 and fibroblast 10T 1/2, albeit at significantly lower levels than those in HeLa and placenta extracts. The specificity of TSR binding was confirmed by oligonucleotide competition experiments in which the wild-type but not the mutant TSR element could compete for TSR binding. In sharp contrast, when nuclear extracts from HepG2 and Hep3B cells as well as from rat liver were tested for TSR binding, no TSR-DNA complexes were observed even with 15 mg of nuclear proteins in the binding reaction. To ensure that the absence of a defined DNA-protein complex with nuclear extracts from liver or liver-derived cell lines truly reflects the absence of TSR-binding activities and not due to the quality of the nuclear extract preparation, these nuclear extracts were tested for C/EBP-binding activities with a radiolabeled consensus C/EBP-binding site. For all three extract preparations, specific C/EBP-DNA complexes were detected (data not shown). Taken together, our results demonstrate that while TSRbinding activities could be detected in several nonliver cells, they are either absent in liver-derived cells or are present in amounts below the sensitivity of our EMSA. DISCUSSION It is increasingly clear that the control of eukaryotic gene transcription involves both positive and negative regulatory mechanisms and that different mechanisms exist whereby the expression of certain genes is restricted to specific cell type or types. There are examples of positive-acting transcription factors that regulate lineage-specific genes and can function as master regulators for cell-type determination or differentiation (2, 5, 47). Similarly, negative regulators that confer repression on a battery of cell type-specific genes have also been reported (10, 12, 48, 49). An example of such negative regulator is the neuron-restrictive silencer factor that may silence many neuron-specific
399
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Functional assay of TSR element. (A) (Top) Schematic representation of plasmid constructs containing wild type or mutated TSR elements. Wild type or mutant TSR-binding sequences were inserted at the HindIII site of pTK/SAA1(2138/273) plasmid to generate p-138/273(TSR)1x, p-138/273(TSR)3x, and p-138/273(mTSR)1x. Orientations of the inserts are indicated by the direction of the arrows. (Bottom) Effects of TSR element on promoter activity. HepG2 and HeLa cells were transfected with 10 mg of p-138/273(TSR)1x, p-138/ 400
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Tissue distribution of TSR binding activities. 32P-labeled TSR probe was incubated with nuclear extracts from HeLa (2 mg), 10T 1/2 fibroblast (5 mg), C2 myoblast (10 mg), human placenta (2 mg), and with 15 mg of nuclear extracts from HepG2, Hep3B, and rat liver. Specificity of DNA–protein complex was determined by competition without competitor (2) or with a 100-fold molar excess of unlabeled wild type (WT) or mutant (mt) TSR-binding site oligonucleotides. DNA-protein complexes were resolved on a native 5.5% polyacrylamide gel. Positions of specific DNA–protein complexes are indicated by the solid arrows and the open arrows indicate positions of the free probe.
promoters in non-neuronal cells (10, 11). In the case of liver-specific gene expression, results of somatic cell fusion experiments and the phenomenon of extinction
of gene expression have led to the interpretation that liver tissue-specific negative trans-acting factors exist and that they play a dominant role in restricting the expression of their target genes in specific cell types (50). Fournier and colleagues have reported that the expression of liver-specific gene tyrosine aminotransferase, in hepatoma 3 fibroblast hybrid cells, is repressed by the presence of a genetic locus Tse-1 (tissuespecific extinguisher-1) that mapped to mouse chromosome 11 (51–53). Further, they showed that Tse-1 acts, in a trans dominant fashion, by affecting protein binding at the cAMP response element located in the tissue-specific enhancer of the tyrosine aminotransferase gene (54). A second negative regulatory locus Tse-2 was also found to selectively repress the expression of albumin and alcohol dehydrogenase genes in somatic hybrid cells (55). In the case of developmentally regulated expression of mouse a-fetoprotein which is repressed in adult livers, genetic experiments in mice have revealed at least one locus raf that might encode the repressor (14). We describe here a 29-bp TSR element in the rat SAA1 gene promoter that could confer repression, in an orientation- and distance-independent manner, on the transcription of its own promoter as well as a heterologous promoter in HeLa cells. Our initial observation that the parental construct pTK/SAA1(2289/273) showed striking contrast in its ability to respond to cytokine stimulation when transfected into HepG2 and HeLa cells. While the CAT reporter gene activity was induced by CM and TPA in HepG2 cells, no increase in CAT activity was observed in HeLa cells despite the presence of positive-acting NFkB and C/EBP transcription factors. The absence of CAT reporter expression in HeLa cells may be explained by two opposing models. One possibility is that for SAA1 gene transcription, additional positive factors or coactivators may be necessary for its activation. These factors, however, are low in abundance or are lacking in HeLa cells, thereby leading to the inability to activate transcription. Alternatively, all the necessary positive factors are present in HeLa cells but are unable to activate transcription due to the presence of a dominant inhibitory factor or factors. Results of our EMSA with HepG2 and HeLa nuclear extracts strongly suggested that the latter possibility is likely to be the mechanism responsible for the repressed CAT expression in HeLa cells. How this DNA-protein interaction represses SAA1 gene transcription in HeLa cells remains unclear. Several mechanisms of transcription repression may be postulated (56, 57). Transcription repression may be
273(TSR)3x, and p-138/273(mTSR)1x plasmids. After transfection, cells were treated with basal medium (2) or 50% CM (1) for 18 h before harvested for CAT assays. (B) HepG2 and HeLa cells were transfected as above. Transfected cells were stimulated with basal medium (control) or 100 ng/ml TPA (1TPA). Plasmids pTK/SAA1(2289/273) and pBLCAT81 were included as positive and negative controls, respectively. CAT activities were quantitated by using a PhosphorImager. 401
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
achieved through competition, quenching, direct repression, or squelching of transcription factors. Although there is no evidence to support a particular mechanism by which TSR represses SAA1 gene transcription, it is conceivable that TSR might function through direct repression in which its binding is sufficient to affect the assembly or function of transcription initiation complexes, regardless of the binding of other positive transcription factors in the neighboring regulatory regions. Alternatively, an occupied TSR-binding site could inhibit the binding of positive regulatory factors or block the activating signal of a bound positive factor from transmitting it effects to the transcription initiation complex. Together with the observation that TSR-binding activities are detected only in nonliver cells, TSR has all the characteristics of a tissuespecific extinguisher described in the somatic cell hybrid studies. Thus, it is tempting to speculate that TSR may play an important role in repressing or extinguishing SAA1 gene expression in nonliver cells that express TSR and thus contribute to SAA1’s liverspecific expression pattern. Together with our earlier studies, a model for the regulatory mechanisms that control SAA1’s tissuespecific and inflammation-induced expression characteristics may be formulated. In the liver cells, inflammatory mediators, such as interleukin-1 and tumor necrosis factor, dramatically increase the activities of positive transcription factors NFkB and C/EBP in the nucleus. Since TSR is not expressed in liver cells, these two potent transactivators could function cooperatively to activate SAA1 gene transcription. In HeLa cells, however, because TSR is constitutively expressed, it can bind to the TSR element and perhaps operate dominantly to inhibit the activities of the neighboring NFkB and C/EBP. Consequently, SAA1 promoter cannot respond to cytokine induction despite the presence of activated NFkB and C/EBP in the nucleus. Thus, the inverse correlation between TSR and SAA1 expression is consistent with the model that TSR may function as a repressor in SAA1 gene regulation and may contribute to SAA1’s liver-specific expression pattern by restricting its expression in nonliver cells. ACKNOWLEDGMENT This research was supported in part by Public Health Service Grant AR 38858 from the National Institutes of Health to W.S-L.L.
REFERENCES 1. Bodner, M., Castrillo, J. L., Theill, L. E., Deerinck, T., Ellisman, M., and Karin, M. (1988) Cell 55(3), 505–518. 2. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., et al. (1991) Science 251(4995), 761–766. 3. Herbst, R. S., Nielsch, U., Sladek, F., Lai, E., Babiss, L. E., and Darnell, J. E., Jr. (1991) New Biol. 3(3), 289 –296.
4. Zhou, M. D., Wu, Y., Kumar, A., and Siddiqui, M. A. (1992) Gene Expr. 2(2), 127–138. 5. Corcoran, L. M., Karvelas, M., Nossal, G. J., Ye, Z. S., Jacks, T., and Baltimore, D. (1993) Genes Dev. 7(4), 570 –582. 6. Herbst, R. S., Friedman, N., Darnell, J. E., Jr., and Babiss, L. E. (1989) Proc. Natl. Acad. Sci. USA 86(5), 1553–1557. 7. Herbst, R. S., Boczko, E. M., Darnell, J. E., Jr., and Babiss, L. E. (1990) Mol. Cell. Biol. 10(8), 3896 –3905. 8. Flanagan, J. R., Murata, M., Burke, P. A., Shirayoshi, Y., Appella, E., Sharp, P. A., and Ozato, K. (1991) Proc. Natl. Acad. Sci. USA 88(8), 3145–3149. 9. Petersen, R., Kempler, G., and Barklis, E. (1991) Mol. Cell. Biol. 11(3), 1214 –1221. 10. Schoenherr, C. J., and Anderson, D. J. (1995) Science 267(5202), 1360 –1363. 11. Schoenherr, C. J., Paquette, A. J., and Anderson, D. J. (1996) Proc. Natl. Acad. Sci. USA 93(18), 9881–9886. 12. Ohsako, S., Hyer, J., Panganiban, G., Oliver, I., and Caudy, M. (1994) Genes Dev. 8(22), 2743–2755. 13. Vacher, J., and Tilghman, S. M. (1990) Science 250(4988), 1732– 1735. 14. Vacher, J., Camper, S. A., Krumlauf, R., Compton, R. S., and Tilghman, S. M. (1992) Mol. Cell. Biol. 12(2), 856 – 864. 15. Colantuoni, V., Pirozzi, A., Blance, C., and Cortese, R. (1987) EMBO J. 6(3), 631– 636. 16. Faraonio, R., Musy, M., and Colantuoni, V. (1990) Nucleic Acids Res. 18(24), 7235–7242. 17. Bulla, G. A., DeSimone, V., Cortese, R., and Fournier, R. E. (1992) Genes Dev. 6(2), 316 –327. 18. Imagawa, M., Osada, S., Okuda, A., and Muramatsu, M. (1991) Nucleic Acids Res. 19(1), 5–10. 19. Getman, D. K., Mutero, A., Inoue, K., and Taylor, P. (1995) J. Biol. Chem. 270(40), 23511–23519. 20. Yan, C., Costa, R. H., Darnell, J. E., Jr., Chen, J. D., and Van Dyke, T. A. (1990) EMBO J. 9(3), 869 – 878. 21. Pierce, J. W., Gifford, A. M., and Baltimore, D. (1991) Mol. Cell. Biol. 11(3), 1431–1437. 22. Benezra, R., Davis, R. L., Lassar, A., Tapscott, S., Thayer, M., Lockshon, D., and Weintraub, H. (1990) Ann. N.Y. Acad. Sci. 599, 1–11. 23. Lee, K. C., Crowe, A. J., and Barton, M. C. (1999) Mol. Cell. Biol. 19(2), 1279 –1288. 24. Baumann, H., and Gauldie, J. (1990) Mol. Biol. Med. 7(2), 147– 159. 25. Rienhoff, H. Y., Jr., Huang, J. H., Li, X. X., and Liao, W. S. (1990) Mol. Biol. Med. 7(3), 287–298. 26. Pannen, B. H., and Robotham, J. L. (1995) New Horiz. 3(2), 183–197. 27. Moshage, H. (1997) J. Pathol. 181(3), 257–266. 28. Lowell, C. A., Stearman, R. S., and Morrow, J. F. (1986) J. Biol. Chem. 261(18), 8453– 8461. 29. Edbrooke, M. R., Burt, D. W., Cheshire, J. K., and Woo, P. (1989) Mol. Cell. Biol. 9(5), 1908 –1916. 30. Li, X. X., and Liao, W. S. (1991) J. Biol. Chem. 266(23), 15192– 15201. 31. de Beer, M. C., Kindy, M. S., Lane, W. S., and de Beer, F. C. (1994) J. Biol. Chem. 269(6), 4661– 4667. 32. Butler, A., and Whitehead, A. S. (1996) Immunogenetics 44(6), 468 – 474. 33. Lowell, C. A., Potter, D. A., Stearman, R. S., and Morrow, J. F. (1986) J. Biol. Chem. 261(18), 8442– 8452.
402
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
34. Meek, R. L., and Benditt, E. P. (1986) J. Exp. Med. 164(6), 2006 –2017. 35. Meek, R. L., Eriksen, N., and Benditt, E. P. (1989) Am. J. Pathol. 135(2), 411– 419. 36. Meek, R. L., Eriksen, N., and Benditt, E. P. (1992) Proc. Natl. Acad. Sci. USA 89(17), 7949 –7952. 37. Li, X., and Liao, W. S. (1992) Nucleic Acids Res. 20(18), 4765– 4772. 38. Lu, S. Y., Rodriguez, M., and Liao, W. S. (1994) Mol. Cell. Biol. 14(9), 6253– 6263. 39. Huang, J. H., Rienhoff, H. Y., Jr., and Liao, W. S. (1990) Mol. Cell. Biol. 10(7), 3619 –3625. 40. Kawai, S., and Nishizawa, M. (1984) Mol. Cell. Biol. 4(6), 1172– 1174. 41. Huang, J. H., and Liao, W. S. (1994) Mol. Cell. Biol. 14(7), 4475– 4484. 42. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 43. Cato, A. C., Miksicek, R., Schutz, G., Arnemann, J., and Beato, M. (1986) EMBO J. 5(9), 2237–2240. 44. Gorman, C., Padmanabhan, R., and Howard, B. H. (1983) Science 221(4610), 551–553. 45. Shapiro, D. J., Sharp, P. A., Wahli, W. W., and Keller, M. J. (1988) DNA 7(1), 47–55.
46. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11(5), 1475–1489. 47. Gehring, W. J. (1996) Genes Cells 1(1), 11–15. 48. Chen, T. T., Wu, R. L., Castro-Munozledo, F., and Sun, T. T. (1997) Mol. Cell. Biol. 17(6), 3056 –3064. 49. Chong, J. A., Tapia-Ramirez, J., Kim, S., Toledo-Aral, J. J., Zheng, Y., Boutros, M. C., Altshuller, Y. M., Frohman, M. A., Kraner, S. D., and Mandel, G. (1995) Cell 80(6), 949 –957. 50. Chin, A. C., and Fournier, R. E. (1986) Ann. N.Y. Acad. Sci. 478, 120 –130. 51. Killary, A. M., and Fournier, R. E. (1984) Cell 38(2), 523–534. 52. Gourdeau, H., Peterson, T. C., and Fournier, R. E. (1989) Mol. Cell. Biol. 9(5), 1813–1822. 53. Lem, J., Chin, A. C., Thayer, M. J., Leach, R. J., and Fournier, R. E. (1988) Proc. Natl. Acad. Sci. USA 85(19), 7302–7306. 54. Boshart, M., Weih, F., Schmidt, A., Fournier, R. E., and Schutz, G. (1990) Cell 61(5), 905–916. 55. Chin, A. C., and Fournier, R. E. (1989) Mol. Cell. Biol. 9(9), 3736 –3743. 56. Herschbach, B. M., and Johnson, A. D. (1993) Annu. Rev. Cell Biol. 9, 479 –509. 57. Johnson, A. D. (1995) Cell 81(5), 655– 658.
403
An Upstream Repressor Element That Contributes to Hepatocyte-Specific Expression of the Rat Serum Amyloid A1 Gene Lei Li and Warren S.-L. Liao 1 Department of Biochemistry and Molecular Biology, Box 117, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030
Received September 14, 1999
Serum amyloid A (SAA) is a major acute-phase protein whose expression can be dramatically induced in response to tissue damage, infection, and inflammation. Its expression is highly tissue-specific, restricted almost exclusively to liver hepatocytes. We have shown that a 320-bp fragment of the rat SAA1 promoter could confer liver-cell-specific expression on a reporter gene when transfected into cultured cells. Here we report the identification of a 29-bp regulatory element that possesses inhibitory activities on SAA1 promoter in HeLa cells but has no such effects in liver cells. Moreover, this regulatory element has properties of a transcriptional repressor; in that, it can function with a heterologous promoter and is independent of orientation and distance from the transcription initiation site. Protein binding studies showed that this regulatory element can form specific protein–DNA complexes with nuclear proteins from several nonliver cell lines (HeLa, 10T 1/2 , and C2) and placenta. However, the same DNA–protein complex was not detected in extracts from liver or liver-derived cell lines (HepG2 and Hep3B). Taken together, our results demonstrate the presence of a DNA-binding protein, termed tissuespecific repressor, found only in nonhepatocytes which may function to repress SAA1 gene expression by interacting with a repressor element. Thus, liverspecific expression of the SAA1 gene is apparently regulated by both positive and negative regulatory elements and their interacting transcription factors to ensure that it is expressed only in suitable cell types. © 1999 Academic Press
In a multicellular organism, the expression of cellular genes is highly regulated in specific spatial and temporal patterns. A major control of cell type-specific gene expres1
To whom correspondence should be addressed. Fax: (713) 7919478. E-mail: [email protected].
sion is at the level of gene transcription. Studies of cell type-specific gene regulation have led to the identification of both positive and negative regulatory elements. Among the positive regulatory sequences, some interact with widely distributed cellular transcription factors while others interact with cell-specific factors (1–5). Therefore, a gene that employs multiple cellular transcription factor-binding sites in the promoter region could display distinct transcription rate, inducibility, and tissue specificity by the combinatorial use of these regulatory elements and their binding factors. Additionally, the gene expression patterns can also be controlled by negative regulatory elements and their DNA-binding transcription repressors. For example, in the mouse albumin gene far-upstream enhancer, a negative regulatory element has been found that is capable of negating the effects of both homologous and heterologous enhancers (6, 7). Thus, this repressor element modulates the expression levels of albumin by suppressing its promoter activities in nonhepatic cells (7). Negative regulatory elements have also been shown to control the developmental appearance or disappearance of gene expression in a stagespecific manner (8–12). Vacher et al. (13, 14) have identified a dominant negative element that acts as a repressor in a position-dependent manner to extinguish mouse a-fetoprotein gene transcription in adult liver. Similarly, a negative regulatory element in MHC class I promoter was found to be active only in undifferentiated F9 embryonal cells (8). Moreover, tissue-specific genes with their distinct tissue specificity, such as liver-specific retinol binding protein (15, 16), a 1-antitrypsin (17), glutathione transferase P (18), acetylcholinesterase (19), transthyretin (20), and B cell-specific immunoglobin (21) genes, all contain either silencer that allows only the expression of the genes in the correct cell type or repressors that contribute to the tissue specificity of gene transcription. In some cases, specific DNA sequences and interaction with their cognate DNA-binding proteins have been delineated (10, 22, 23).
395
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Serum amyloid A (SAA), one of the major acutephase proteins, is synthesized and secreted primarily by the liver (24 –27). During normal conditions, the plasma concentration of SAA is very low or undetectable. However, in response to tissue damage, infection, or inflammation, its plasma concentration rises several hundredfold as a consequence of increased production of SAA mRNA at the transcriptional level (28). In cultures of primary hepatocytes and hepatoma cells, SAA gene expression can be induced by inflammatory cytokines, interleukin-1 and tumor necrosis factor, and by the phorbol ester, 12-O-tetradecanoyl-phorbol-13acetate (TPA) (25, 29, 30). In mice, the SAA gene family consists of three acutephase genes (SAA1, 2, and 3), one constitutively expressed gene (SAA5) and a pseudogene (31–33). Among the three acute-phase SAA genes, SAA1 and 2, are highly homologous to each other having over 95% sequence homology over a region that includes four exons, introns, and 59 and 39-flanking DNA sequences. The SAA3 DNA sequence, however, shows significant homologies only to the protein coding exons of SAA1 and 2; otherwise, this sequence has diverge considerably (33). Consistent with their DNA sequence divergence, the expression patterns of these three acutephase genes also differed. At the peak of inflammatory response, all three SAA genes are expressed in the liver with each gene accounting for approximately onethird of total SAA mRNA (28). However, while SAA1 and 2 genes are expressed almost exclusively in the liver, expression of SAA3 gene is less restricted where high levels of expression are also found in the macrophages, and at lower levels in the kidney and intestine (34 –36). Thus, the expression of SAA1 and 2 are tightly regulated in a liver cell-specific manner and provide good experimental models to study the control of tissue-specific gene expression. Our previous studies on the molecular mechanisms of rat SAA1 gene regulation have identified several regulatory regions that are essential for its dramatic response to inflammatory cytokines (30, 37, 38). Transient transfection analysis of the promoter demonstrated that a 66-bp fragment (positions 2138 to 273) could confer cytokine responsiveness onto a heterologous promoter in liver and nonliver cells (37). This fragment thus functions as a non-cell-specific cytokine response unit (CRU). Within the CRU resided binding sites for two positive transcription factors C/EBP and NFkB that function synergistically to transactivate the target promoter (37). A third transcription factor YY1 also binds to the CRU and competes with NFkB for overlapping binding sites. Thus, in the rat SAA1 promoter, YY1 functions as a transcriptional repressor, not only contribute to SAA1’s low basal expression, but also perhaps play a role in the transiency of its expression in response to acute inflammation (38).
In this report, we provide evidence for a second negative regulatory element in the rat SAA1 promoter. This regulatory element, however, differs from YY1 in that, while YY1 participates in modulating the cytokine responsiveness of the SAA1 promoter, this negative regulatory element contributes to its liver-specific expression. We show that a 29-bp regulatory element located upstream of the CRU could repress the expression of SAA1/CAT reporter gene in HeLa cells, but has no inhibitory effect in liver cells. Furthermore, it can function with a heterologous promoter and is independent of orientation and distance from the transcription start site. Protein binding studies revealed DNA sequence-specific binding activities in several nonliverderived cell lines; however, this binding activity is completely absent in liver or liver-derived cell lines. Thus, this DNA-binding protein, termed tissue-specific repressor (TSR), may function to repress SAA1 gene expression in nonliver cells, contributing to SAA1’s liverspecific expression pattern. MATERIALS AND METHODS Plasmid construction and oligonucleotides. Two restriction enzyme fragments KpnI to AvaII (2289/273) and HinfI to AvaII (2138/ 273) from the rat SAA1 gene promoter were isolated from pSAA1/ CAT(2304) (30) and inserted as blunt-ended fragments into the SmaI site of pBLCAT81 to generate pTK/SAA1(2289/273) and pTK/SAA1(2138/273), respectively. A PCR fragment from bp 2226 to 273 was amplified and inserted similarly to yield pTK/ SAA1(2226/273) construct. The double-stranded wild type TSR-binding site oligonucleotides (59-CTTTCACTCTATACCTCAGGCAGCTAAGG-39) corresponding to bp 2285 to 2257 and the mutated TSR-binding site oligonucleotides (59-CTTTCACTCTATAggaCAGGCAGCTAAGG-39) were synthesized and used as radiolabeled probes or as competitors in gel shift assays. Plasmids p-138/273(TSR)1x and p-138/273(TSR)3x, containing one and three copies of wild type TSR-binding site oligonucleotides, respectively, were constructed by inserting annealed oligonucleotides into the HindIII site of pTK/SAA1(2138/273). p-138/273(mTSR)1x was constructed by inserting mutated TSRbinding site oligonucleotides into pTK/SAA1(2138/273). Cell culture and transient transfection assay. Human hepatoma HepG2 cells and HeLa cells were cultured as monolayers in modified Eagle’s medium and Waymouth MAB (3:1, vol/vol) plus 10% fetal calf serum (39) and passaged at confluence by trypsinization approximately once a week. DNA transfection was performed by the Polybrene procedure (40). Approximately 18 h after transfection, cells were stimulated with basal medium, 50% conditioned medium (CM), or 100 ng/ml TPA (Sigma) (30, 41). Cell extracts were assayed for protein content by the Bradford assay (42), and chloramphenicol acetyltransferase (CAT) activity was determined by modifications (43) of procedures described by Gorman et al. (44). To determine CAT activities, [ 14C]chloramphenicol spots corresponding to acetylated and nonacetylated forms were quantitated using a PhosphorImager (Molecular Dynamic). CM was prepared from mixed human lymphocyte cultures stimulated with 0.75% phytohemagglutinin as described previously (30, 41) and was used as a mixture with an equal volume of basal medium. Nuclear extracts. Nuclear extracts from cultured cells were prepared as described (45). Briefly, isolated nuclei were resuspended at a concentration of 10 9 nuclei in 6 ml of final nuclear resuspension
396
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
buffer (9 vol of nuclear resuspension buffer and 1 vol of saturated ammonium sulfate; nuclear resuspension buffer consists of 20 mM N-2-hydroxyethylpiperazine-N9-2ethane-sulfonic acid (Hepes), pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 0.2 mM EGTA, 2 mM dithiothreitol (DTT), and 25% glycerol. The resulting suspension was mixed by gentle rocking at 4°C for 30 min. The chromatin was subsequently removed by sedimentation at 150,000g for 90 min at 2°C. The supernatant was collected and the protein precipitated by the addition of powdered ammonium sulfate (0.33 g/ml), and collected by centrifugation at 85,000g in SW28 rotor for 20 min at 4°C. Protein pellets were dissolved in 1 ml of nuclear dialysis buffer (20 mM Hepes, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, and 2 mM DTT) per 10 9 nuclei and dialyzed for 4 h at 4°C against 300 ml of nuclear dialysis buffer, with one change of buffer. The dialyzed nuclear extract was then clarified by centrifugation at 10,000g for 10 min, and frozen in small aliquots in dry ice and stored at 280°C. Nuclear extracts from human placenta were prepared by a modification of the method described by Dignam et al. (46). Electrophoretic mobility shift assay (EMSA). Reactions for EMSA were performed in a 20-ml reaction mixture consisting of 12 mM Hepes (pH 7.9), 60 mM KCl, 1.2 mM DTT, 0.12 mM EDTA, 12% glycerol, 4 mg poly(dI:dC), nuclear protein extracts, and 2 3 10 4 cpm (;1 ng) of 32P-labeled DNA fragment. The reaction mixture was preincubated for 5 min at room temperature before the radioactive probe was added. The reaction mixture was then incubated for 20 min at room temperature. Samples were loaded onto a low-ionic strength, 6% acrylamide gel (19:1 crosslinking ratio) containing 0.253 TBE (13 TBE: 89 mM Tris, pH 7.8, 89 mM boric acid, 1 mM EDTA) and subjected to electrophoresis at 200 V for 2 h. The gel was then dried and autoradiographed.
RESULTS Rat SAA1 promoter contains a regulatory element that inhibits its cytokine responsiveness in HeLa cells. We have previously reported that the rat SAA1 promoter is highly responsive to cytokine induction when transiently transfected into hepatoma HepG2 and Hep3B cells (30, 37, 38). This dramatic induction by cytokines is mediated primarily by the activation and cooperative interactions of transcription factors NFkB and C/EBP that bind at adjacent sites within a 66-bp CRU in the proximal region of the SAA1 promoter (37, 38). Interestingly, while the SAA1 promoter can direct accurate transcription in liver or liver-derived cells in response to stimulation, it is totally nonresponsive in HeLa cells (37). To examine the cis regulatory elements responsible for this differential expression in HepG2 and HeLa cells, we inserted the 217-bp KpnI–AvaII fragment from the SAA1 promoter into the unique SmaI site of plasmid pBLCAT81 to generate the CAT reporter construct pTK/SAA1(2289/273). This reporter gene construct which contains the CRU showed pronounced response upon stimulation by CM or TPA when transfected into HepG2 cells (Fig. 1). However, there was no increase in CAT activity when the same construct was introduced into HeLa cells. Thus, despite the presence of transcription factors NFkB and C/EBP in HeLa cells, these two transcription factors are nevertheless unable to transactivate the pTK/ SAA1(2289/273) reporter construct. This result indicates that the regulatory region between bp 2289 and
FIG. 1. Identification of a cell-specific regulatory element in the rat SAA1 promoter. (Top) Schematic diagrams of the proximal region of the rat SAA1 promoter and the 59 deletion constructs used for transfection. Deletion constructs containing various promoter regions of SAA1 gene were generated as described under Materials and Methods. The SAA1 promoter fragments were inserted into the pBLCAT81 vector with the minimum thymidine kinase (TK) promoter. The arrow denotes the transcription initiation site. (Middle and bottom) CAT assay analysis. Cultured HepG2 and HeLa cells were transfected with 10 mg of pTK/ SAA1(2289/273), pTK/SAA1(2226/273), and pTK/SAA1(2138/273) DNAs. As a negative control, pBLCAT81 vector (TK) was also included. Transfected cells were not stimulated (2), or stimulated with 50% CM (CM) or 100 ng/ml TPA (TPA). After treatment, cell extracts were prepared and the protein content and the CAT activities determined. Consistent results were obtained from three independent experiments, one of which is shown.
273 in the rat SAA1 promoter contains sequences that contributes to its hepatocyte-specific expression. To further delineate sequences within this regulatory region that inhibits SAA1 promoter function in
397
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HeLa cells, we examined two 59 deletion mutants, pTK/ SAA1(2226/273) and pTK/SAA1(2138/273), with deletions of 63 and 151 bp, respectively, from the KpnI site (Fig. 1). These two constructs, along with the parental plasmid pTK/SAA1(2289/273) and the promoterless pBLCAT81 vector, were tested for CM- and TPA-induced expression in HepG2 and HeLa cells. As shown in Fig. 1, when compared with the parental pTK/SAA1(2289/273) construct, both deletion mutants showed comparable levels of CAT expression in HepG2 cells in response to induction by CM and TPA. Interestingly, these two deletion constructs also showed dramatic response to CM- and TPA-mediated induction in HeLa cells. This is in sharp contrast to the parental pTK/SAA1(2289/273) construct in which there was no response to cytokine induction. As expected, the promoterless vector construct had no CAT activity with or without stimulation. Taken together, these results indicate that while the regulatory region from bp 2138 to 273 in the SAA1 promoter is necessary and sufficient to confer cytokine responsiveness onto a heterologous thymidine kinase promoter, the region from 2289 to 2226 may be crucial for SAA1’s tissue-specific expression by restricting its promoter function in HeLa cells. Binding of a HeLa nuclear factor to the TSR element. Deletion of bp 2289 to bp 2226 in the rat SAA1 promoter resulted in dramatic increase in CM- and TPAinduced transcription of the CAT reporter gene in HeLa cells. This might be taken to represent deletion of a binding region for a transacting factor capable of suppressing the positive effects of NFkB and C/EBP. To determine whether any transcription factor could bind to this region of the SAA1 promoter, two DNA fragments, a 29-bp (2285/2257) fragment and a 31-bp fragment (2256/2226) were generated and used as probes in EMSA with nuclear extracts from HeLa and HepG2 cells (Fig. 2A). When the 31-bp 2256/2226 fragment was used as a probe, no protein-DNA complexes were formed with either HeLa or HepG2 nuclear extracts (data not shown). In contrast, an intense protein-DNA complex was detected with the 29-bp (2285/2257) fragment (Fig. 2B). Interestingly, this protein-DNA complex was only detected with HeLa nuclear extracts while the same complex was not found with HepG2 nuclear extracts. This HeLa nuclear protein that possesses the DNA-binding activity was termed the tissue-specific repressor (TSR) and the 29-bp DNA regulatory element that it interacts with was denoted the TSR element. To test whether the lack of complex formation with HepG2 nuclear extracts might be due to the presence of inhibitory components that interfere with protein binding to the probe, we mixed equal amounts of HepG2 and HeLa nuclear extracts and incubated this mixture with the DNA probe in EMSA. As shown in Fig. 2B, a
FIG. 2. Tissue-specific repressor binding activity in HeLa but not in HepG2 cells. (A) Location of tissue specific repressor element in the rat SAA1 promoter. Nucleotide sequences (bp 2285/2257) corresponding to wild type (TSR) or mutant (mTSR) TSR-binding sites are indicated. (B) End-labeled TSR element was incubated with 5 mg of nuclear extracts from HeLa and HepG2 cells. For the mixing experiment, equal amounts (2.5 mg 1 2.5 mg) of nuclear extracts from each cell line were mixed before addition of the 32P-labeled TSR probe. (C) Specific competition of TSR-binding activities. End-labeled TSR probe was incubated with HeLa nuclear extracts (2 mg) in the presence of a 25- or 100-fold molar excess of unlabeled oligonucleotides corresponding to wild-type (TSR) and mutated (mTSR) TSRbinding sequence, and the binding sequence for NFkB (NFkB). Position of specific DNA-protein complex formed is indicated by the solid arrow and the open arrow denotes the position of the free probe. (D) Heat stability of TSR-binding activity. HeLa nuclear extracts were not heated or heated at 42, 65, and 90°C for 10 min before incubated with radiolabeled probe. Position of specific complex formed is indicated by the solid arrow. The open arrow denotes the position of the free probe.
protein-DNA complex that is indistinguishable from that with HeLa nuclear extract alone was formed. This result strongly indicates that the TSR-binding activity is present only in HeLa cells and that the lack of binding in HepG2 nuclear extracts is due to the absence of TSR in HepG2 cells rather than the presence of an inhibitory factor. To examine the sequence specificity of TSR binding, wild type and mutant TSRbinding site oligonucleotides were used in competition experiments. In addition, an oligonucleotide corresponding to NFkB-binding site was also included as a
398
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
nonspecific competitor. As shown in Fig. 2C, the TSRDNA complex was specifically competed by an excess of unlabeled wild type TSR element but not by mutated TSR element or by the unrelated NFkB competitors. To examine the heat stability of TSR-binding activity, aliquots of HeLa nuclear extracts were heated at 42, 65, or 90°C for 10 min before incubation with labeled DNA probe. No significant change was observed in TSR–DNA complex when HeLa nuclear extract was heated at 42°C. However, at temperatures higher than 65°C, the TSR-binding activities were completely inactivated (Fig. 2D). The TSR element confers inhibition on cytokine responsiveness in HeLa but not in HepG2 cells. Our earlier transient transfection experiments have shown that a 63-bp deletion, that includes the TSR element, from the 59 region of the parental pTK/SAA1(2289/ 273) construct resulted in loss of its liver-specific expression pattern (Fig. 1). To demonstrate that it is the TSR element that is responsible for the repressed cytokine responsiveness in HeLa cells, we inserted one or three copies of wild type TSR element into the HindIII site of the pTK/SAA1(2138/273) construct, yielding p-138/273(TSR)1x and p-138/273(TSR)3x constructs, respectively (Fig. 3A). A p-138/273(mTSR)1x construct that contained one copy of the mutated TSR element was also generated for use as a negative control and to examine the correlation between TSR’s DNA binding activity and its repression function in HeLa cells. These reporter constructs were transfected into HeLa and HepG2 cells and CAT activities determined following stimulation with CM or TPA. While insertion of either wild type or mutant TSR element did not affect the promoter’s cytokine responsiveness in HepG2 cells, they had profound effects when transfected into HeLa cells. In HepG2 cells, all three constructs, p-138/273(TSR)1x, p-138/273(TSR)3x, and p-138/ 273(mTSR)1x, showed essentially identical responsiveness to CM and TPA induction and were indistinguishable from that of the parental pTK/SAA1(2138/ 273) construct (Figs. 3A and 3B). In HeLa cells, however, constructs containing the wild type TSR element showed reduced CM- and TPA-mediated induction, regardless of its orientation and copy number. In contrast, insertion of a mutated TSR element did not have such inhibitory effects (Figs. 3A and 3B). These data demonstrated that one copy of the TSR element is sufficient to confer repression on a heterologous promoter in HeLa cells and that the repression may be independent of its orientation and distance from the transcription initiation site. Results from these functional studies further support the notion that the liverspecific expression of SAA1 gene is regulated, at least in part, by the inhibitory activities of the negative regulatory TSR element. The fact that TSR-binding activity was found in HeLa cells but not in HepG2 cells
is consistent with its role in conferring SAA1’s differential expression patterns in these two cell types. Tissue distribution of TSR-binding activities. Our results showed that the presence of TSR-binding activity in HeLa cells is correlated with the absence of SAA1 gene transcription, suggesting that TSR may play a role in repressing SAA1 expression in nonliver cells and contribute to its cell-type-specific expression. To examine whether the correlation between TSR-binding and absence of SAA1 expression may be extended to other cell types, nuclear extracts were prepared from several nonliver and liver cell lines and tissues and the TSR-binding activities determined by EMSA. As shown in Fig. 4, when compared with HeLa nuclear extracts comparable levels of TSR-binding activities were detected in human placenta nuclear extracts. TSR-binding activities were also detected in two nonliver-derived cell nuclear extracts, mouse myoblast C2 and fibroblast 10T 1/2, albeit at significantly lower levels than those in HeLa and placenta extracts. The specificity of TSR binding was confirmed by oligonucleotide competition experiments in which the wild-type but not the mutant TSR element could compete for TSR binding. In sharp contrast, when nuclear extracts from HepG2 and Hep3B cells as well as from rat liver were tested for TSR binding, no TSR-DNA complexes were observed even with 15 mg of nuclear proteins in the binding reaction. To ensure that the absence of a defined DNA-protein complex with nuclear extracts from liver or liver-derived cell lines truly reflects the absence of TSR-binding activities and not due to the quality of the nuclear extract preparation, these nuclear extracts were tested for C/EBP-binding activities with a radiolabeled consensus C/EBP-binding site. For all three extract preparations, specific C/EBP-DNA complexes were detected (data not shown). Taken together, our results demonstrate that while TSRbinding activities could be detected in several nonliver cells, they are either absent in liver-derived cells or are present in amounts below the sensitivity of our EMSA. DISCUSSION It is increasingly clear that the control of eukaryotic gene transcription involves both positive and negative regulatory mechanisms and that different mechanisms exist whereby the expression of certain genes is restricted to specific cell type or types. There are examples of positive-acting transcription factors that regulate lineage-specific genes and can function as master regulators for cell-type determination or differentiation (2, 5, 47). Similarly, negative regulators that confer repression on a battery of cell type-specific genes have also been reported (10, 12, 48, 49). An example of such negative regulator is the neuron-restrictive silencer factor that may silence many neuron-specific
399
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Functional assay of TSR element. (A) (Top) Schematic representation of plasmid constructs containing wild type or mutated TSR elements. Wild type or mutant TSR-binding sequences were inserted at the HindIII site of pTK/SAA1(2138/273) plasmid to generate p-138/273(TSR)1x, p-138/273(TSR)3x, and p-138/273(mTSR)1x. Orientations of the inserts are indicated by the direction of the arrows. (Bottom) Effects of TSR element on promoter activity. HepG2 and HeLa cells were transfected with 10 mg of p-138/273(TSR)1x, p-138/ 400
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Tissue distribution of TSR binding activities. 32P-labeled TSR probe was incubated with nuclear extracts from HeLa (2 mg), 10T 1/2 fibroblast (5 mg), C2 myoblast (10 mg), human placenta (2 mg), and with 15 mg of nuclear extracts from HepG2, Hep3B, and rat liver. Specificity of DNA–protein complex was determined by competition without competitor (2) or with a 100-fold molar excess of unlabeled wild type (WT) or mutant (mt) TSR-binding site oligonucleotides. DNA-protein complexes were resolved on a native 5.5% polyacrylamide gel. Positions of specific DNA–protein complexes are indicated by the solid arrows and the open arrows indicate positions of the free probe.
promoters in non-neuronal cells (10, 11). In the case of liver-specific gene expression, results of somatic cell fusion experiments and the phenomenon of extinction
of gene expression have led to the interpretation that liver tissue-specific negative trans-acting factors exist and that they play a dominant role in restricting the expression of their target genes in specific cell types (50). Fournier and colleagues have reported that the expression of liver-specific gene tyrosine aminotransferase, in hepatoma 3 fibroblast hybrid cells, is repressed by the presence of a genetic locus Tse-1 (tissuespecific extinguisher-1) that mapped to mouse chromosome 11 (51–53). Further, they showed that Tse-1 acts, in a trans dominant fashion, by affecting protein binding at the cAMP response element located in the tissue-specific enhancer of the tyrosine aminotransferase gene (54). A second negative regulatory locus Tse-2 was also found to selectively repress the expression of albumin and alcohol dehydrogenase genes in somatic hybrid cells (55). In the case of developmentally regulated expression of mouse a-fetoprotein which is repressed in adult livers, genetic experiments in mice have revealed at least one locus raf that might encode the repressor (14). We describe here a 29-bp TSR element in the rat SAA1 gene promoter that could confer repression, in an orientation- and distance-independent manner, on the transcription of its own promoter as well as a heterologous promoter in HeLa cells. Our initial observation that the parental construct pTK/SAA1(2289/273) showed striking contrast in its ability to respond to cytokine stimulation when transfected into HepG2 and HeLa cells. While the CAT reporter gene activity was induced by CM and TPA in HepG2 cells, no increase in CAT activity was observed in HeLa cells despite the presence of positive-acting NFkB and C/EBP transcription factors. The absence of CAT reporter expression in HeLa cells may be explained by two opposing models. One possibility is that for SAA1 gene transcription, additional positive factors or coactivators may be necessary for its activation. These factors, however, are low in abundance or are lacking in HeLa cells, thereby leading to the inability to activate transcription. Alternatively, all the necessary positive factors are present in HeLa cells but are unable to activate transcription due to the presence of a dominant inhibitory factor or factors. Results of our EMSA with HepG2 and HeLa nuclear extracts strongly suggested that the latter possibility is likely to be the mechanism responsible for the repressed CAT expression in HeLa cells. How this DNA-protein interaction represses SAA1 gene transcription in HeLa cells remains unclear. Several mechanisms of transcription repression may be postulated (56, 57). Transcription repression may be
273(TSR)3x, and p-138/273(mTSR)1x plasmids. After transfection, cells were treated with basal medium (2) or 50% CM (1) for 18 h before harvested for CAT assays. (B) HepG2 and HeLa cells were transfected as above. Transfected cells were stimulated with basal medium (control) or 100 ng/ml TPA (1TPA). Plasmids pTK/SAA1(2289/273) and pBLCAT81 were included as positive and negative controls, respectively. CAT activities were quantitated by using a PhosphorImager. 401
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
achieved through competition, quenching, direct repression, or squelching of transcription factors. Although there is no evidence to support a particular mechanism by which TSR represses SAA1 gene transcription, it is conceivable that TSR might function through direct repression in which its binding is sufficient to affect the assembly or function of transcription initiation complexes, regardless of the binding of other positive transcription factors in the neighboring regulatory regions. Alternatively, an occupied TSR-binding site could inhibit the binding of positive regulatory factors or block the activating signal of a bound positive factor from transmitting it effects to the transcription initiation complex. Together with the observation that TSR-binding activities are detected only in nonliver cells, TSR has all the characteristics of a tissuespecific extinguisher described in the somatic cell hybrid studies. Thus, it is tempting to speculate that TSR may play an important role in repressing or extinguishing SAA1 gene expression in nonliver cells that express TSR and thus contribute to SAA1’s liverspecific expression pattern. Together with our earlier studies, a model for the regulatory mechanisms that control SAA1’s tissuespecific and inflammation-induced expression characteristics may be formulated. In the liver cells, inflammatory mediators, such as interleukin-1 and tumor necrosis factor, dramatically increase the activities of positive transcription factors NFkB and C/EBP in the nucleus. Since TSR is not expressed in liver cells, these two potent transactivators could function cooperatively to activate SAA1 gene transcription. In HeLa cells, however, because TSR is constitutively expressed, it can bind to the TSR element and perhaps operate dominantly to inhibit the activities of the neighboring NFkB and C/EBP. Consequently, SAA1 promoter cannot respond to cytokine induction despite the presence of activated NFkB and C/EBP in the nucleus. Thus, the inverse correlation between TSR and SAA1 expression is consistent with the model that TSR may function as a repressor in SAA1 gene regulation and may contribute to SAA1’s liver-specific expression pattern by restricting its expression in nonliver cells. ACKNOWLEDGMENT This research was supported in part by Public Health Service Grant AR 38858 from the National Institutes of Health to W.S-L.L.
REFERENCES 1. Bodner, M., Castrillo, J. L., Theill, L. E., Deerinck, T., Ellisman, M., and Karin, M. (1988) Cell 55(3), 505–518. 2. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., et al. (1991) Science 251(4995), 761–766. 3. Herbst, R. S., Nielsch, U., Sladek, F., Lai, E., Babiss, L. E., and Darnell, J. E., Jr. (1991) New Biol. 3(3), 289 –296.
4. Zhou, M. D., Wu, Y., Kumar, A., and Siddiqui, M. A. (1992) Gene Expr. 2(2), 127–138. 5. Corcoran, L. M., Karvelas, M., Nossal, G. J., Ye, Z. S., Jacks, T., and Baltimore, D. (1993) Genes Dev. 7(4), 570 –582. 6. Herbst, R. S., Friedman, N., Darnell, J. E., Jr., and Babiss, L. E. (1989) Proc. Natl. Acad. Sci. USA 86(5), 1553–1557. 7. Herbst, R. S., Boczko, E. M., Darnell, J. E., Jr., and Babiss, L. E. (1990) Mol. Cell. Biol. 10(8), 3896 –3905. 8. Flanagan, J. R., Murata, M., Burke, P. A., Shirayoshi, Y., Appella, E., Sharp, P. A., and Ozato, K. (1991) Proc. Natl. Acad. Sci. USA 88(8), 3145–3149. 9. Petersen, R., Kempler, G., and Barklis, E. (1991) Mol. Cell. Biol. 11(3), 1214 –1221. 10. Schoenherr, C. J., and Anderson, D. J. (1995) Science 267(5202), 1360 –1363. 11. Schoenherr, C. J., Paquette, A. J., and Anderson, D. J. (1996) Proc. Natl. Acad. Sci. USA 93(18), 9881–9886. 12. Ohsako, S., Hyer, J., Panganiban, G., Oliver, I., and Caudy, M. (1994) Genes Dev. 8(22), 2743–2755. 13. Vacher, J., and Tilghman, S. M. (1990) Science 250(4988), 1732– 1735. 14. Vacher, J., Camper, S. A., Krumlauf, R., Compton, R. S., and Tilghman, S. M. (1992) Mol. Cell. Biol. 12(2), 856 – 864. 15. Colantuoni, V., Pirozzi, A., Blance, C., and Cortese, R. (1987) EMBO J. 6(3), 631– 636. 16. Faraonio, R., Musy, M., and Colantuoni, V. (1990) Nucleic Acids Res. 18(24), 7235–7242. 17. Bulla, G. A., DeSimone, V., Cortese, R., and Fournier, R. E. (1992) Genes Dev. 6(2), 316 –327. 18. Imagawa, M., Osada, S., Okuda, A., and Muramatsu, M. (1991) Nucleic Acids Res. 19(1), 5–10. 19. Getman, D. K., Mutero, A., Inoue, K., and Taylor, P. (1995) J. Biol. Chem. 270(40), 23511–23519. 20. Yan, C., Costa, R. H., Darnell, J. E., Jr., Chen, J. D., and Van Dyke, T. A. (1990) EMBO J. 9(3), 869 – 878. 21. Pierce, J. W., Gifford, A. M., and Baltimore, D. (1991) Mol. Cell. Biol. 11(3), 1431–1437. 22. Benezra, R., Davis, R. L., Lassar, A., Tapscott, S., Thayer, M., Lockshon, D., and Weintraub, H. (1990) Ann. N.Y. Acad. Sci. 599, 1–11. 23. Lee, K. C., Crowe, A. J., and Barton, M. C. (1999) Mol. Cell. Biol. 19(2), 1279 –1288. 24. Baumann, H., and Gauldie, J. (1990) Mol. Biol. Med. 7(2), 147– 159. 25. Rienhoff, H. Y., Jr., Huang, J. H., Li, X. X., and Liao, W. S. (1990) Mol. Biol. Med. 7(3), 287–298. 26. Pannen, B. H., and Robotham, J. L. (1995) New Horiz. 3(2), 183–197. 27. Moshage, H. (1997) J. Pathol. 181(3), 257–266. 28. Lowell, C. A., Stearman, R. S., and Morrow, J. F. (1986) J. Biol. Chem. 261(18), 8453– 8461. 29. Edbrooke, M. R., Burt, D. W., Cheshire, J. K., and Woo, P. (1989) Mol. Cell. Biol. 9(5), 1908 –1916. 30. Li, X. X., and Liao, W. S. (1991) J. Biol. Chem. 266(23), 15192– 15201. 31. de Beer, M. C., Kindy, M. S., Lane, W. S., and de Beer, F. C. (1994) J. Biol. Chem. 269(6), 4661– 4667. 32. Butler, A., and Whitehead, A. S. (1996) Immunogenetics 44(6), 468 – 474. 33. Lowell, C. A., Potter, D. A., Stearman, R. S., and Morrow, J. F. (1986) J. Biol. Chem. 261(18), 8442– 8452.
402
Vol. 264, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
34. Meek, R. L., and Benditt, E. P. (1986) J. Exp. Med. 164(6), 2006 –2017. 35. Meek, R. L., Eriksen, N., and Benditt, E. P. (1989) Am. J. Pathol. 135(2), 411– 419. 36. Meek, R. L., Eriksen, N., and Benditt, E. P. (1992) Proc. Natl. Acad. Sci. USA 89(17), 7949 –7952. 37. Li, X., and Liao, W. S. (1992) Nucleic Acids Res. 20(18), 4765– 4772. 38. Lu, S. Y., Rodriguez, M., and Liao, W. S. (1994) Mol. Cell. Biol. 14(9), 6253– 6263. 39. Huang, J. H., Rienhoff, H. Y., Jr., and Liao, W. S. (1990) Mol. Cell. Biol. 10(7), 3619 –3625. 40. Kawai, S., and Nishizawa, M. (1984) Mol. Cell. Biol. 4(6), 1172– 1174. 41. Huang, J. H., and Liao, W. S. (1994) Mol. Cell. Biol. 14(7), 4475– 4484. 42. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 43. Cato, A. C., Miksicek, R., Schutz, G., Arnemann, J., and Beato, M. (1986) EMBO J. 5(9), 2237–2240. 44. Gorman, C., Padmanabhan, R., and Howard, B. H. (1983) Science 221(4610), 551–553. 45. Shapiro, D. J., Sharp, P. A., Wahli, W. W., and Keller, M. J. (1988) DNA 7(1), 47–55.
46. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11(5), 1475–1489. 47. Gehring, W. J. (1996) Genes Cells 1(1), 11–15. 48. Chen, T. T., Wu, R. L., Castro-Munozledo, F., and Sun, T. T. (1997) Mol. Cell. Biol. 17(6), 3056 –3064. 49. Chong, J. A., Tapia-Ramirez, J., Kim, S., Toledo-Aral, J. J., Zheng, Y., Boutros, M. C., Altshuller, Y. M., Frohman, M. A., Kraner, S. D., and Mandel, G. (1995) Cell 80(6), 949 –957. 50. Chin, A. C., and Fournier, R. E. (1986) Ann. N.Y. Acad. Sci. 478, 120 –130. 51. Killary, A. M., and Fournier, R. E. (1984) Cell 38(2), 523–534. 52. Gourdeau, H., Peterson, T. C., and Fournier, R. E. (1989) Mol. Cell. Biol. 9(5), 1813–1822. 53. Lem, J., Chin, A. C., Thayer, M. J., Leach, R. J., and Fournier, R. E. (1988) Proc. Natl. Acad. Sci. USA 85(19), 7302–7306. 54. Boshart, M., Weih, F., Schmidt, A., Fournier, R. E., and Schutz, G. (1990) Cell 61(5), 905–916. 55. Chin, A. C., and Fournier, R. E. (1989) Mol. Cell. Biol. 9(9), 3736 –3743. 56. Herschbach, B. M., and Johnson, A. D. (1993) Annu. Rev. Cell Biol. 9, 479 –509. 57. Johnson, A. D. (1995) Cell 81(5), 655– 658.
403