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J Biol Chem, Vol. 273, Issue 52, 35208-35215, December 25, 1998


Transcriptional Up-regulation of the Delayed Early Gene HRS/SRp40 during Liver Regeneration
INTERACTIONS AMONG YY1, GA-BINDING PROTEINS, AND MITOGENIC SIGNALS*

Keyong Du, Julia I. Leu, Yong Peng, and Rebecca TaubDagger

From the Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Arg-Ser-rich domain-containing proteins (SR proteins), a family of splicing factors, can regulate pre-mRNA alternative splicing in a concentration dependent manner. Thus, the relative expression of various SR proteins may play an important role in alternative splicing regulation. HRS/SRp40, an SR protein and delayed early gene in liver regeneration, can mediate alternative splicing of fibronectin mRNA. Here we determined that transcription of the HRS/SRp40 gene is induced about 5-fold during liver regeneration, similar to the level of steady-state mRNA. We found that both mouse and human HRS promoters lack TATA and CAAT boxes. The mouse promoter region from -130 to -18, which contains highly conserved GA-binding protein (GABP) and YY1 binding sites, conferred high transcriptional activity. While GABPalpha /GABPbeta heterodimer transactivated the HRS promoter, YY1 functioned as a repressor. During liver regeneration, the relative amount of GABPalpha /GABPbeta heterodimer increased 3-fold, and YY1 changed little, which could partially account for the increase in HRS gene transcription. Interleukin-6, a critical mitogenic component of liver regeneration, was able to relieve the repressive activity of the YY1 site within the HRS promoter. The combined effect of small changes in the level of existing transcription factors and mitogenic signals may explain the transcriptional activation of the HRS gene during cell growth.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SR proteins1 are a conserved family of splicing factors that function both as alternative splicing regulators and basal splicing factors (1, 2). At present, eight SR proteins have been identified and characterized (2). All of these proteins share two common characteristics: one or two RNA binding domains at the amino terminus and one Arg-Ser-rich domain at the carboxyl terminus. As alternative splicing regulators, SR proteins have been shown to regulate various forms of alternative splicing including exon inclusion, alternative 5' splicing site selection, and alternative 3' splicing site selection in both in vitro and in vivo assays (3-13).

In our initial studies of hepatic growth, we isolated the HRS/SRp40 (hereafter referred to as HRS) cDNA, which encodes an SR protein and is a highly induced immediate early gene in insulin-treated rat hepatoma H35 cells (14). HRS mRNA is induced as a delayed early gene during liver regeneration with peak expression in rat liver at 6-8 h and approximately 5-fold induction (14). Furthermore, HRS mRNA expression is high in developing liver, especially prior to birth, when it is elevated 100-fold relative to normal adult liver (15). We showed that HRS expression correlates with the expression of EIIIB+ fibronectin pre-mRNA in proliferating liver. HRS can directly bind to a purine-rich enhancer in the fibronectin EIIIB exon and mediate fibronectin EIIIB exon inclusion in in vivo splicing assays (15). Changes in the level of fibronectin isoforms could play a critical role in reorganizing liver architecture during liver regeneration and development.

Distinct patterns of SR protein expression during development, cell differentiation, and cell proliferation determine alternative splicing decisions (15, 16). The expression of SR genes has been shown to be regulated at the transcriptional level (1, 17). However, the mechanism of transcriptional regulation of SR genes remains largely unknown. To understand how HRS gene expression is regulated, we analyzed the mouse HRS promoter. We found that the region from -130 to -18 of the HRS promoter contained high promoter activity in HepG2 cells. We further demonstrated that GABPalpha /GABPbeta heterodimer and YY1 regulate HRS gene expression positively and negatively, respectively. Whereas the level of YY1 changes little during liver regeneration, the relative level of GABPalpha /beta heterodimer increases. Interleukin-6, which confers a positive growth stimulus in liver regeneration (18), can relieve YY1 repression. The combination of these effects provides a potential mechanism for HRS gene induction during liver regeneration.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rat Tissue Preparation-- For regenerating liver, female Fisher rats (160-220 g, Charles River) or normal C57Bl6 mice were metofane (Pitman-Moore, Inc.)-anesthetized and subjected to midventral laparotomy with approximately 70% liver (left lateral and median lobes) (19).

Cell Culture and Transient Transfections-- Human hepatoma HepG2 and mouse fibroblast NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 units of penicillin and 50 units of streptomycin (Life Technologies Inc.). The transient transfection was performed by the calcium phosphate method as described (20). Cells were plated on 60-mm plates at a density of 2 × 105 cells/plate and transfected with the indicated amount of reporter plasmids and expression plasmids along with 2 µg of pRSV-beta -galactosidase for normalization of transfection efficiency. The DNA precipitate was incubated with the cells overnight (usually 14-16 h). The cells were harvested with 1× Reporter Lysis Buffer (Promega), and the luciferase assay was performed on a Luminat luminometer (Wallace Inc.). IL-6 was added at 50 ng/ml for 4 h where indicated.

Nuclear Run-on Assays-- Nuclei were prepared as reported previously (21). Nuclear run-on assays were carried out by following the method described (22). We used beta 2-microglobulin as a positive control and Bluescript SK(-) as negative control.

RNA, Northern Blot, and RNase Protection Assays-- The RNase protection assay was carried out by using the RPA II kit from Ambion. To prepare the riboprobe, the EcoRI-TthIII fragment, which covers the first exon and putative 700 bp of 5'-flanking sequence, was cloned into the pGEM4 vector, linearized with EcoRI, and in vitro transcribed with T7 RNA polymerase by adding [32P]UTP. The labeled probe was purified on urea-denatured 5% polyacrylamide gel. 2 × 104 cpm was hybridized with 10 µg of total RNA from NIH 3T3 cells. Primer extensions analyses were performed as we have described (23) using a primer, 5'-GTAGTCTGCGACGGAGCTGTCTGCCT-3', from the first exon region of the mouse gene and mouse regenerating liver RNA.

Immunoblots-- The total liver nuclei were prepared as described (15). Briefly, the liver nuclei were resuspended in 1× SDS/sample buffer (62.5 mM Tris, pH 6.8, 0.1% SDS, 72 mM 2-mercaptoethanol, and 10% glycerol, without bromphenol blue). The protein concentration was determined by A280. For immunoblots, 10 µg of total nuclear proteins in each lane were separated on 8% SDS-polyacrylamide gel. The proteins were transferred onto a nitrocellulose membrane and probed with 1:1000 diluted antibodies. Horse rabbit peroxidase-conjugated goat anti-rabbit immunoglobulin secondary antibody and a chemiluminescence detection system (Amersham Corp.) were used.

Plasmid Constructs-- To clone the mouse HRS promoter into pGL2-basic, the 1.5-kb fragment between -1.5 kb and +100 bp was amplified by PCR with primer 5'- CGGCTGAGATCTGGAGAGGTCCG-3 and Sp6 primer from pG7-6 (24). The amplified fragment was filled in with T4 DNA polymerase and digested with BglII. The resulting DNA fragment was cloned into pGL2-basic between the BglII and NheI (blunted) sites. To construct pGL-0.4 and -0.8, the amplified fragment was digested with BglII and NheI or digested with EcoRI first and then filled in and digested with BglII. To construct pGL-r0.8, the BglII/EcoRI-blunted fragment was ligated into pGL2-basic BglII/HindIII-blunted sites. To construct pGEM-0.8, the amplified fragment was digested with EcoRI and BglII and cloned into the EcoRI and BamHI sites of pGEM4. The plasmid pGL-5.3 was constructed as follows. Mouse genomic DNA from the HRS gene phage clone was digested with EcoRI. The 5'-flanking 4.6-kb EcoRI fragment was cloned into pGEM4. The 5' EcoRI site was then blunted, and the resulting blunted EcoRI fragment was cloned into the KpnI (blunted) and EcoRI site in pGL-1.5. To construct pGL-2.7, pGL-5.3 was digested with SmaI and KpnI, and the KpnI site was blunted. Then the resulting 8.7-kb fragment was religated. Further deletions of pGL-0.4 were made using Bal-31 nuclease. SstI-linearized pGL-0.4 was incubated with Bal-31 nuclease at 30 °C. The reaction was stopped at 3-min intervals and then digested with BglII. The resulting DNA fragments were separated on an 8% native polyacrylamide gel, and DNA fragments were eluted into 0.5 M NH4Ac and 1 mM EDTA. After ethanol precipitation, the deletion fragments were cloned into pGL-basic between BlgII and NheI (blunted) sites.

Site-directed mutagenesis at GABP and YY1 sites was also performed by overlapping extension PCR. All of the mutations were made in pGL-0.1 (Fig. 2). The oligonucleotides were as follows: for mutating the YY1 site, mYY1 (5'-AGGACGTCCTTAAATTTTGTGCAACG-3') and mYY2 (5'-ACGTTGCACAAAATTTAAGGACGTCC-3'); for mutating the GABP site, GA-M1 (5'- ACCGCCCTGCAGTGACGCCAGG-3') and GA-M2 (5'-TCCTGGCGTCACTGCAGGGCGG-3'); and constant primers, HRSBgl (5'-CGGCTGAGATCTGGAGAGGTCCG-3') and pGLp1 (5'-TGTATCTTATGGTACTGGTACTGTAACTG-3').

pGL-0.1 was first amplified with primers mYY1/HRSBgl or mYY2/pGLp1 and GA-M1/HRS40Bgl or GA-M2/pGLp1. The resultant PCR products were purified and mixed and further amplified with pGLp1 and HRSBgl. The final PCR products were digested with MluI-BglII and cloned into pGL-basic between MluI and BglII sites. All of the mutations were verified by DNA sequence analysis.

Preparation of Nuclear Extracts-- HepG2 cells were grown as described above, and about 1-2 × 108 cells were used to prepare nuclear extract exactly as described by Kramer (25). Briefly, HepG2 cells were collected in 25 ml of cold PBS and pelleted. After washing with 1 ml of buffer A (10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol), the cells were resuspended into 0.5 ml of buffer A. The cells were lysed with a 1-ml syringe with a 25-gauge × <FR><NU>5</NU><DE>8</DE></FR>-inch hypodermic needle. The nuclei were resuspended into 0.2 ml of buffer C (20 mM HEPES-KOH (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepsin). The nuclear extracts were dialyzed against 400 ml of buffer D (20 mM HEPES-KOH (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol). The liver nuclear extract was prepared as reported (18). Briefly, the liver nuclei were suspended into 0.5 ml of buffer C, and the nuclear extracts were dialyzed against buffer D. The protein concentration of nuclear extracts was determined by a method from Bio-Rad. The nuclear extract was divided into 50-µl aliquots and then stored at -80 °C.

Gel Mobility Shift Assays-- EMSAs were performed with nuclear extract from HepG2 cells or rat liver (26). Typically, 5 µg of nuclear extracts were incubated with 2 µg of poly(dI-dC), 104 cpm of labeled probe in 12 mM HEPES, pH 7.6, 12% glycerol, 4 mM Tris, 60 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol in a 20-µl volume at room temperature for 20 min. For competition experiments, a 50-fold excess of cold oligonucleotides was added to the reaction mixture. For supershift, 1 µl of the indicated antibodies were added to the reaction mixture. The DNA-protein complex was separated on 5% native polyacrylamide gel in 0.5× TBE at 4 °C. Anti-YY1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-GABPalpha and GABPbeta antibodies were gifts from Steven McKnight (University of Texas Southwestern, Dallas, TX).

The sequences of double-stranded oligonucleotides were as follows: wild type YY1, 5'-AGGACGTCCTTCCATTTTGTGCAACG-3'; mutant YY1, 5'-AGGACGTCCTTAAATTTTGTGCAACG-3'; wild type GABP, 5'-ACCGCCCCGGAAGTGACGCCAGG-3'; mutant GABP, 5'-ACCGCCAACGAAGTGACGCCAGG-3'.

The oligonucleotides were synthesized by Life Technologies, Inc. The two complementary oligonucleotides were annealed and purified through 12% native polyacrylamide gel and then labeled with 32P by T4 nucleotide kinase.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transcriptional Control of HRS Gene Expression-- HRS mRNA is induced during liver regeneration with peak expression at 6-8 h in rat liver and approximately 5-fold induction (14, 15). Since RNA abundance may be controlled by both transcriptional and posttranscriptional mechanisms, the transcriptional activity of the HRS gene was assessed using nuclear run-on assays (Fig. 1). Transcription of HRS was increased at 2 and 6 h posthepatectomy (about 5-fold) and not increased at 30 min, consistent with the delayed early induction of HRS (14). As a control, the transcription of beta 2-microglobulin did not change appreciably during this time period, whereas that of the IGFBP-1 gene, an immediate early gene in liver regeneration, was increased about 14-fold at 30 min, consistent with earlier studies (22).


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  		Fig. 1.   Transcriptional regulation of HRS expression in regenerating liver. A nuclear run-on assay was performed on nuclei from regenerating rat liver. Bluescript and beta 2-microglobulin were used as negative and positive control, respectively. Times after hepatectomy are indicated. Time 0 is normal liver.

Lack of TATA and CAAT Boxes in the HRS Promoter-- RNase protection and primer extension assays were used to map the transcription initiation site of the mouse HRS gene (Fig. 2A), which is indicated as +1 (Fig. 2B). We sequenced a 1.5-kb upstream region of the predicted transcription initiation site in both the human and mouse HRS genes (GenBankTM accession numbers are AF020307 (human HRS gene) and AF020308 (mouse HRS gene)). Based on the location of the RNA initiation site, neither human nor mouse HRS promoters appear to contain TATA or CAAT boxes. With extensive alignment analysis between the human and mouse HRS genes, two regions of strong homology were found upstream of the RNA initiation site and not in more 5' upstream regions (Fig. 2B). Two regions of identity contained a putative YY1 binding site (27) and an EFT4 or GABP binding site (28, 29). Additionally, there were two identical regions between -18 and +44, and there are putative GABP and Sp1 sites in the mouse promoter that were incompletely conserved in the human gene.


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  		Fig. 2.   Localization of HRS gene RNA initiation site and mouse and human HRS promoter region sequence. A, left, RNase protection assay to map the HRS gene transcription initiation site. The RNase protection probe is indicated at the bottom, and described under "Materials and Methods." The sequencing ladder shown is of HRS-LF rat cDNA primed with GATTACGTCTAGACCGGC (24). The RNA used was from NIH 3T3 cells, and yeast tRNA served as control. Right, primer extension analysis of RNA from regenerating mouse liver at indicated times posthepatectomy as described under "Materials and Methods." In mouse liver, peak expression of the HRS gene extends to 16 h posthepatectomy (41). The sequencing ladder is M13mp18 with universal primer. B, alignment of human and mouse HRS promoters. The conserved GABP and YY1 binding sites are boxed, and the nonconserved Sp1 and reverse GABP sites are indicated by hatched boxes.

The Region of the HRS Promoter between -130 and -18 bp Demonstrates High Transcriptional Activity in HepG2 Cells-- We chose HepG2 cells to further analyze the transcriptional control of the HRS gene, because HRS is highly expressed in HepG2 cells, and HepG2 cells represent a fetal liver phenotype (Fig. 3A). To test the ability of the predicted region to function as a promoter, we first cloned the 0.7 kb of 5'-flanking sequence of the HRS gene which included part of the first exon into a pGL2-basic luciferase reporter vector in both sense and antisense orientations (Fig. 3B). As shown (Fig. 3B), only sense pGL-0.8kb showed high luciferase activity, indicating that this region contains the HRS promoter and that the HRS promoter is orientation-dependent (comparing pGL-0.8 and pGL-r0.8). To further examine HRS promoter activity, we cloned up to 5 kb of 5'-flanking sequence and performed a series of deletion mutations. There was no significant change in promoter activity when the deletion extended from -5.3 kb to -130 bp, suggesting that there are no major regulatory element(s) in this region, at least in HepG2 cells. When the deletion was extended to -60 bp, the promoter activity dropped about 2.5-fold, which could be explained by loss of the upstream antisense GABP site and predicted Sp1 site (see Fig. 2B). Further deletion (to -56 bp) virtually abolished HRS promoter activity. These data predict that the sequences between -130 and -18 bp of the HRS promoter contain critical regulatory element(s), consistent with the high sequence homology of this region. Using these deletion constructs in transiently transfected mouse NIH 3T3 fibroblasts, similar results were obtained, implying that this regulation was not limited to liver cells (data not shown).


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  		Fig. 3.   5' to 3' deletion analysis of HRS promoter activity. A, Northern blot analysis of HRS gene expression showing relative expression in HepG2, H35, and normal liver. 32P-Labeled rat HRS--SF was used as probe. Hybridization was under high stringency. B, deletion analysis of the HRS promoter activity in HepG2 cells. The plasmids were constructed as described under "Materials and Methods." 2 µg of luciferase constructs were transiently transfected by the calcium phosphate precipitation method along with 2 µg of pRSV-beta -galactosidase construct as an internal control for transfection efficiency. Luciferase activity was normalized to beta -galactosidase activity in the same transfection. The luciferase activity is presented as relative percentage, which that of pGL-5.3 set as 100. The value is from at least three separate transfections. The error bars represent the S.D. pGL-B, pGL2-Basic; pGL-C, pGL2-Control (SV40).

Regulation of HRS Promoter by YY1 and GABP-- The -130/+100 promoter construct (pGL-0.1) exhibited significant promoter activity in transfected cells. Sequence analysis revealed conserved YY1 and GABP binding sites in this region of the HRS promoter. We analyzed these two binding sites in further detail. YY1 is a zinc finger transcription factor that was initially identified as a transcriptional repressor of the P5 promoter. YY1-mediated repression can be converted to activation in the presence of adenoviral protein E1A (30-32). Depending on the promoter context and cell type, YY1 can be either a transcriptional activator or repressor (33-35). To test how YY1 regulates the HRS promoter, the YY1 binding site was mutated, with the CC changed into AA (Fig. 4A). Previous studies of the P5 promoter showed that this mutation abolishes YY1 regulatory activity (27). This mutation increased HRS promoter activity about 3-fold, suggesting that the YY1 site confers transcriptional repression of the HRS promoter in HepG2 cells. Again, this effect was not cell type-specific in that the YY1 mutation increased reporter activity by about 10-fold in transfected NIH 3T3 cells (not shown).


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  		Fig. 4.   YY1 functions as a transcriptional repressor of the HRS promoter. A, mutated YY1 binding site increases HRS promoter activity. The mutation of the YY1 binding site and the sequence of oligonucleotides used in EMSAs are depicted. The positions of oligonucleotides are indicated relative to the mouse HRS promoter (transcription initiation site is designated as +1). Transfection and luciferase assays were performed as in Fig. 3. The data are shown as -fold activity relative to wild type promoter (pGL-0.1), which was set at 1. The data were from three different cotransfection experiments. B, EMSAs demonstrated that YY1 specifically binds to the YY1 binding site in the HRS promoter. Lane 1, no nuclear extract. In competition assays, binding reactions were performed with either a 50× molar excess of wild type oligonucleotide (lane 3) or mutant oligonucleotide (lane 4). In supershift assays, the binding reactions were carried out either with anti-YY1 antibody (lane 5) or anti-Sp1 antibody (lane 6). C, overexpression of YY1 represses the HRS promoter in HepG2 cells. The data are shown as percentage of control activity relative to each reporter cotransfected with pCMV vector, set at 100%. HepG2 cells were cotransfected with 1 µg of each reporter and different amounts of indicated expression vector (pCMV-YY1) or 2 µg of pCMV vector along with 2 µg of pRSV-beta -galactosidase as an internal control. D, overexpression of E1A activates the HRS promoter. The data are shown as -fold activity relative to the activity set at 1 of each reporter control when cotransfected with pCMV vector alone. For C and D, the data were from three different transfection assays with S.D. values shown.

To confirm that HepG2 nuclear extracts contain YY1, which is able to interact with the predicted YY1 binding site in the HRS promoter, we carried out EMSAs using HepG2 cell nuclear extracts and an oligonucleotide probe corresponding to the YY1 binding site in the HRS promoter. One specific DNA-protein binding complex was detected (Fig. 4B, lane 2). This complex could be competed off by excess cold wild type probe but not by mutant oligonucleotides (lanes 3 and 4). Anti-YY1 antibody, not anti-Sp1 antibody, partly supershifted but primarily disrupted this band (lanes 5 and 6). Taken together, these data suggested that YY1 specifically binds to the YY1 binding site in the HRS promoter and functions as a transcriptional repressor.

To further demonstrate the repression of the HRS promoter by YY1 in HepG2 cells, we cotransfected the HRS promoter construct (pGL-0.1) with a pCMV-YY1 expression plasmid to determine whether overexpression of YY1 can further repress the HRS promoter. Overexpression of YY1 efficiently repressed the HRS promoter construct (Fig. 4C). As a control, overexpression of YY1 did not affect pGL2-basic or pGL2-promoter luciferase activity. Furthermore, YY1-mediated repression was dependent on the presence of an intact YY1 binding site, because the activity of the pGL-0.1mYY1 reporter was not affected by cotransfection with pCMV-YY1. Since E1A can transform YY1 from a transcriptional repressor to an activator, we predicted that overexpression of E1A would activate the HRS promoter. As shown (Fig. 4D), overexpression of E1A activated the HRS promoter about 5-fold, and activation of the HRS promoter by E1A was abolished when the YY1 binding site was mutated. As a control, E1A did not affect pGL2-basic and pGL2-promoter luciferase activity. All of these data are consistent with the finding that YY1 functions as a repressor of the HRS promoter.

GABP is an ETS transcription factor initially isolated as a transcription factor that recognizes a site important for herpes simplex virus type I immediate early gene activation. GABP is composed of two subunits: alpha  and beta  (36, 37). GABPalpha , which contains the DNA binding domain, interacts with GABPbeta to form an active heterodimer (36-39). Initial deletion mutation studies showed that deleting part of the putative GABP binding site dramatically decreased HRS promoter activity (Fig. 3). To test the possible role of GABP in HRS promoter activity, we mutated the GABP binding site in the HRS promoter in plasmid pGL-0.1 and examined how this mutation affected HRS promoter activity in HepG2 cells. As shown (Fig. 5A), this mutation decreased basal HRS promoter activity by about 15-fold. To examine whether GABP from HepG2 nuclear extract bound to the predicted GABP site in the HRS promoter, EMSAs were performed with an oligonucleotide probe corresponding to the HRS GABP binding site (Fig. 5B). A major retarded band was detected (lane 2). This band could be competed off by wild type but not mutant oligonucleotides. In EMSA experiments performed with antibodies specific to GABPalpha and -beta as well as anti-Sp1 antibody, only anti-GABPalpha and -GABPbeta antibodies (lanes 5 and 6) disrupted the DNA binding complex. These data indicated that GABP bound to the site as a heterodimer.


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  		Fig. 5.   GABP functions as a transcriptional activator of the HRS promoter. A, mutation of the GABP binding site decreases HRS promoter activity. Site-directed mutation of the GABP binding site (mGABP) and the sequence of oligonucleotides used in EMSAs are shown. The position of oligonucleotides is indicated in the mouse HRS promoter (transcription initiation site is designated as +1). The data are shown relative to pGL0.1 reporter, which was set at 100. The data were from three different co-transfection experiments. B, EMSAs demonstrate that GABP specifically binds to the HRS GABP site. A radiolabeled HRS GABP site (A) was used as probe. The experiments were performed essentially as in Fig. 4. Antibodies were as follows: GABPbeta (beta ; lane 5); GABPalpha (alpha ; lane 6); Sp1 (lane 7). C, GABPalpha and beta  synergistically activate the HRS promoter. The data are shown as -fold activity relative to each reporter plus pCMV vector, which was set at 1. HepG2 cells were cotransfected with 1 µg of indicated reporter vector and either 1 µg of indicated GABP expression plasmids or the pCMV vector. The data were from three different cotransfection experiments with S.D. values shown.

Together pCMV-GABPalpha and -beta activated the HRS promoter up to 5-fold. GABPalpha alone slightly inhibited HRS promoter activity, and GABPbeta alone slightly activated the HRS promoter (Fig. 5C). These data are consistent with the previous finding that GABPalpha and -beta synergistically activate gene expression (38). The fact that GABPalpha alone slightly inhibited the HRS promoter while GABPbeta alone slightly activated the HRS promoter suggested that there is a limiting amount of GABPbeta in HepG2 cells.

The Expression of GABP and YY1 in Regenerating Liver-- HRS is induced as a delayed early gene with approximately 5-fold transcriptional induction during liver regeneration (Fig. 1). Since both GABP and YY1 regulate HRS gene expression, we wondered whether there were changes in GABP or YY1 levels during liver regeneration that might explain the increase in HRS gene transcription. We did not detect any change in the overall binding activity or the level of YY1 protein (Fig. 6, A and B). Of note, a smaller YY1 DNA binding complex was detected in EMSAs (Delta YY1), and a 23-kDa peptide was detected in immunoblots of regenerating liver nuclear extracts (data not shown). The Delta YY1 DNA binding complex is probably an experimental artifact, since inconsistent results were obtained from different regenerating nuclear extracts (data not shown), and a single undegraded YY1 peptide was detected in an immunoblot of total liver nuclear protein that had been immediately suspended in SDS loading buffer (Fig. 6B).


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  		Fig. 6.   Expression of YY1 and GABP during liver regeneration. A, YY1 binding activity in regenerating liver. The EMSAs were carried out with 5 µg of liver nuclear extracts at the indicated times posthepatectomy. B, immunoblot of regenerating liver total liver nuclei probed with an anti-YY1 antibody. C, GABP binding activity in regenerating liver at indicated times posthepatectomy (lanes 6-10). The EMSAs were performed with 5 µg of liver nuclear extracts. Antibody supershifts and competitions are as in Fig. 4. D, immunoblot of total liver nuclear proteins probed with a mixed anti-GABP antibody that detects both GABPalpha and -beta .

Two DNA binding complexes were detected in liver nuclear extracts when the GABP binding site was used as a probe (Fig. 6C). These two complexes corresponded to the GABPalpha /beta heterodimer and GABPalpha monomer (alpha :beta and alpha  in Fig. 6C), because both complexes were competed off by wild type oligonucleotides and the lower band supershifted only with anti-GABPalpha antibody (lanes 2 and 3 in Fig. 6C). The GABPalpha monomer band decreased during liver regeneration by about 3-fold. An immunoblot of nuclear proteins from regenerating liver was probed with a mixture of anti-GABPalpha and GABPbeta antibodies (Fig. 6D) indicated that an increase in the level of GABPbeta by 3-8 h posthepatectomy could explain the relative increase in the GABPalpha /beta heterodimer. As predicted by Fig. 5C, an increase in the ratio of GABPalpha /beta heterodimer to GABPalpha homodimer could explain at least some of the transcriptional up-regulation of the HRS gene.

The Effect of YY1, GABP, and C/EBPbeta on the Net Activation of the HRS Promoter-- Like E1A, C/EBPbeta may transform YY1 from a dominant repressor into a transcriptional activator (40). This activity is dependent on an interaction among C/EBPbeta , YY1, and the DNA binding site. We showed that C/EBPbeta protein expression increases in the regenerating liver in a time course that is consistent with the transcriptional activation of the HRS gene (21) and have found a decrease in HRS expression in CEBPbeta -/- livers posthepatectomy (41). However, little direct effect of C/EBPbeta on HRS promoter constructs was observed in transfected HepG2 cells. In transfected NIH 3T3 cells, C/EBPbeta was partially able to relieve YY1-mediated repression (not shown). The complex posttranscriptional regulation of C/EBPbeta in HepG2 cells may explain the inability to demonstrate a positive effect of CEBPbeta on HRS promoter reporters (42).

IL-6 mediates an early signal that is required for normal liver regeneration (18), and IL-6 is able to activate intracellular signals via both STAT and mitogen-activated protein kinase pathways. HRS mRNA expression is reduced by about 2-fold from 4-16 h posthepatectomy in IL-6-/- livers (not shown). IL-6 treatment of transfected HepG2 cells eliminated YY1-mediated repression, and this effect was dependent on an intact YY1 element (Fig. 7).


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  		Fig. 7.   Effects of YY1 and IL-6 in the regulation of the HRS promoter. HepG2 cells were cotransfected with 1 µg of either pGL-0.1 or pGL-0.1mYY1 as indicated. The value of the pGL-0.1 reporter was set at 1 in one of the duplicate samples in two separate experiments, and S.D. values are shown. IL-6 was added as described under "Materials and Methods" for 4 h prior to harvesting. Normalization was to beta -galactosidase.


    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The region from -130 to -18 bp of the HRS promoter had high transcriptional activity. Deletion of the region from -130 to -60 bp reduced promoter activity by 2.5-fold and could be explained by the loss of an antisense GABP element at -130 and Sp1 element adjacent to the -59 GABP site. Sp1 has been found to potentiate GABP transcription (43). Two conserved transcription factor binding sites in this region, YY1 and GABP, strongly regulated HRS gene expression, YY1 acting as a repressor and GABP as an activator.

GABP specifically bound the GABP binding site within the HRS promoter and transactivated the HRS promoter. Our data support previous reports that GABPalpha and -beta synergistically transactivate gene expression. GABP belongs to the ETS family of transcription factors, which bind to the consensus sequence (CGGAAGTG), an important element in the polyoma virus enhancer and viral late gene initiator element (29). GABP has been shown to be important in the expression of several genes such as the IL-2 receptor beta -chain gene (44), cytochrome c oxidase Vb (45, 46), and gamma -chain gene (47). In all these genes, the GABP binding site is found close to the transcription initiation sites, which lack TATA boxes. In this promoter context, as postulated, GABP may function as both a transcription activator and a basal transcription factor (48). Most SR proteins have been shown to be highly expressed in spleen and thymus (49). Coincidentally, GABP has been implicated in activating genes such as CD18 (48, 50) and T cell receptor beta  (51) and is necessary for thymus-specific expression of the gamma -chain promoter (47). It has been reported that both SC35 and 9G8 promoters contain putative GABP binding sites (52, 53). Therefore, GABP could function as a common transcription factor to enforce the high expression of SR proteins in hematopoietic tissues. However, unlike the SC35 and 9G8 SR protein promoters, which have typical TATA boxes, the HRS promoter lacks both TATA and CAAT boxes (52, 53). Distinct differences in the regulatory sequences controlling the HRS, SC35, and 9G8 genes provide a basis for differential expression.

At least in some cells (HepG2, NIH 3T3), YY1 functions as a transcriptional repressor of the HRS promoter. Mutation of the YY1 binding site in the HRS promoter increased HRS promoter activity, and cotransfection of a YY1 expression vector with the HRS promoter reduced HRS promoter activity. YY1-mediated repression was reversed by oncoprotein E1A via an intact YY1 site. After adenovirus infection, viral proteins are produced largely due to alternative splicing of viral transcripts. It is likely that changes in relative expression of SR proteins mediate viral pre-mRNA alternative splicing (7, 54). Coincidentally, SV40 T antigen has similar activity (55), and it is possible that T antigen could also activate HRS expression. Of note, HRS protein expression is elevated in SV40-transformed human WI38 fibroblasts (56).

Since there is an excess amount of GABPalpha subunit in liver that acts as a negative transcriptional regulator, we predicted that increased GABPbeta expression would lead to increased HRS gene expression during liver regeneration. In fact, the GABPbeta expression increased only slightly but sufficiently to increase the relative level of GABPalpha /beta heterodimer by 3-fold, eliminating most of the GABPalpha homodimer. We did not detect a change in the YY1 level during liver regeneration. Therefore, the changes in levels of GABP and YY1 appeared to explain some, but not all, of the up-regulation of the HRS gene during liver regeneration.

YY1 can interact with different cellular proteins, and its repression or activation function appears to be modulated by specific protein-protein interactions (57, 58). The activity of YY1 may be modulated by growth factor action, cytokines, and other types of cellular induction such as viral infection. For example, the interaction between YY1 and CREB is required by YY1 to repress the c-fos promoter (59), and the interaction between YY1 and CEBPbeta is required to activate human papillomavirus type 18 promoter (40). The conversion of YY1 from repressor to activator by E1A appears to be dependent on a member of the coactivator family, p300/CBP (31, 60, 61). Both E1A and YY1 bind to p300 at different sites, and it is postulated that the addition of E1A to the p300-YY1 complex masks the repressor domain of YY1. p300/CBP can interact with various cellular and viral proteins such as CREB (62), p53 (63), pp90RSK (64), E1A (31, 65), T antigen (55), and the C/EBPs (66), many of which also interact with YY1.

IL-6 confers a positive mitogenic signal during liver regeneration and is able to relieve YY1 site-mediated repression of the HRS promoter. It is possible that intracellular signals generated by IL-6 and other growth factors that play a role in liver regeneration allow YY1 to interact with other transcription factors and coactivators converting YY1 into a activating factor or at least eliminate its negative effects. Immediate early genes show dramatic increases in transcription posthepatectomy, which are mediated by major changes in signal-induced transcription by serum response factor, AP-1, STAT3, and other transcription factors, which are themselves increased severalfold (67). However, as with the HRS gene, the increase in transcription of delayed early genes is only a few fold and results from small changes in the levels of preexisting transcription factors, combined with posttranslational effects of growth factor-generated signals.

    ACKNOWLEDGEMENTS

We thank S. McKnight for GABP expression vector and antibodies and Mike Atchison for the YY1 expression plasmid.

    FOOTNOTES

* This work was supported in part by a Juvenile Diabetes Foundation Award and National Institutes of Health Grants R01 DK44237 and P50 DK49210 (to R. T.).

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF020307 and AF020308.

Dagger To whom correspondence should be addressed: Dept. of Genetics, University of Pennsylvania School of Medicine, 705a Stellar-Chance, 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-9131; Fax: 215-573-2195; E-mail: taubra{at}mail.med.upenn.edu.

The abbreviations used are: SR, Arg-Ser-rich domain-containing protein; HRS and HRS, hepatic Arg-Ser protein and gene, respectively; PCR, polymerase chain reaction; EMSA, eletrophoretic gel mobility shift assay; GABP, GA-binding protein; C/EBP, CAAT enhancer-binding protein; IL, interleukin; bp, base pair(s); kb, kilobase pair(s).
    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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