The Gene Encoding Human Nuclear Protein Tyrosine Phosphatase, PRL-1
CLONING, CHROMOSOMAL LOCALIZATION, AND IDENTIFICATION OF AN INTRON ENHANCER*

Yong PengDagger , Anna GeninDagger §, Nancy B. SpinnerDagger §, Robert H. Diamond, and Rebecca TaubDagger parallel

From the Dagger  Department of Genetics and the § Department of Pediatrics, Division of Genetics, Children's Hospital of Philadelphia and the  Department of Medicine and Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of the rat PRL-1 gene, which encodes a unique nuclear protein tyrosine phosphatase, is positively associated with cellular growth during liver development, regeneration, and oncogenesis but with differentiation in intestine and other tissues. Here, we analyzed the structure of the human PRL-1 gene and localized it to chromosome 6 within band q12. Human, rat, and mouse PRL-1 are 100% conserved at the amino acid level and 55% identical to a newly identified Caenorhabditis elegans PRL-1. The presence of two promoter activities, P1 and P2, in the human PRL-1 gene were identified by primer extension and RNase protection assays. A functional TATA box was identified in promoter P1 upstream of the non-coding first exon. A non-canonical internal promoter, P2, was found in the first intron that results in PRL-1 transcripts beginning 8 base pairs downstream of the 5'-end of exon 2 and causes no alteration in the encoded protein. The first 200-base pair region of either promoter P1 or P2 conferred high basal transcriptional activity. An enhancer that bound a developmentally regulated factor, PRL-1 intron enhancer complex (PIEC), was localized to the first intron of the human PRL-1 gene. The presence of PIEC correlated with the ability of the intron enhancer to confer transcriptional activation in HepG2 and F9 cells. The intron enhancer contributed significantly to PRL-1 promoter activity in HepG2 cells which contain PIEC but not to NIH 3T3 cells which do not.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein tyrosine kinases and phosphatases play important roles in the regulation of cell growth, development, and differentiation. One of the most interesting immediate early or primary growth response genes first identified in regenerating liver encodes a novel 20-kDa nuclear protein tyrosine phosphatase, PTPase,1 PRL-1 (phosphatase of regenerating liver). Other than the signature sequence for PTPases, PRL-1 is not homologous to either the dual specificity PTPases (cdc25 and MKP-1) or monospecific PTPases (1, 2). A PRL-1-like protein, OV-1, was isolated from human cells (3) and found to be 95% identical to rat PRL-1 at the amino acid level but is not the human PRL-1 homologue. Human PRL-1 (CAAX-1) and human OV-1 (CAAX-2) cDNAs were isolated as encoding proteins containing isoprenylation sites (4). The finding of at least two members of this PTPase family, PRL-1 and OV-1, with a high degree of conservation suggests that PRL-1 and family members have important cellular functions.

PRL-1 mRNA is elevated throughout the major proliferative phase of liver regeneration when hepatocytes and nonparenchymal cells in the liver are rapidly proliferating. PRL-1 is expressed at high levels in other proliferating cells including fetal liver and a number of tumor cell lines such as hepatomas (1). PRL-1-transfected cells showed altered growth characteristics, including a faster doubling time, growth to a greater saturation density, altered morphology, and evidence of anchorage-independent growth. Overexpression of human PRL-1 and OV-1 in epithelial cells resulted in tumor formation in nude mice (4). Taken together, these results suggest a positive role for PRL-1 in the growth response.

However, in addition to regenerating liver, PRL-1 is expressed at significant levels in brain and skeletal muscle, two terminally differentiated tissues that are notable for their absence of ongoing cellular proliferation. In intestinal epithelia which contain both terminally differentiated and proliferating cells, PRL-1 is expressed in the terminally differentiated villus but not proliferating crypt enterocytes (5).

The correlation of PRL-1 expression with growth in some cellular systems and differentiation in others suggests that PRL-1 may have different roles depending on the cell type. Because of the interesting pattern of regulation of this gene during cellular growth and differentiation and its association with cellular transformation, we cloned and characterized the human PRL-1 gene and determined its chromosomal localization. Moreover, we performed an analysis of the regulatory regions of the human PRL-1 gene that are necessary for its expression.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of Human PRL Genomic DNA-- A human lymphocyte genomic library established in lambda -FIX II (gift from E. Rand) was used to isolate human PRL-1 gene clones. Approximately 1 × 106 plaques were screened with a 243-bp EcoRI/SstI fragment derived from the 5'-end of rat PRL-1 cDNA using high stringency conditions. Several positive isolates proved to contain intronless PRL-1 genes and were believed to be pseudogenes. Two intron-containing positive phage clones were further characterized by restriction endonuclease mapping and Southern blotting. SstI fragments from the positive phage clone, hprl1, were subcloned into the vector Bluescript KS(+) and denoted pPRL/SstI, pPRL/Sst2, etc. 12 kb of continuous sequence was determined using ABI automated cycle sequencer with Stretch upgrade (DNA Sequencing Core, Department of Genetics, University of Pennsylvania School of Medicine) and was analyzed with Intelligenetics (Intelligenetics, Inc., Mountain View, CA) and GCG sequence analysis software (Genetics Computer Group, Inc., Madison, WI).

Chromosomal Localization-- To determine the chromosomal localization of the human PRL-1, the 17.5-kb genomic DNA clone hprl1 was used for fluorescence in situ hybridization. The probe was labeled by nick translation with biotin-11-dUTP and hybridized to human metaphase chromosomes. After fluorescence in situ hybridization, the slide was destained and G-banded for chromosomal identification (6).

RNA and RNA Analysis-- RNAs were prepared exactly as reported using guanidinium isothiocyanate extraction (7, 8). Primer extension was performed following the reported protocols (9) with minor modifications. For P1 a 22-nucleotide antisense primer, corresponding to bases +57-78 relative to the transcription start site (+1), and for P2 a 25-bp antisense primer from +3542-3567 were used. Primers were end-labeled with T4 polynucleotide kinase and [gamma -32P]ATP and purified by Sephadex G-25 spin column. 50 µg of human HepG2 total cellular RNA was hybridized with the radiolabeled primer (105 cpm) by heating at 80 °C for 10 min and then cooled to room temperature in a solution containing 50 mM Tris·HCl, pH 8.3, at 42 °C, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM dNTP mixture, and 0.5 mM spermidine. Then 2 units of avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc.) were added to the mixture, and the reaction was incubated at 42 °C for 60 min. Following phenol/chloroform extraction, and ethanol precipitation with 50 µg/ml tRNA, the primer-extended products were analyzed on 6% polyacrylamide gel containing 8 M urea. Sequencing reactions were performed using Sequenase 2.0 according to the manufacturer's protocol (U. S. Biochemical Corp.).

Ribonuclease Protection Assay-- The antisense RNA probe was generated from an EcoRI-linearized Bluescript KS plasmid template (plasmid pP2-B) containing sequence 3269-3567 of human PRL genomic DNA by transcription with T7 RNA polymerase in the presence of [alpha -32P]UTP using an in vitro transcription kit (Stratagene, La Jolla, CA). The probe used contained 130 bp of exon 2 and 169 bp of intron 1 (see Fig. 3). 50-70 µg of total HepG2 RNA was used for the assay. After overnight hybridization of 0.5-1 × 106 cpm of probe to the RNA, the hybrids were digested according to the described protocol (9). The protected fragments were then analyzed on 6% polyacrylamide gel containing 8 M urea.

Human PRL-1 Gene/Luciferase Fusion Plasmid Construction-- Chimeric human promoter-luciferase plasmids were constructed by cloning various lengths of 5'-flanking sequence and intron sequence from the PRL-1 gene into the polylinker region of the luciferase reporter plasmids pGL2-Basic or pGL2-SV40-promoter (Promega). Unless indicated, the nomenclature used for the constructs is based upon the succession of 5' restriction sites within the fragments. The plasmid pP1-BglS was constructed by ligating the 2219-bp (BglII-BglI blunted) fragment from plasmid pPRL/SstI, which contains 6.1 kb of the 5'-flanking region and 818 bp of the PRL-1 gene, into the BglII and HindIII (blunted) sites of the vector pGL2-Basic. A series of 5'-end deletion mutant plasmids were then generated from the plasmid pP1-BglS (see Fig. 4A) by use of various convenient restriction endonuclease sites. For construction of the plasmid pP1-KpnS, pP1-Hind, pP1-Pst, and pP1-Sma, the plasmid pP1-BglS was digested with KpnI, KpnI/HindIII, KpnI/PstI, SmaI, respectively, and blunted by incubating with T4 DNA polymerase and dNTP, and then the fragments were self-ligated to generate corresponding plasmids. To construct the plasmids pP1-KpnL and pP1-BglL, the plasmid pPRL/SstI was cut by BglII and KpnI respectively; the 3.8-kb KpnI fragment and 1-kb BglII fragment were gel-purified and ligated to KpnI and BglII sites of the plasmids pP1-KpnL and pP1-BglL, respectively. Orientation was determined by DNA sequence.

For construction of plasmid pP2-Hind (see Fig. 4B), the 1834-bp region of PRL-1 gene (+1733 to +3567) was first amplified by PCR and subcloned into the KpnI and BglII sites of the plasmid pGL2-Basic. The sequences of the primers for the PCR were 5'-GATTCATGGTACCGAAGCTTT-3' (upstream) and with KpnI and BglII sites (underlined) for cloning. "pP2-Sst" was generated by inserting the SstI/HindIII fragment from PRL-1 gene subclone pPRL/Sst 2 (containing PRL-1 exons 2-4, Fig. 1) into the site between SstI and HindIII of plasmid "pP2-Hind." To generate plasmid "pP2-Sst-anti-," plasmid pP2-Sst was partially digested with HindIII after digestion by SstI, and the 2705-bp SstI/HindIII fragment was gel-purified and subcloned into the site between SstI and HindIII of the plasmid Bluescript KS, which was digested with SstI, blunt-ended, and redigested with XhoI. The SstI (blunted)/XhoI fragment was then cloned into the site between XhoI and HindIII (blunted) of plasmid pGL2-Basic. P2 mutant plasmids pP2-A and pP2-B were generated by PCR amplification of plasmid pPRL/Sst 2 and subcloned into the site between KpnI and BglII of plasmid pGL2-basic. P2 plasmids pP2-C and pP2-D were constructed by exonuclease Bal31 digestion of pP2-B. The sequences of the primers for the PCR were as follows: upstream, 5'-GATTCATGGTACCGAAGCTTT-3' (pP2-Hind); 5'-CTTCAGAGGATCCTGGAGT-3' (pP2-A); 5'-ATCTGGATCCTCACCCCTTGAG-3' (pP2-B); and downstream, 5'-GCTAGATCTTGGTGGAGCAGTAA-3'.

For construction of plasmids Pst-P2, Pst-P2del862-1733, Pst-P2del862-2954, and Pst-P2del862-3269, a 4.2-kb BamHI/SstI (blunted) fragment from pGL-Sst containing 1614-base pair human PRL-1 sequence from -752 to +862 was subcloned into the site between BamHI and KpnI (blunted) of plasmids pP2-Sst, pP2-Hind, pP2-A, and pP2-B, respectively. To generate plasmid Pst-P2/SmaI, plasmid Pst-P2 was cut with SmaI, and 9.02- and 0.542-kb SmaI fragments were gel-purified and religated. The insert orientation was confirmed by DNA sequencing. Chimeric human PRL 1 intron enhancer-promoter luciferase plasmids pBglI/SmalI, pSmaI/SmaI, and pSmaI/SstI were constructed by blunt-ended cloning the first intron into blunted XhoI site of the SV40 promoter luciferase-containing plasmid pGL2-promoter (Promega). The deletion of pSma I/SmaI was done by Bal-31 nuclease mutation. The HindIII-linearized pSma I/SmaI was incubated with Bal-31 nuclease at 30 °C. The reaction was stopped at 3-min intervals and then digested with SstI. The resulting DNA fragments were gel-purified 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 the site between SstI and BglII (blunted) of pGL2 promoter. The sequence and orientation of the inserts were confirmed by DNA sequencing using T7 DNA sequencing (U. S. Biochemical Corp.).

Cell Culture, DNA Transfection-- Human hepatoma HepG2 cells, mouse embryo testicular carcinoma cell line F9 cells, and mouse fibroblast NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 2 mM L-glutamine (Flow Laboratories), 100 units of penicillin, and 50 units of streptomycin (Flow Laboratories) as described previously (1, 10). Human cervical carcinoma HeLa cells were grown in Iscove's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units of penicillin, and 50 units of streptomycin. Calcium phosphate-mediated transient transfections were performed as described previously with minor modifications (10). Twenty four hours before transfection, the cells were plated in 60-mm dishes at 1-5 × 105 cells/cm per dish and then cotransfected with various human PRL-luciferase chimeric plasmids (5 µg per dish) and 1 µg of the pSV-beta -galactosidase reference plasmid (Promega) as an internal standard for transfection efficiency. After incubation with DNA-calcium phosphate coprecipitation buffer for 16 h, the cells were washed twice with serum-free culture medium; complete medium was added, and then the cells were incubated for an additional 24 h before harvesting for luciferase activity assays performed with luciferase assay system (Promega) and the recommended procedure by the supplier. Relative luciferase activity was reported after normalization to beta -galactosidase activity.

Preparation of Nuclear Protein Extracts-- All steps were performed at 4 °C, and 2 µg/ml proteinase inhibitors antipain, aprotinin, bestatin, and leupeptin were added into all buffers except phosphate-buffered saline, pH 7.4. Crude nuclear extracts from regenerating liver were prepared as reported (11). Crude nuclear extracts from cell lines HepG2, H35, HeLa, F9, and NIH 3T3 were prepared using the procedure of Seal et al. (12) with minor modifications. Cells were washed twice and scraped into phosphate-buffered saline solution. The cells were pelleted by centrifugation at 500 × g for 10 min and lysed by homogenization in approximately 9 volumes of Buffer A (10 mM HEPES, pH 7.6, 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.5% Triton X-100, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride). Aliquots of cell lysate were layered over 5 ml of Buffer B (same as Buffer A except that the concentration of sucrose was 1 M) in 15-ml tubes and centrifuged at 10,000 × g for 10 min. The supernatant was removed by aspiration, and the nuclei pellet was resuspended in Buffer C (20 mM HEPES, pH 7.9, 0.2 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 20% glycerol). Nuclei were lysed by the dropwise addition of 1/9 volume of 4.2 M NaCl before being centrifuged at 50,000 × g for 50 min. The supernatant was dialyzed against Buffer D (20 mM HEPES, pH 7.6, 0.2 mM EDTA, 0.1 M KCl, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 20% glycerol) for at least 6 h. Finally, the extract was centrifuged at 12,000 × g for 10 min, and aliquots were quick-frozen in dry ice, 95% ethanol mixture and stored at -70 °C. Protein concentrations were determined using the Bio-Rad protein determination method per manufacturer's instructions.

Electrophoretic Gel Mobility Shift Assays (EMSA)-- The synthesized single polynucleotides were annealed in 10 mM Tris·HCl, pH 8.0, 1 mM EDTA, 0.3 M NaCl at 90-95 °C for 10 min, and then cooled to room temperature. After being kept at room temperature for 24 h, the annealed oligonucleotide was purified by 15% native polyacrylamide. End labeling of oligonucleotides was carried with T4 polynucleotide kinase and [gamma -32P]ATP and purified by Sephadex G-25 spin column. Binding reactions were performed essentially as reported (11, 13). Nuclear protein extracts (3-6 µg) were incubated with 2 µg of poly(dI-dC) in 10 µl of binding buffer containing 20 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM NaCl, 1 mM DTT, 1.5 mM NaCl, 10% glycerol for 15 min at room temperature. 32P-Labeled probe (0.5-1 × 104 cpm) was then added to the reaction, and incubation was continued for another 20 min. DNA-protein binding complexes were analyzed on 6% native polyacrylamide gel at 300 V using 0.5× TBE as electrophoresis buffer. Gels were dried and exposed to Kodak X-Omat AR films overnight. The sequence of the oligonucleotide used in the EMSA was as follows, 5'GCCGCCTCCGCCCTTGGCCCTTTGGTCTG3'.

For UV cross-linking experiments, a gel mobility shift experiment was performed as described above using 20 µg of nuclear protein extract in 20 µl of binding reaction buffer. After electrophoresis, the wet gel was UV-irradiated for 60 min at 305 nm, 7000 microwatts/cm2 using a fotodyne UV transilluminator, and exposed to film for 30-60 min. The specific DNA-protein complexes were cut out and eluted into a solution containing 10 mM Tris·HCl, pH 8.0, 1 mM EDTA, 0.5 M ammonium acetate at 4 °C overnight. After precipitation with 3 volume of 95% ethanol, the specific DNA-protein complexes were dissolved in 20 µl of 1× Laemmli buffer, electrophoresed on 12% SDS-polyacrylamide gels, and then subjected to autoradiography.

DNase I Footprinting-- DNase I footprinting was carried out as described in Ohlsson and Edlund (13). Briefly, 0-150 µg of nuclear protein extract was preincubated for 20 min at room temperature with 4 µg of poly(dI-dC) in 50 µl of a mixture containing 10 mM HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 0.25 mM fluoride. 1-2 × 104 cpm of 32P-end-labeled DNA probe was then added. After the binding reaction was incubated for 40 min at room temperature, MgCl2 and CaCl2 were added to a final concentration of 5 and 2 mM, respectively. An empirically adjusted amount of freshly diluted DNase I was added, and the reaction was allowed to proceed for exact 45 s at room temperature. The reaction was stopped by the addition of 100 µl of buffer containing 100 mM Tris·HCl, pH 8.0, 100 mM NaCl, 1% SDS, 20 mM EDTA, 50 mg/ml yeast tRNA, and 100 mg/ml proteinase K and was incubated for 15 min at 37 °C. Nucleic acids were extracted twice with phenol/chloroform, precipitated by ethanol/sodium acetate, and fractionated on 6% polyacrylamide, 8 M urea gel, alongside sequencing ladder G + A that was produced by chemical cleavage of aliquots of the probe DNA (14).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation and Characterization of Human PRL-1 Gene-- Two overlapping genomic clones, designated as hprl1 (17.5 kB) and hprl2 (14.5 kB), containing the human PRL-1 gene were isolated (Fig. 1A). Results from detailed restriction mapping and Southern hybridization demonstrated that both clones spanned the entire human PRL-1 gene but had different 5' and 3' boundaries (Fig. 1). The clone hprl1, with a 17.5-kb insert, was subcloned, and 12 kb were sequenced from the upstream BglII site (-2179) to 0.8 kb past the 3'-end of the second polyadenylation signal. The human PRL-1 gene is composed of 6 exons and 5 introns. The boundaries of each exon display the canonical splice donor (GT) and splice acceptor (AG) consensus sequences (Fig. 1A). The first exon contains no coding sequence and is separated from the second exon by the largest intron (3.2 kb) in the gene. An ATG initiation codon is located in the middle of the second exon. Based on GenBankTM analyses, rat, human, and mouse PRL-1 are identical at the amino acid level. We found that human PRL-1 had 55% amino acid identity with a Caenorhabditis elegans "similar to the protein tyrosine phosphatase (U42846)" sequence identified by C. elegans genomic sequencing (15) (Fig. 1B). Interesting features of this conservation include virtual identity within the enzymatic domain (around amino acid 120), size similarity, and absolute conservation of the C-terminal CAAX (CCIQ) sequence.


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Fig. 1.   Structure and sequence of the human PRL-1 gene. A, top, structure of the human PRL-1 gene. Key endonuclease restriction sites are shown. Intron and flanking sequences are represented by solid lines and exons by boxes; the open boxes and the solid boxes indicate the noncoding regions and coding regions, respectively. hprl1 and hprl2 represent two overlapping genomic clones from human lymphocyte genomic library. Bottom, nucleotide sequence of human PRL-1 exon, partial intron (lowercase letters), and 73-bp 5'-flanking region. The underlined sequences represent the TATA box, transcription initiation site, translational start site, translational stop site, and AATAAA polyadenylation signals. B, alignment of the amino acid sequences of human PRL-1 and the protein tyrosine phosphatase from C. elegans (GenBankTM accession number U42840). Top, the amino acid sequence of C. elegans protein tyrosine phosphatase. Bottom, the human (also rat/mouse) PRL-1 amino acid sequence.

Chromosomal Localization-- The human hprl1 genomic probe specifically hybridized to the centromeric region of the long arm of a C group chromosome in 20 out of 30 metaphases that were scored. Sequential G-banding localized the probe to chromosome 6, within band q12 (Fig. 2).


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Fig. 2.   Chromosomal localization of human PRL-1. A, fluorescence in situ hybridization of a genomic probe for PRL-1 on a metaphase spread from a normal individual. The signal (arrows) is seen on the long arm just distal to the centromere of a C group chromosome. B, sequential G-banding demonstrated the signals lie on chromosome 6 within band q12.

Identification of PRL-1 Promoters P1 and P2-- Examination of the nucleotide sequence of the human PRL-1 gene 5'-flanking region revealed that the promoter region was highly GC-rich and contained a canonical TATA box. The transcription start site of the gene was identified from total RNA of human hepatoma cell line HepG2 using primer extension analysis with a primer contained within exon 1 (Fig. 3A). A single major primer extension product was identified at a position 25 bp 3' to the TATA box. We designated it as promoter P1.


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Fig. 3.   Identification of transcription initiation sites in the human PRL-1 gene. Primer extension analysis of the human hepatoma cell line HepG2 mRNA. A, to identify P1, a 22-bp human PRL-1 exon 1-specific primer was annealed to 50 µg of total RNA or tRNA, and the cDNA was extended and resolved on an 8% urea-denatured sequencing gel. The 78-bp product was identified. B, an exon 2-specific primer was used in a similar primer extension analysis to identify P2. See "Materials and Methods" for specific primer locations. C, RNase protection analysis of the transcripts of human PRL-1. Top, schematic diagram of the relevant portion of the human PRL-1 gene along with the RNase protection probe used for detection of the indicated gene transcripts. Bottom, RNase protection assay of human hepatoma cell line HepG2 total RNA or tRNA hybridized to the 299-bp 32P-labeled RNA template extending 5' of the exon 2. DNA sequencing ladders were used to map the sites accurately.

The protein coding sequence was predicted to begin within the second exon suggesting that the first intron could contain an additional promoter site(s) for initiation of PRL-1 mRNA. A primer extension analysis of HepG2 RNA using an oligonucleotide primer from within exon 2 revealed a specific extended fragment of 123 bp (Fig. 3B). A confirmatory RNase protection assay using an antisense probe (299 bp) containing the 3' 169 bp of the first intron and the 5' 130 bp of the second exon was performed with HepG2 total RNA. As shown (Fig. 3C), two RNase-resistant duplexes of approximately 123 and 130 bp were detected. The 130-bp fragment, which was the same size as the region of exon 2 contained in the probe, likely derived from RNA initiated at the P1 promoter. The 123-bp product detected on both primer extension and RNase protection analyses could be explained by an mRNA initiation site 8 bases 3' to the 5' boundary of exon 2. A similar internal RNA initiation site was detected in rat liver PRL-1 mRNA.2 A non-TATA sequence just upstream of the exon 2 initiation site was designated promoter P2.

Functional Analysis of Promoters P1 and P2 of the Human PRL-1 Gene-- To establish that P1 and P2 are functional and begin to delineate the sequences essential for transcription of the human PRL-1 gene in cell culture systems, we first constructed a series of the human PRL-1 P1 luciferase chimeric plasmids in which various lengths of the 5'-flanking region of the human PRL-1 gene including the P1 RNA initiation site were fused with the coding region of the bacterial luciferase gene (Fig. 4A). A plasmid containing the SV40 promoter and enhancer (pGL-C) was used as a positive control for transfection. Sequences necessary for high level basal transcription of the PRL-1 P1 promoter in both HepG2 and mouse NIH 3T3 cells were found within the first 200 bp of the P1 promoter, and a negative element may exist in the region between -3200 and -2179 bp.


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Fig. 4.   Functional analysis of human PRL-1 promoters P1 and P2. A, deletion mapping analysis of the promoter P1. Left panel, schematic diagram of the upstream region and the first exon of the human PRL-1 gene. The indicated restriction endonuclease sites were used to construct pGL2-Basic luciferase reporter plasmids containing various lengths of human PRL-1 5'-flanking sequences (described under "Materials and Methods"). Right panel, graphical presentation of relative luciferase activity after normalization for beta -galactosidase; standard deviations are shown. B, deletion mapping analysis of the promoter P2. Left panel, schematic diagram of the first intron and exon 2 of the human PRL-1 gene. Delineation of the 5' and 3' boundary of the plasmids containing various lengths of the first intron were indicated. Right panel, graphical presentation of relative luciferase activity after normalization for beta -galactosidase. Transfections were performed with HepG2 and NIH 3T3 cell lines by the calcium phosphate precipitation method. For A and B, four independent determinations were made for each construct in each cell line by performing duplicates in two separate experiments. Standard deviations were determined from the duplicate values in a single experiment and were representative of the deviation in all four determinations.

To delineate the sequences essential for promoter P2 function in the human PRL gene, we constructed a series of the human PRL-1 P2-luciferase chimeric plasmids in which various lengths of the first intron of the human PRL-1 and the first 130 bp of exon 2 were fused with the coding region of bacterial luciferase gene. As shown (Fig. 4B), except plasmid P2-D (without the internal transcription start site), all P2 luciferase constructs with the correct orientation had luciferase activity. When the 2538 bp intron was inserted into the vector in reverse orientation, no luciferase activity was detected after transfection. Deletion mapping analysis revealed the sequence between +3269 and +3330 in PRL-1 intron 1 was responsible for a significant amount of P2 activity in HepG2 cells (compare pP2-B with pP2-C). Results were similar when these constructs were analyzed in NIH 3T3 cells (data not shown).

Identification of a Transcriptional Enhancer in Intron 1-- The fact that the first exon of the PRL-1 gene is non-coding and the gene contains two transcription start sites in exons 1 and 2 suggested that transcriptional regulatory sequences may reside in intron 1. The sequence of the first intron shows a high content of GC bases in the first 900 bp downstream of exon 1 and contains several consensus sites for transcription factor binding, including SP-1, AP-2, Myo D, NF-kappa B, HNF5, and C/EBP elements. To test for the presence of an enhancer sequence within the first intron, constructs were made that contained promoters P1, various lengths of the first intron, and the first 130 bp of exon 2 (including P2) inserted upstream of the luciferase gene. Analyses suggested that removal of the BglI/SstI fragment (+40 to +862) greatly diminished transcriptional activity in HepG2 cells (Fig. 5A). This could be explained by the fact that this deletion removed the splice donor for exon 1 and destabilized P1-derived transcripts, and/or the region contained a transcriptional enhancer. This region was subdivided into three fragments to test for enhancer activity. Only the SmaI/SmaI fragment significantly enhanced transcription from the PRL-1 P1 or SV40 promoter luciferase constructs (Fig. 5B). As shown, this fragment conferred orientation independent activity. Sequence analysis of the region showed a high percent of GC composition (76.8%). Detailed deletion with exonuclease Bal 31 narrowed the enhancer activity to a 90-bp region (Fig. 5C).


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Fig. 5.   Enhancer activity within a region of the first intron of the human PRL-1 gene. Transfections were performed in HepG2 cells. A, deletion analysis of intron 1 of the PRL-1 gene. The localization of deletions spanning intron 1 and the relative luciferase activity in HepG2 cell transfections are shown. B, localization of the first intron enhancer. Left panel, schematic diagram of 5'-flanking region of the first intron of the human PRL-1 gene and the structure of the chimeric luciferase reporter plasmids. Right panel, graphical presentation of relative luciferase activity after normalization for beta -galactosidase. C, top, nucleotide sequence of the first intron enhancer region. The relative positions of the intron enhancer and deletion mapping sites are shown. Bottom, deletion mapping of the first intron enhancer. Left panel, schematic diagram of 3' and 5' unidirectional mapping of the intron enhancer. Right panel, graphical presentation of relative luciferase activity after normalization for beta -galactosidase. Six independent determinations were made for each construct by performing duplicates in three separate experiments. Standard deviations were determined from the duplicate values in a single experiment and were representative of the deviation in all six determinations.

DNase footprinting experiments were performed to localize the sites of interaction between DNA sequences within the SmaI/SmaI fragment and nuclear proteins from HepG2 cells (Fig. 6A). A large protected region (25 bp) was located in the same region with potential enhancer activity. Gel mobility shift assays using the oligonucleotides corresponding to the DNase I-protected region were performed to define further the DNA-protein interaction. As shown (Fig. 6B), EMSA resulted in one major complex in HepG2 cells that appeared to be a doublet, designated PIEC (PRL-1 intron enhancer complex). PIEC was competed off by self but not other oligonucleotides containing known DNA-binding sites. Competition with a series of mutated oligonucleotides from the region allowed the delineation of an enhancer core sequence (Fig. 6C) that contained an internal SP1 sequence but was otherwise distinct from SP1. No known transcription factor binding sequences aligned with the core sequence. However, because of the similarity to consensus sequences for zinc finger transcription factor binding sites, zinc and metal dependence of PIEC binding was assessed by EMSA. No metal dependence was found (data not shown). When mutation 3 (M3) was inserted within the enhancer site in the SmaI fragment upstream of SV40 luciferase, most (80%) enhancer activity was lost (Fig. 6D), confirming that activation occurs via the core binding site.


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Fig. 6.   Identification of an enhancer DNA binding complex, PIEC. A, DNase I footprinting analysis of the first intron enhancer region. The enhancer-containing SmaI/SmaI fragment was end-labeled and incubated with increasing amounts (0-50 mg) of crude nuclear extract from HepG2 cells in the presence of poly(dI-dc)·poly(dI-dc) as a nonspecific competitor. After 40 min of incubation at room temperature, the samples were treated with DNase I, and the DNA was analyzed on 8% urea-denatured sequencing gels. The Maxam-Gilbert sequencing ladder G + A, using same 32P-labeled DNA fragment, is displayed in the leftmost lane. The protected region is indicated, and the Gs contained within the enhancer core (C) are delineated by asterisks. B, determination of the first intron enhancer binding specificity. The synthetic double-strand oligonucleotide covering the DNase I-protected region was labeled and incubated with HepG2 nuclear extracts. The binding reactions were performed as described under "Materials and Methods." The complex is designated PIEC (PRL-1 intron enhancer complex). One hundred-fold excess molar ratios of the corresponding unlabeled double-stranded oligonucleotide and some unrelated double-stranded oligonucleotides were added to the reaction mixture to compete for binding as indicated. C, delineation of the first intron enhancer core. Top, gel mobility shift assays were performed with the labeled wild-type double-strand oligonucleotides and 50-fold molar excesses of the unlabeled wild type and a series of the mutated double-strand oligonucleotides as competitors. The binding complex (PIEC) is indicated. Bottom, upper strand nucleotide sequences of wild-type and mutated intron enhancer oligonucleotides. The nucleotides mutated are indicated by bold letters. The "Gs" corresponding to footprinted sites are indicated by asterisks. D, functional analysis of the first intron enhancer mutation. The mutated containing enhancer region (SmaI/SmaI fragment), generated by overlapping PCR amplification with the mutated oligonucleotide M3, was cloned into the SV40 promoter pGL2 luciferase chimeric plasmid (Promega). For transfections, four independent determinations were made for each construct in HepG2 cells by performing duplicates in two separate experiments. Standard deviations were determined from the duplicate values in a single experiment and were representative of the deviation in all four determinations.

PIEC was detected in nuclear extracts from HepG2 and F9 cells but not NIH 3T3, HeLa, or H35 cells which contained other specific complexes of different mobility (Fig. 7A). The presence of PIEC correlated with enhancer activity in transfection experiments, because the SmaI-SV40 luciferase construct conferred significant enhancer activity only in HepG2 (7.2-fold) and F9 (4.6-fold) cells and not NIH 3T3 or HeLa cells (Fig. 7B). The enhancer region contained within the SmaI fragment contributed significantly to PRL-1 promoter activity in the context of the whole promoter in HepG2 cells which contain PIEC but not NIH 3T3 cells which do not (Fig. 7C). There was an approximately 7-fold decrease in promoter activity in HepG2 cells when the enhancer-containing fragment was removed from the luciferase construct (Pst-P2delSmaI) and only a small decrease in activity in NIH 3T3 cells. Unlike Pst-P2del40-862 (Fig. 5A), the P2delSmaI construct retained all of the splicing consensus sequences for exon 1 and functioned nearly as well as P2-Pst in NIH 3T3 cells. In contrast, Pst-P2del40-862 wich removed the exon 1 splice donor showed an almost complete loss of luciferase activity in both HepG2 (Fig. 5A) and NIH 3T3 cells (not shown). Interestingly, the Pst-P2delSmaI construct showed roughly equal activity in the two cell types, whereas constructs containing the enhancer fragment had severalfold higher activity in HepG2 than NIH 3T3 cells.


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Fig. 7.   Characterization of cell-specific and developmentally regulated binding activity of the first intron enhancer. A, binding activities of nuclear extracts from different cell lines with the enhancer oligonucleotide. The position of PIEC is indicated. B, cell-specific functional analysis of the first intron enhancer. Transient transfections were performed using the PRL-1 SmaI-SmaI enhancer fragment upstream of the SV40 promoter with four different cell lines. The results represent fold induction of the relative luciferase activities compared with the enhancer-less vector after normalization for beta -galactosidase. C, binding activities of nuclear extracts from developing rat livers, adult liver and brain, and HepG2 cells. D, UV cross-linking analysis of the first intron enhancer binding protein(s). EMSA was performed by incubating the 32P-labeled double-stranded oligonucelotides and HepG2 nuclear extracts at room temperature for 15 min. After native polyacrylamide gel electrophoresis and UV irradiation of the wet gel, the specific DNA-protein complexes were cut out, eluted, and subjected to 12% SDS-polyacrylamide gel electrophoresis and autoradiography. The relative positions of molecular mass markers are illustrated on the right. For transfections, six independent determinations were made for each construct by performing duplicates in three separate experiments in the indicated cells. Standard deviations were determined from the duplicate values in a single experiment and were representative of the deviation in all six determinations.

Both F9 and HepG2 cells which contain PIEC have characteristics of fetal cells. PIEC was detected in developing fetal rat liver but rapidly disappeared after birth and was not detected in adult rat liver cells (Fig. 7D). The adult tissues (rat liver and brain) contained complexes with slower mobility similar to those detected in NIH 3T3 cells. No reappearance of PIEC was detected in regenerating rat liver (not shown). Finally, a UV cross-linking experiment was performed to determine the molecular mass of PIEC proteins from HepG2 cells. The strongest specific cross-linking of the probe was found in two proteins which had apparent molecular masses of 47 and 67 kDa (Fig. 7E), and this result was obtained twice.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The rat PRL-1 gene is highly expressed in proliferating liver tissues in development, regeneration, and oncogenesis and is associated with differentiation in other cell types. As an initial step in understanding the regulation of the PRL-1 gene, we isolated the human PRL-1 genomic clone. We showed that the PRL-1 gene is highly conserved in mammals and contains a C. elegans homologue. We identified its chromosomal position as 6q12. We demonstrated the presence of two functional promoters near exons 1 and 2 of the PRL-1 gene and identified a developmentally regulated enhancer site within the first intron of the PRL-1 gene.

The 100% conservation of the mammalian PRL-1 protein, strong similarity of a family member OV-1, and conservation within simple eucaryotes (C. elegans) suggest that the PRL-1 protein has a critical function in cellular regulation. Of particular interest is the near identity of the active site region between mammalian and C. elegans PRL-1 and the 100% conservation of the unusual isoprenylation sequence, CCIQ. This suggests that prenylation indeed plays an important role in posttranslational regulation of PRL-1 activity.

The localization of the PRL-1 gene to chromosomal segment 6q12 by in situ hybridization correlates with the recent mapping of the PRL-1 ESTs to between the D6S1695 and D6S21 markers in this region (NCBI, the Human Gene Map). This region of chromosome 6 does not have any known closely linked markers to disease genes. Although chromosome 6 is frequently abnormal in solid tumor malignancies, these abnormalities usually involve segment q15 to the terminus (17).

Both P1 and P2 promoters of the PRL-1 gene contribute to the activity of the gene. In HepG2 cells P1 contributes more to the steady state transcript (Fig. 3B), but both promoters show high activity in transfection studies. In HepG2 and NIH 3T3 cells, major basal regulation is still retained within 200 bp of the RNA initiation site. We have identified several transcription factor binding sites including Egr-1 and SP1 elements within this sequence. Because Egr-1 is strongly up-regulated during liver regeneration, we have explored the contribution of Egr-1 to regulation of the PRL-1 gene in liver regeneration.2 A major regulatory site of the TATA-less P2 promoter has been identified within a 39-bp segment upstream of P2. This segment contains a C/EBP-binding site, and C/EBPbeta like Egr-1 is also increased during liver regeneration (18).2

PRL-1 is also expressed at a high level in terminally differentiated intestinal cells (5). The Cdx class of homeodomain proteins has been shown to be of critical importance in the regulation of genes that are expressed in differentiated intestinal cells (16). Of interest is the significant number of consensus Cdx elements found upstream of PRL-1 promoter P1 including several within a few hundred bases of the TATA element. Subsequent studies examining the ability of Cdx to regulate the PRL-1 promoter will be of interest.

Promoter P1, upstream of untranslated exon 1, and P2, upstream of exon 2, are separated by the large intron 1. We assessed the possible contribution of sequences within intron 1 to promoter regulation. We localized a novel enhancer sequence within intron 1. This sequence fulfilled the requirement for an enhancer because it was position-independent and acted upstream of various promoters. A specific complex (PIEC) bound to the strongly footprinted region within the enhancer sequence and allowed us to derive a consensus binding sequence. In different cells, different specific protein complexes were observed that bound this sequence, and thus far PIEC has only been detected in cells of a fetal lineage including HepG2 and F9. In addition, fetal but not adult liver contained PIEC, and PIEC was not induced in regenerating liver. There was a correlation between the presence of PIEC in four cell lines tested and the ability of this sequence to function as an enhancer. Moreover, the intron enhancer contributed significantly to total PRL-1 promoter activity in HepG2 which contain PIEC but not NIH 3T3 cells which do not. However, the presence of PIEC does not necessarily correlate with the level of steady state PRL-1 mRNA in every tissue or cell line. HeLa cells and regenerating liver express high levels of PRL-1 mRNA but do not contain PIEC. Other regions of the PRL-1 promoter are likely to contribute to regulation in these cells; post-transcriptional controls could play a role as well.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants DK49629 (to R. T.) and DK44237 (to R. T. and R. H. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF051160 and U42840.

parallel 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 or 215-573-9411; E-mail: taubra{at}mail.med.upenn.edu.

1 The abbreviations used are: PTPase, protein tyrosine phosphatase; PRL-1, phosphatase of regenerating liver; PIEC, PRL-1 intron enhancer complex; EMSA, electrophoretic gel mobility shift assay; kb, kilobase pairs; bp, base pair(s); DTT, dithiothreitol; PCR, polymerase chain reaction.

2 Y. Peng, K. Du, S. Ramirez, and R. Taub, submitted for publication.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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