From the Division of Endocrinology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048
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ABSTRACT |
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The rat pituitary tumor transforming gene
(PTTG) genomic structure was characterized in this study.
Northern blot analysis showed that PTTG mRNA is highly
expressed in testicular cell lines. Transfection of testicular cell
lines with fusion constructs containing various portions of PTTG
5'-flanking sequences linked to luciferase showed that at least
745 base-pair (bp(s)) 5'-flanking sequences are required for PTTG
transcriptional activation. DNaseI footprinting assays indicated
that nuclear protein(s) from testicular cell lines interacts with
PTTG 5'-flanking sequence between 509 and
624 bp,
including two consensus Sp1 binding sites. Western and Southwestern
blot analysis showed that three nuclear proteins in addition to Sp1
protein specifically interact with this DNA sequence and that two of
these proteins are testicular cell-specific. Deletion of this 115-bp
sequence from PTTG promoter resulted in complete loss of
promoter function. Site-directed mutagenesis within the Sp1 consensus
sequences indicated that the Sp1 binding sites are not critical
components of the enhancer sequence for PTTG trancriptional
activation in testicular cell lines. Finally, the 115-bp enhancer
sequence was shown to be able to activate transcription from a
heterologous promoter. These results suggest that PTTG
transcriptional activation in testicular cell lines involves
interactions of multiple nuclear factors with the PTTG 5'
enhancer sequence.
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INTRODUCTION |
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Recently, we have isolated and characterized a pituitary tumor transforming gene (PTTG)1 from rat pituitary tumor cell lines (1). PTTG encodes a novel protein of 199 amino acids and does not contain any known functional motifs. Overexpression of PTTG protein in 3T3 fibroblasts resulted in cell transformation in vitro, and injection of transfected 3T3 cells into nude mice resulted in tumor formation, indicating that PTTG is a transforming gene (1). In addition to pituitary tumor cell lines, rat PTTG mRNA is also expressed in a variety of tumor cell lines, including lung carcinoma, melanoma, leukemia, lymphoma, and HeLa cell lines.2 However, among adult rat tissues, PTTG mRNA is only expressed in testis (1), suggesting that PTTG may play a role in testicular biological functions. PTTG transcript in testis is shorter than that of tumor cells, indicating that PTTG mRNA is either differentially spliced or it is using an alternate promoter or an alternative polyadenylation signal. The pattern of expression exhibited by PTTG is similar to proto-oncogene c-mos and c-abl. Both also exhibit testis-specific transcripts (2).
To begin to understand the molecular mechanisms that regulate
PTTG expression in testis and tumor cells, I have focused on characterizing the rat PTTG gene. I report here the
isolation and structural characterization of the entire rat
PTTG gene and its expression in different testicular cell
lines. By transient transfection of fusion constructs containing
various portions of the PTTG 5'-flanking region and
luciferase gene into testicular cell lines, I have identified a
potential enhancer sequence between 462 and
745 bp. I show by
DNaseI footprint assays that nuclear protein(s) from testicular cells
interacts with DNA sequences between
509 and
642 bp of
PTTG gene. Using Western and Southwestern analysis, I show
that four nuclear proteins, including Sp1 protein, specifically
interact with this DNA sequence and that two of these proteins are
specific to testicular cells. I demonstrate by mutagenesis studies that
deletion of this binding site for multiple testicular proteins results
in loss of PTTG transcriptional activation, whereas point
mutations within the Sp1 binding sites do not reduce transcription remarkably. Finally, I show that the sequence between
509 and
624
bp is able to confer transcriptional activation to a heterologous promoter. These results indicate that the sequence between
509 and
624 bp constitutes the core enhancer that is responsible for
PTTG transcriptional activation in testicular cell
lines.
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MATERIALS AND METHODS |
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Isolation of the Rat PTTG Gene--
A rat genomic library (using
genomic DNA from Sprague-Dawley rat testis) in DASHTM
vector (Stratagene) was screened using the rat PTTG cDNA
(1) as a probe. Southern blot analysis identified two SstI
fragments that contain the entire PTTG gene and were
subcloned into PGEM3Z (Promega) for further characterization.
Dideoxy-DNA sequencing was performed using the Sequenase kit (U. S.
Biochemical Corp.). Both strands of DNA were sequenced using either
internal primers or sequencing subcloned fragments. DNA restriction and
modification enzymes were purchased from Life Technologies, Inc.
Polymerase Chain Reactions (PCR)-- Phage DNA was isolated using the Qiagen lambda phage DNA isolation kit. DNA was resuspended in TE to a final concentration of 1 mg/ml. For each PCR reaction, 100 ng of DNA was used. The PCR reaction mix contains 200 µM each of dNTP, 200 nM each of primer, 60 mM Tris-SO4, pH 9.1, 18 mM (NH4)2SO4, 2 mM MgSO4, and 2 units of Elongase (Life Technologies, Inc.) in a total volume of 50 µl. PCR reactions were performed in s DNA Thermal Cycler 480 (Perkin-Elmer) in the following conditions: 94 °C, 30 s; 55 °C, 30 s; and 68 °C, 6 min; for 35 cycles. Thirteen pairs of PCR primers were used as listed below: P(1)5', 5'-CACGAGCCAACCTTGAGCATCTGATCC-3'; P(1)3', 5'-CCTTATCAACAAAGATCAGAGTAGCCA-3'; P(2)5', 5'-CAGGATGGCTACTCTGATCTTTGTTGAT-3'; P(2)3', 5'-GGCGTTGAAACCTGCAATTTCCCATCT-3'; P(3)5', 5'-CTTAGATGGGAAATTGCAGGTTTCAAC-3'; P(3)3', 5'-TTCACTGGCTTTTCAGTAACTCTGTTG-3'; P(4)5', 5'-GCCACGAGGAAGGCTCTGGGAACT-3'; P(4)3', 5'-TCATCAGGAGCAGGAGCAGAGCCTTG-3'; P(5)5', 5'-TCCTGCTCCTGATGATGCCTACCCAG-3'; P(5)3', 5'-GAAGAGCCAGGCAGCCGTTTGG-3'; P(6)5', 5'-GCTTGAGAAGCTGCTGCACCTGGACC-3'; P(6)3', 5'-TAAGACGTTTAAATATCTGCATCGTAACAA-3'; P(7)5', 5'-GATGTTGAATTGCCGCCTGTTTGTTAC-3'; P(7)3', 5'-GAGTTTTTTTTTTTTTTTTTACACACAAATGC-3'. PCR products were analyzed on 1.5% agarose gels and were sequenced using PCR Sequenase sequencing kit (U. S. Biochemical Corp.) following manufacturer's instructions.
RNase Protection Assays and Northern Blot Analysis--
Total
RNA was isolated from rat testis (Leydig, germ, and Sertoli) and
pituitary GH4 cell lines, using RNAeasy isolation kit (Qiagen),
following manufacturer's instructions. Rat testis poly(A)+
RNA was purchased from CLONTECH. A 533-bp fragment
spinning +72 to 461 bp was synthesized by PCR and subcloned into TA
vector (Invitrogen). The antisense probe was synthesized from T7
promoter using the MAXIscriptTM kit (Ambion). RNA
protection assays were performed with 20 µg of total RNA or 0.5 µg
of poly(A)+ RNA and 105 cpm of the probe, using
the RPA IITM ribonuclease protection assay kit (Ambion). A
X174 HinfI DNA marker was labeled with
[
-32P]ATP and T4 DNA polynucleotide kinase. The RNase
protection assay products were analysis on 6% sequencing gel alongside
the labeled DNA marker and a DNA sequencing ladder.
Plasmid Construction-- To construct promoter and reporter fusion constructs, different portions of the PTTG 5'-flanking sequences were synthesized by PCR using conditions described above. The downstream primer was 5'-TGGATTACTGAGGGGAGAGACCTG-3' (+41 to +17 bp). The upstream primer included U0, 5'-GCTGAGCTGGTAGGCTGGAGACAG-3'; U1, 5'-GTCGTTGCCAAAGATGTCTTGGGC-3'; U2, 5'-CAGTAGATTGGTGCCTCTGACTTT-3'; U3, 5'-AGGAGACTAAGGAACTTTTAAGCT-3'; U4, 5'-GTGTCTAGACTGAGGAAGAGTCAC-3'; U5, 5'-GTCTTGTATAGCTTCATGAACTCA-3'; U6, 5'-TATACTCTTCTACGGTAAGCTATC-3'.
The PCR products were cloned into TA vector (Invitrogen). A SstI-XhoI fragment was isolated from the plasmid and cloned at the corresponding sites in the pGL3 luciferase reporter vector (Promega). To clone the PTTG enhancer sequence in front of a heterologous promoter, the enhancer sequence was amplified by PCR using the following primers: P1, 5'-TCCGGGAGGGGGCGGTGGTTGAAAACTTCC-3'; P2, 5'-TCACCCAAGACTACGCCACCAACGACC-3'. The PCR product was cloned into TA vector, and the insert was excised by digesting the plasmid with SstI and EcoRV. The insert was clone at the SstI/SmaI site of the thymidine kinase (TK)-luciferase vector.Cell Cultures and Transfection-- Mouse testis germ GC2 and rat Leydig cell line were grown in Dulbecco's modified Eagle's medium or minimal essential medium, respectively, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.11 mg/ml sodium pyruvate, and nonessential amino acids. Cells were transfected with 5 µg of fusion construct DNA by calcium phosphate precipitation (3). After a 24-h incubation, cells were assayed for luciferase and CAT activities. Each construct was transfected using triplicate plates for each experiment, and each construct was tested in at least three independent experiments. In all experiments, cells were co-transfected with 1 µg of TKCAT (thymidine kinase promoter and chloramphenicol acetyl transferase fusion gene) to monitor transfection efficiency. A negative control plasmid containing a promoterless luciferase gene (pGL3) and a positive control plasmid containing Rous sarcoma virus 3' long terminal repeat promoter fused to luciferase were included in all experiments.
Luciferase Assays and CAT Assays-- Cell lysates were prepared by freezing and thawing cells in 0.25 M Tris, pH 7.8. Protein concentration was determined using a dye binding assay (Bio-Rad). Luciferase activity were determined by adding 100 µl of 1 mM luciferin (Analytical Luminescence Laboratory) to the cell lysate (50 µg of protein) in assay buffer (4 mM ATP, 0.25 M Tris, pH 7.8, 15 mM MgSO4, 1 µg/ml bovine serum albumin). Light emission was integrated over 15 s using a luminometer (Autolumat LB953). For CAT assays, 50 µg of cell lysate were incubated with 20 µl of 4 mM acetyl-CoA, 1 µCi of [14C]chloramphenicol in a total volume of 100 µl at 37 °C for 90 min. CAT activity was determined by ascending chromatography on TLC plates and quantified using an Ambis Radioanalytic Imaging System (Ambis).
DNaseI Footprinting Assay--
Nuclear extracts were prepared
from GC2 cells as described by Dignam et al. (4). The probe
used in DNase I footprinting assays is the 366-bp PTTG
5'-flanking sequence from 379 to
745 bp. To generate the probe,
primer 5'-CAGTAGATTGGTGCCTCTGACT-3' was end-labeled using
[
-32P]ATP and T4 polynucleotide kinase and was then
used in PCR with downstream primer 5'-TCAGCAGCGATTTCGTACTTGGATTTTTTG-3'
and
745LUC as the template. 10,000 cpm of the probe was used in each
reaction. Binding reactions were performed in 25 µl of binding buffer
(20 mM Tris, pH 7.5, 1 mM EDTA, 2 mM MgCl2, 50 mM NaCl, 20 mM dithiothreitol, and 20% glycerol) with 50 ng of
purified Sp1 protein (Promega) or with increasing amounts of GC2 cell
nuclear extracts. Competitor DNA was mixed with the probe before
receptor addition. Binding was for 30 min at room temperature. DNase I
digestion was carried out at room temperature for 1 min using 1-10
units of DNase I (Ambion), depending upon protein concentrations.
Reactions were terminated by adding 30 µl of 2 × stop buffer
containing 15 mM EDTA, 0.2% SDS, and 40 µg/ml salmon
sperm DNA. Nucleic acids were extracted with phenol/chloroform (1:1),
ethanol-precipitated, and electrophoresed on 6% sequencing gels
alongside of a G+A DNA sequencing ladder.
Western and Southwestern Blot Analysis-- Cell nuclear extracts (50 µg) were separated on a 10% polyacrylamide-SDS gel alongside a protein standards (Amersham Life Science, Inc.). The protein was transferred onto a nitrocellulose membrane. For the Western blot, the membrane was blocked with 5% nonfat milk in Tris-buffered saline-Tween, washed in Tris-buffered saline-Tween, and incubated with a affinity-purified polyclonal antibody corresponding to residues 520-538 of the human Sp1 protein (Santa Cruz Biotechnology) at 4 °C overnight. After incubating with the secondary antibody and washing, the signal was detected using ECL detection system (Amersham).
For the Southwestern blot, the membrane was treated with decreasing concentrations of guanidine HCl (6, 3, 1.5, 0.75, 0.375, and 0.1875 M) and prebound in 10 mM Na2(PO4), pH 7.4, 5% nonfat milk, 150 mM NaCl, 1% bovine serum albumin, 2.5% polyvinylpyrrolidone 40, 0.1% Triton X-100 at 4 °C for 1 h. Binding was performed at 4 °C overnight in a buffer containing 10 mM Tris, pH 7.5, 0.5% nonfat milk, 0.5% bovine serum albumin, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 2 mM dithiothreitol, 0.1% Triton X-100, 5% glycerol, and 10 µg/ml salmon sperm DNA. 106 cpm/ml of the above probe used for footprinting analysis was used. The membrane was washed twice for 30 min in the binding buffer less single-stranded DNA at 4 °C and exposed to x-ray films for 2 h.Mutagenesis-- The deletion mutant of the PTTG promoter was generated using Exsite PCR-based site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The PCR primers include the upstream primer, 5'-GGAAAAAAAAAAGCTTAGTAGTAGAGACCAACAT-3' and the downstream primer, 5'-GGTGGCGTAGTCTTGGGTGAGTTTTAGCTGCGA-3'.
The point mutations in the two consensus Sp1 binding sites were generated using QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. The mutagenesis primers include Sp1MI5', 5'-TTCCGGGATGGGGTCGTGGTTGAAAACTTCCG-3'; Sp1 MI3', 5'-CGGAAGTTTTCAACCACGACCCCATCCCGGAA-3'; Sp1MII 5', 5'-GACACTAGGCCCATCCCCCCATCGGATGGACC-3'; Sp1MII3', 5'-GGTCCATCCGATGGGGGGATGGGCCTAGTGTC-3'. The double Sp1 mutant was generated using plasmid containing the first Sp1 site mutations as template and primers containing the second Sp1 site mutations in the PCR reaction. All mutants were sequenced to confirm the desired mutations. ![]() |
RESULTS |
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Genomic Organization of PTTG Gene-- The rat PTTG gene was isolated from a genomic library. The exon-intron boundaries and the size of introns were determined by PCR, using primers derived from PTTG cDNA sequence (see "Materials and Methods") to synthesize DNA from phage DNA. The size of the introns were determined by subtracting exon sequences from each PCR fragment (Fig. 1). The exon-intron boundaries were sequenced, and all the exon/intron splice junctions of PTTG gene showed the characteristic splice site consensus sequence GTAG (Fig. 1). These results together with results from Southern blot analysis and restriction mapping indicated that the PTTG gene contains five exons separated by four introns ranging from 640 bp to 1.8 kilobase pairs. Fig. 2 depicts the genomic structure of the rat PTTG gene.
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Characterization of PTTG Gene 5' Region-- To map the transcription start site of the PTTG gene, a 533-bp fragment containing part of exon 1 was used to generate an antisense probe (Fig. 2). As shown in Fig. 3, hybridization of this probe to RNA derived from rat testis, testis Leydig, and pituitary GH4 cell lines results in a protected band of 71 bp (adjusted to reflect the mobility difference between RNA and DNA) in size, whereas no protected band was apparent when the probe was hybridized to yeast RNA. When the protection assay was performed alongside a sequencing ladder, the transcription start site was mapped to a thymidine residue, 44 bp upstream from the ATG initiation codon (Fig. 4).
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PTTG mRNA Expression in Testicular Cell Lines-- Because testis is the only adult rat tissue that expresses PTTG mRNA, the expression of PTTG mRNA was examined in cell lines representing its three major cell types. Fig. 5 shows that all three cell lines, including Leydig, Sertoli, and germ (GC2) cells expressed PTTG mRNA to high levels. These cell lines provide a convenient tool to study transcriptional regulation of PTTG in the testicular cell culture system.
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Activity of the 5'-flanking Region of the PTTG
Promoter--
To identify the DNA sequences involved in
transcriptional regulation of the PTTG gene in testicular
cells, various portions of the PTTG 5'-flanking region were
cloned in front of a luciferase reporter gene and transiently
transfected into testis germ GC2 and Leydig cells. Constructs beginning
at 1779,
1530,
1315,
1054,
745,
462, and
194 bp relative
to the transcription start site were tested. The downstream boundary of
each construct was located at +43 bp relative to the start site of
transcription. As shown in Fig. 6,
constructs containing up to 462 bp of PTTG upstream
sequences were not able to induce luciferase activity significantly
over a promoterless control in GC2 cells (germ cells). However,
construct containing 745 bp of the PTTG 5'-flanking region induced luciferase activity approximately 43-fold over control. The
luciferase activity reached maximal (79-fold over control) with the
construct containing 1,054 bp of the PTTG 5' sequences. Inclusion of more upstream sequences resulted in a slight reduction in
luciferase activity (Fig. 6, constructs
1315LUC,
1530LUC, and
1779LUC). Similar results were obtained when these fusion constructs
were transfected into Leydig cells (Fig. 6), although the overall
induction of luciferase activity was lower than that of the GC2 cells.
These results indicate that a minimal 745 bp of the 5'-flanking
sequences are required for transcriptional activation of
PTTG in testicular germ and Leydig cells and that a
transcriptional enhancer sequence is present between
462 and
745
bp.
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GC2 Cell Nuclear Protein(s) Interacts with PTTG
5'-flanking Sequences--
The above cell transfection data indicates
that a potential transcriptional enhancer sequence is present between
462 and
745 bp of the PTTG 5'-flanking region. To
determine whether any testicular cell nuclear proteins specifically
interact with the DNA sequences in this region, DNaseI footprinting
assays were performed. As shown in Fig.
7, panel A (plus strand), a
footprint between
509 and
624 bp on the plus strand was present
when GC2 nuclear extracts was added. The protection from DNaseI became more evident with increasing amounts of GC2 nuclear extracts, and the
protection was competed by the unlabeled homologous DNA fragment. When
purified Sp1 protein was used in the assay, the footprints were
confined to the two consensus Sp1 binding sequences between
638 and
624 bp and between
572 and
551 bp (Fig. 7, panel A).
Similar results were observed when the minus strand DNA was labeled
(Fig. 7, panel B). These results suggest that GC2 cell
nuclear protein(s), in addition to Sp1, specifically binds to PTTG
5'-flanking region between
509 and
624 bp and that the interaction
between these proteins and the DNA sequence may result in
transcriptional activation of PTTG gene in testis germ cells.
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Deletion between 509 and
624 bp Results in Loss of PTTG
Promoter Function--
To determine whether the binding site for
multiple testicular nuclear proteins is responsible for PTTG
transcriptional activation, the DNA sequence between
509 and
624 bp was deleted from the fusion construct
745LUC. As shown in
Fig. 9, when transiently transfected into
GC2 cells, the luciferase activity of the deletion mutant was reduced
to background level, suggesting that the sequence between
509 and
624 bp constitutes the core enhancer sequence that is critical for
PTTG transcriptional activation in testicular cell lines.
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Point Mutations with the Sp1 Binding Sites Has Little Effect on PTTG Transcriptional Activation-- To determine the role of Sp1 protein in PTTG transactivation, point mutations were made within the two consensus Sp1 binding sites in the core enhancer sequence (see "Materials and Methods"). Fig. 9 shows that mutations within each individual Sp1 site resulted in a 20-40% reduction in luciferase activity. When both Sp1 sites were mutagenized, luciferase activity decreased to about 50% that of the wild type level (Fig. 9). However, the luciferase activity of the double Sp1 mutant was still 34-fold over the construct, in which the entire 115-bp core enhancer sequence was deleted. These results suggest that the Sp1 binding sites do not play a crucial role in PTTG transcriptional activation.
The PTTG Enhancer Is Capable of Transcriptional Activation of a Heterologous Promoter-- To determine whether the 115-bp sequence in the PTTG 5'-flanking region is able to confer transcriptional activation to a heterologous promoter, this sequence was cloned in front of a minimal TK promoter linked to luciferase reporter gene (TKLUC) in both orientations. When transfected into GC2 cell, the enhancer sequence in either orientation induced a 10-fold increase in TK transcription (Fig. 10), suggesting that the PTTG enhancer was able to confer transcriptional activation to a heterologous promoter.
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DISCUSSION |
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The structural organization of the entire rat PTTG gene was characterized in this study. Previous study showed that PTTG transcript in testis is different in size from that of tumor cells (1). In this study, the same transcription start site was observed in both normal testis and in testicular and pituitary cell lines, suggesting that the different transcript size in testis is not a result of using alternative promoters. Whether the different transcript size is a result of differential splicing or using an alternative polyadenylation site needs to be clarified in future studies. The testis commonly gives rise to transcripts that are differentially processed or are derived from alternate promoters, compared with somatic tissues (5-13). For example, alternative transcriptional initiation gives rise to a different transcript encoding proopiomelanocortin in testis, whereas alternative splicing is responsible for the generation of testis-specific transcripts of prodynorphin and proenkephalin (13).
The original PTTG cDNA was isolated from a rat pituitary somatotroph tumor cell line (1). Surprisingly, PTTG transcript is also present in many other immortalized and malignant cell lines I have examined, regardless of cell lineage (e.g. lymphoid, myeloid, mesenchymal, and epithelial). However, in rat adult tissues, PTTG displays a very selective pattern of expression. PTTG transcript is present only in testis but not in most adult tissues we have analyzed (1). This distinct expression pattern of PTTG mRNA is similar to several other genes including proto-oncogenes c-mos and c-abl (2) and PEM, a homeobox gene (14). Testis consists of multiple cell types, and PTTG mRNA is expressed to high levels in both germ (GC2) and supporting Sertoli and Leydig cell lines. Whether this expression pattern represents PTTG mRNA expression in intact testis will be determined by in situ hybridization in future studies.
Transient transfection of testicular cell lines showed that PTTG
transcriptional activation requires at least 745 bp of the 5'-flanking
sequences. Although a TATA box-like sequence is present near the
transcription start site, it is not sufficient for transcriptional activation of PTTG gene; sequences further upstream (i.e. 5'
of 462 bp) are required. Several transcription activator binding sites including AP-2 (15, 16) and EGR-1 (17, 18) are present in this
region. Both AP-2 and EGR-1 are inducible enhancers. AP-2 mediates
transcriptional activation by cAMP and phorbol esters (16). EGR-1 can
be induced by a variety of stimuli and activates transcription of
target genes whose products are required for mitogenesis and
differentiation (17-20). Although neither AP-2 nor EGR-1 site seems to
be involved in basal promoter activity of PTTG gene, their roles in
hormonal or growth factor-induced transactivation will be investigated
in the future.
Results from transient transfection studies also indicated that a
potent transcriptional enhancer is present between 462 and
745 bp
of the PTTG. There are two consensus Sp1 binding sites in this region.
Sp1 is an ubiquitously expressed transactivator that binds to GC box
sequence and regulates basal transcription of a variety of housekeeping
genes (21). Sp1 also plays a role in directing tissue-specific,
hormonal, and developmental regulation of gene expression. Sp1 has been
shown to regulate expression of erythroid (22)-, lymphocyte (23)-, and
monocyte (24)-specific genes and as a modulator of the retinoic
acid/cAMP-dependent transcription of the tissue-plasminogen
activator gene (25). DNaseI footprinting assays demonstrated that
although purified Sp1 protein binds to its consensus binding sequence
in this region, the region protected by testicular cell nuclear
extracts is much larger, indicating that additional proteins in
testicular cells other than Sp1 may interact with the DNA sequence.
Western and Southwestern blot analysis showed that in addition to Sp1
protein, three distinct proteins of 35-, 46-, and 60-kDa also
interacted with this DNA sequence and that the 46- and 60- kDa proteins
were testicular cell-specific. These results support the observation
from DNaseI footprint assays that multiple nuclear proteins interact
with PTTG enhancer sequence and that the two testicular cell-specific proteins may play a role in testis-specific expression of the PTTG
gene.
Deletion of this multiple protein binding site completely abolished
PTTG transcriptional activation in testicular cells, indicating that
the DNA sequence between 509 and
624 bp constitutes the core
enhancer sequence that interacts with multiple proteins to activate
PTTG transcription. That this 115-bp sequence is a true enhancer was
demonstrated further by its ability to activate transcription from a
heterologous promoter in an orientation-independent manner. Regulation
of gene expression by Sp1 through its interactions with transcription
factors bound to adjacent cis-acting elements has been demonstrated.
Sp1 has been found to interact with nuclear factor-kB (26), Ets (27),
and steroid receptors (28, 29). My results showed that point mutations
within each individual or both Sp1 consensus sequences in the enhancer
sequence did not have a remarkable effect on PTTG transcription,
suggesting that the Sp1 binding sites are not critical for PTTG
transcriptional activation; binding sites for other testicular nuclear
proteins within the enhancer sequence are required.
In summary, I have isolated and characterized the structure of the rat PTTG gene. I have also identified an enhancer element in the PTTG 5'-flanking region that is the binding site for multiple testicular nuclear proteins. The interaction of these proteins with PTTG promoter may activate its transcription in testicular cells.
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ACKNOWLEDGEMENT |
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The author would like to thank Dr. Shlomo Melmed for his support.
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FOOTNOTES |
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* This work is supported by National Institutes of Health Grant DK-02346 (to L. P.).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) AF021802.
To whom correspondence should be addressed: Div. of Endocrinology,
Cedars-Sinai Medical Center, 8700 Beverly Blvd., D-3066, Los Angeles,
CA 90048. Tel.: 310-855-7682; Fax: 310-559-2357; E-mail:
Pei{at}CSMC.edu.
1 The abbreviations used are: PTTG, pituitary tumor-transforming gene; bp, base pair(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; AP, activation protein; EGR, early growth response.
2 L. Pei, unpublished data.
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REFERENCES |
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