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Endocrinology Vol. 141, No. 2 763-771
Copyright © 2000 by The Endocrine Society

ARTICLES

Characterization of the Murine Pituitary Tumor Transforming Gene (PTTG) and Its Promoter1

Zhiyong Wang and Shlomo Melmed

Cedars-Sinai Research Institute, University of California School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Academic Affairs 2015, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently isolated rat pituitary tumor transforming gene (PTTG) complementary DNA and showed its potent in vitro and in vivo transforming activity. We now characterize the mouse PTTG gene and its promoter. The entire gene is composed of five exons and four introns and spans about 7 kb. Northern analysis showed that PTTG was expressed in several tumor cell lines examined, but not in all normal tissues, implying a correlation between PTTG and tumorigenesis. Using rapid amplification of 5'-cDNA ends, the transcription start site was localized at -303 nucleotides upstream to the ATG codon in both F9 and AtT20 cells. An approximately 4.3-kb upstream region demonstrated promoter activity in AtT20 cells as well as other cell lines tested, and in vivo, the cloned promoter driving an enhanced green fluorescent protein transgene exhibited transcriptional activation in testis and embryo. Serial deletions showed that -313 bp of the 5'-flanking region was critical for promoter activity. Three elements contribute to promoter activity. Both element A (-313/-293) and element C (-180/-160), in an electrophoretic mobility shift assay using NIH-3T3 nuclear extract, formed three specific complexes, which were competed by a known Sp1 oligo; one complex was supershifted by Sp1 antibody, and the other two complexes were both supershifted by an Sp3 antibody. Two mutants disrupting element A resulted in up to 70% loss of promoter activity and abrogated formation of specific DNA-protein binding complexes, implying a more important role for element A. Element B (-249/-229) shows more than 80% homology to a consensus c-myb element, but formed two specific complexes that differed from that of c-myb in the electrophoretic mobility shift assay. Thus, the integrity and possible cooperation among these elements contribute to the basal promoter activity of the mouse PTTG oncogene homolog.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TUMORIGENESIS is a complex process during which cell population equilibrium is lost due to enhanced proliferative and or apoptotic defects. The earliest event in tumorigenesis is generally believed to be cellular transformation occurring in a single cell, which deviates from normal cellular control. Several categories of genes, including protooncogenes, tumor suppressor genes, and DNA repair enzymes, are involved in the normal control mechanisms that affect cell proliferation pathways, apoptosis, DNA repair, angiogenesis, and metastasis (1). About 200 known oncogenes, under certain conditions, regulate cellular proliferation, death, migration, and adhesion to cause neoplastic transformation. Major oncoproteins include growth factors such as platelet-derived growth factor, tyrosine kinases such as epidermal growth factor receptor and Src; G proteins such as H-Ras and K-Ras, serine kinases such as breakpoint cluster region protein, and transcription factors such as Fos, Jun, and Myb (1).

A novel oncogene, pituitary tumor transforming gene (PTTG), recently cloned from GH4 rat pituitary cells in our laboratory (2), encodes a novel protein of 199 amino acids with no significant similarity to known proteins. Overexpression of rat PTTG in NIH-3T3 fibroblasts induced cellular transformation in vitro, and injection of PTTG-transfected 3T3 cells into athymic nude mice generated tumors, indicating that PTTG is a transforming gene (2). The human homolog of rat PTTG (hPTTG) has also been identified (3, 4), and its expression in human pituitary adenomas has been assayed and implied in early development of prolactinoma (5, 6). hPTTG also demonstrated transforming ability, and its overexpression in 3T3 cells resulted in increased expression of basic fibroblast growth factor-2, a potent angiogenic growth factor (3). In another study, hPTTG was overexpressed in Jurkat cells and hemopoietic malignant tumor samples, and its acidic C-terminal region acts as a trans-activation domain when fused to a heterologous DNA-binding domain in both yeast and mammalian cells (4). More recently, PTTG was shown to behave as a vertebrate sister chromatid separation inhibitor, providing a potential mechanism for PTTG to mediate aneuploidy and genetic instability, thus contributing to cell malignancy (7). hPTTG gene 2, which shows high homology with cloned human PTTG, has recently been reported, suggesting the presence of a PTTG gene family (8).

We now report cloning of the murine homolog of the rat PTTG complementary DNA (cDNA; which is most homologous to h PTTG gene 1), screening of the entire gene, and characterization of its promoter. We found that PTTG is a highly conserved protein, although PTTG is detected in only a few normal tissues, it is expressed in all tumor cell lines examined. The 4.3-kb cloned promoter is active both in vitro and in vivo, and the -313 bp region is critical for promoter activity. Detailed in vitro analysis shows that three elements, including two Sp1 motifs, are required for transcriptional activity, and the other element binds to a novel transcription factor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Homology cloning of murine PTTG cDNA
Total RNA was isolated from murine F9 and AtT20 cells, and 1 µg total RNA was subjected to RT-PCR using a pair of primers designed from the reported rat PTTG cDNA sequence (1): M5PTTG2S (5'-GGAGACAGTTGTTTGGGTGCCAAC) and MPTTG5AS (5'-AATATCTGCATCGTAACAAACAGG). PCR parameters were 94 C for 30 min, 57 C for 30 min, and 72 C for 1 min and 30 sec for 30 cycles. A specific band of about 800 bp was amplified from both F9 and AtT20 samples, subsequently cloned into pCRII (Invitrogen, San Diego, CA), and sequenced. Sequencing revealed that cDNA clones from F9 and AtT20 were identical.

Plasmid and antibodies
A fusion protein vector was constructed to link mPTTG cDNA with a C-terminal His tag using pcDNA3.1-c-myc as backbone (Invitrogen). Plasmid pGL3-U2P harboring an alternative human LIFR promoter was previously described (9). Antibodies to Sp1 and Sp3 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell culture
Mouse fibroblast 3T3 lsqb]American Type Culture Collection (ATCC; Manassas, VA) CCL-92] and pituitary tumor AtT20 (ATCC CCL-89) were maintained in DMEM low glucose medium (Life Technologies, Inc., Gaithersburg, MD) with 10% FBS; mouse MOP8 (ATCC CRL-1709) and mouse embryonal carcinoma F9 (ATCC CRL-1720) were maintained in DMEM high glucose medium (Life Technologies, Inc.) with 10% FBS. All culture media were supplemented with standard antibiotics, and cells were passaged twice weekly.

Genomic library screening
Approximately 1 x 106 plaques of a mouse 129 Sv genomic library (Stratagene, La Jolla, CA) were screened using the cloned murine PTTG cDNA as probe. Five positive clones were obtained and further analyzed. Restriction mapping and Southern blot analysis revealed that two clones contained about 4- and 1.5-kb fragments upstream to the ATG start site. NotI-EcoRI fragments from these two clones were subcloned into pBluescript for subsequent promoter analysis.

Rapid amplification of 5'-cDNA end (5'RACE)
5'RACE was performed as previously described (8). Using total RNA from F9 and AtT20 cells, MPTTG3AS (5'-CAACAAAGATCAGAGTAGCCATCC-3') was used for RT; in the PCR, the murine PTTG-specific primer was MPTTGS2AS (5'-CAGAAATCAGTTTATGTTGGCACCCA-3'), and PCR parameters were 94 C for 30 min, 59 C for 30 min, and 72 C for 1 min for 30 cycles.

Northern blot analysis
Normal murine tissue total RNA was purchased from Ambion, and total RNAs from cell lines were prepared using Trizol reagent (Life Technologies, Inc.). Electrophoresis and transfer were performed as previously described (9). Murine PTTG cDNA fragment was used as a probe and labeled to a specific activity of approximately 1 x 109 cpm/µg with a random labeling kit (Prime-it II, Stratagene). The membrane was prehybridized in QuikHyb solution (Stratagene) at 68 C for 20 min and hybridized with the probe at 68 C for 2 h. After hybridization, the membrane was washed with 2 x SSC (standard saline citrate)-0.1% SDS twice at room temperature for 30 min each time, and then with 0.1 x SSC-0.1% SDS at 60 C for 30 min.

Construction of plasmids for promoter analysis
A genomic clone consisting of an approximately 4-kb fragment upstream of the ATG start site was used. The NotI-EcoRI fragment was subcloned into pBluescript II (Stratagene) and then into pGL3-Basic (Promega Corp., Madison, WI), and pGL3-MP was generated by EcoRI and HindIII digestion to partially eliminate the first exon and first intron region, which now contained the entire 4.3-kb 5'-flanking region and the partial 5'-noncoding region. Serial nested deletions were generated using either exonuclease III and mung bean nuclease (10) or the PCR-based method (Stratagene). All deletions were verified by sequencing.

Transient cell transfections
For promoter activity analysis, pGL3-Basic (Promega Corp.) was used as a negative control. pCMV-ß-galactosidase (ß-Gal; Stratagene) was cotransfected as an internal control. Plasmid DNAs were prepared using the Maxi-prep kit (QIAGEN, Chatsworth, CA). Cells were transfected using standard Lipofectamine (Life Technologies, Inc.) or calcium phosphate precipitation methods. Each plasmid sample was cotransfected with pCMV-ß-Gal in triplicate, and individual transfections were repeated at least twice. Forty-eight hours after transfection, cell lysates were prepared for measurement of luciferase and ß-Gal activities. All transfections were performed in triplicate, and luciferase activities were normalized to ß-Gal activities.

Preparation of nuclear extract, electrophorectic mobility shift assay (EMSA), and supershift assay
Crude nuclear extracts were prepared as previously reported (9). Protein concentrations were quantitated using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Richmond, CA). For EMSA, one strand of the oligonucleotide duplex was element A [-313 nucleotides (nt)/-293 nt 5'-AGGCTGTAGGCCCACCTCCTC-3'), element B (-249 nt/-229 nt 5'-TGTGCCGGGTCGTTGGTGGCG-3'), and element C (-173 nt/-153 nt 5'-GCTTGGGGTGGCGGGGAGGGC-3'). Labeled oligonucleotide duplex (20,000–30,000 cpm) was mixed with about 5 µg nuclear extract, 1 µg poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ) in 25 µl reaction buffer [10 mM Tris-Cl (pH 7.5), 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, and 8% glycerol], and incubated at room temperature for 15 min. For competition assays, a 200-fold excess of cold competitor oligos was added before the addition of labeled probe. For supershift assays, antibody was added after the addition of labeled probe and was incubated at room temperature for 30 min, EMSA samples were resolved on a 5% nondenaturing polyacrylamide gel. Sp1 and Oct-1 oligonucleotide sequences were used in competition assays.

Mutagenesis
Mutagenesis was performed by the ExSite method (Stratagene). For mutations in the -313 nt/-293 nt (element A) region, three mutants were generated, and three pairs of primers were 5'-AAGCTGAGAACCAATGGGAGC-3' and 5'-AAGGGCCCACCTCCTCTCGGA-3', 5'-AAGTACAGCCTGAGAACCAATG-3' and 5'-AAGCCTCCTCTCGGAGGGACC-3', 5'-AAGTGGGCCTACAGCCTGAGA-3', and 5'-AAGCTCGGAGGGACCAATTGAG-3'.

Production of PTTG promoter-driven enhanced green fluorescent protein (EGFP) transgenic mice
The cloned 4.3-kb mPTTG promoter was subcloned from pGL3-MP into promoterless pEGFP-1 vector (CLONTECH Laboratories, Inc., Palo Alto, CA) and designated pmPTTGP-EGFP, the mPTTG promoter-EGFP transgene was linearized by digestion of KpnI and AflII, gel-purified, and microinjected into FVN fertilized eggs. Transgenic mice were bred according to standard protocol, and mouse tail genomic DNA was used to detect positive transgenic mice by PCR and Southern blot. The protocol for animal usage has been approved by institutional animal care and use committee at Cedars-Sinai Medical Center.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of murine PTTG cDNA and its genomic structure
By testing several pairs of degenerate primers designed from the rat PTTG cDNA sequence (1), one pair produced a prominent, approximately 800-bp band during RT-PCR using F9 or AtT20 RNA samples. Cloning and sequencing revealed that PCR products from F9 and AtT20 consisted of identical sequences and contained an entire coding region. As rat and human PTTG cDNA have both been cloned (2, 3, 4), amino acid sequence comparison revealed that murine PTTG protein shared 88% and 66% homology with rat and human PTTG, respectively (Fig. 1Go). Long stretches of conserved amino acids (more than eight amino acids) were found in the internal and C-terminal regions of the protein, but not in the N-terminus.



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  		Figure 1. Protein homology comparison of PTTG. Murine PTTG protein sequence was aligned with rat and human PTTG; different amino acid residues in the same positions are indicated. In comparison to murine PTTG, rat and human PTTG have three and six residue insertions, respectively; inserted residues are underlined.

 
Subsequently, using the murine PTTG cDNA as a probe, a genomic library was screened, and five positive clones were obtained. Exonic sequencing, PCR, and restriction enzyme digestion revealed that the entire murine PTTG gene spans about 7 kb and consists of five exons and four introns, as shown in Fig. 2Go. The sizes of the exons are 391, 179, 94, 150, and 131 bp, respectively, and the sizes of the four introns, as determined by PCR, are about 0.9, 2.1, 2, and 0.8 kb, respectively.



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  		Figure 2. Genomic structure of murine PTTG and its structural correlation with cDNA. For the murine PTTG gene, 0 represents the transcription start site, shaded areas represent exons, and restriction sites are indicated. For murine PTTG cDNA, the coding region is gridded, and 5'- and 3'-nontranslational regions are open.

 
Distribution of PTTG transcripts in normal tissues and tumor cell lines
Using the murine PTTG cDNA as probe, Northern blots were performed to identify the distribution of PTTG transcripts in normal tissues. As depicted in Fig. 3Go, normal murine tissues, including thymus, spleen, testis, and ovary as well as intact embryo, showed detectable PTTG expression, whereas liver, heart, lung, brain, and kidney transcript were undetectable by Northern blotting. RNA samples from F9 embryonic carcinoma and AtT20 pituitary tumor cell lines showed much higher expression of PTTG than the normal tissues. The major transcript detected has a size of about 1 kb; moreover, in several samples, including heart, whole embryo, and F9 and AtT 20 cells, an approximately 1.7-kb band was detected, and in whole embryo and embryonic F9 cell samples, a third, approximately 3.0-kb band was also detected, implying the presence of alternatively spliced transcripts.



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  		Figure 3. PTTG gene expression patterns in normal tissues and tumor cell lines. Northern analysis of murine PTTG messenger RNA in normal tissues and in two tumor cell lines. About 20 µg total RNA from the indicated normal tissues or cell lines were loaded in each lane, and the internal GAPDH control is shown below. The major transcript detected has a size of about 1 kb. 1, Liver; 2, heart; 3, testis; 4, lung; 5, spleen; 6, thymus; 7, ovary; 8, brain; 9, kidney; 10, whole embryo; 11, F9 embryonic carcinoma cells; 12, AtT20 pituitary tumor cells.

 
Determination of the murine PTTG transcription start site and the -313/-150 nt region as the critical region for promoter activity
We performed 5'RACE to locate the transcription start site(s) of murine PTTG, using MPTTG3AS as the RT primer and MPTTGS2AS as the gene-specific primer in PCR. Cloning and sequencing of 5'RACE products in both F9 and AtT20 RNA samples showed that murine PTTG had a single transcription start site, 303 nt upstream of the PTTG ATG translation start site. Subsequent 5'RACE showed that in untransformed NIH-3T3 cells the same transcription start site was located for the murine PTTG gene. Thus, the complete cDNA sequence is 945 nt (deposited in GenBank under accession no. AF069051), and the coding region of murine PTTG spans 304–894 nt, encoding a total of 196 amino acids (Fig. 1Go).

Subsequently, using a reporter vector containing an approximately 4.3-kb sequence upstream to the transcription start site and 226 nt in the 5'-noncoding region, designated pGL3-MP (murine promoter), we detected robust promoter activity in AtT20 and 3T3 cells. This vector contains -4333 nt sequence upstream of the murine PTTG transcription start site, and the -4333 nt promoter sequence has been deposited in GenBank under accession no. AF060887.

A total of nine representative, serially deleted subclones were generated from pGL3-MP, and their corresponding promoter activities in AtT 20 and other cells are shown in Fig. 4Go, a and b. As depicted, sequences up to -313 nt are critical for promoter activity, and sequences deleted from -313 and -150 nt (pGL3-MP-D5) resulted in complete loss of promoter activity. Notably, although this promoter was cloned from a murine source, it is also active in several human cell lines, as shown in Fig. 4bGo, indicating that highly conserved transcription factors are probably responsible for promoter trans-activation. It is also notable that promoter activities in tumorous cells, such as U-2 OS, F9 (Fig. 4bGo), JEG-3, MCF-7, and Hep 3B (data not shown), are significantly higher than those in nontransformed NIH-3T3 and Hs 67 cells, implying that the cellular neoplastic status may correlate with up-regulation of PTTG gene expression. Database searching demonstrated several potential transcription factor-binding sites, including Sp1, activating protein-4, and c-Myb, within -313 nt to 1 nt of PTTG (Fig. 5Go).



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  		Figure 4. Determination of the murine PTTG transcription start site and promoter activity analysis. a, Promoter activity of the cloned 4.3-kb fragment in AtT20 cells. Nine deletion constructs of pGL3-MP were cotransfected by lipofection with pCMV-ß-Gal as transfection control into 3T3 cells. pGL3-MP-D7, pGL3-MP-D8, and pGL3-MP-D9 represent deletion at -313/-293, -252/-222, and -180/-150 nt, respectively. The relative promoter activity of each construct is shown as the fold increase over a promoterless luciferase control pGL3-Basic, whose activity is set at 1, after normalizing to ß-Gal activity. Values represent the mean ± SE of triplicate determinations. b, Promoter activity comparison of pGL3-MP in different cells. Several typical constructs derived from pGL3-MP were transfected into the indicated cell lines, and their promoter activities were measured in triplicate transfection assays. Luciferase activity was normalized to ß-Gal activity, and for each cell, the normalized pGL3-Basic control reading was set at 1. 1, pGL3-Basic; 2, pGL3-MP; 3, pGL3-MP-D4; 4, pGL3-MP-D5.

 


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  		Figure 5. Potential transcription factor binding sites in the -313/+1 region of the murine PTTG promoter. Potential elements are shown in boldface, with corresponding factors shown below.

 
In vivo function of cloned PTTG promoter
To test whether the cloned PTTG promoter is functional in vivo, transgenic mice harboring a transgene consisting of the EGFP gene under the control of the cloned PTTG promoter were produced. Various tissues, including testis, kidney, lung, ovary, liver, spleen, etc., and whole embryo have been examined. Tissue sections of both an adult mouse testis and embryos showed bright green fluorescence (Fig. 6Go), whereas other adult tissues did not show significant green fluorescence (data not shown). These observations show that the cloned PTTG promoter is highly active in testis.



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  		Figure 6. In vivo function of the cloned PTTG promoter. Transgenic mice harboring cloned PTTG promoter-driven EGFP (enhanced green fluorescent protein) genes were produced. Slide sections were prepared from an adult murine testis and an embryonic day 17.5 testis, and photographed under a fluorescent microscope. Cells showing bright green fluorescence are spermatogonia.

 
Three elements contribute to PTTG promoter activity
More detailed deletions within the -313 and -150 nt regions showed that three elements contributed to promoter activity. Deletion of element A (-313/-293), B (-249/-229), and C (-173/-153) reduced the promoter activity by 70%, 40%, and 40%, respectively, showing that element A plays a more significant role than the other two elements. Database searching revealed that elements A and C resembled an Sp1 consensus binding element, whereas element B showed similarity to a c-Myb consensus binding element. EMSA confirmed that elements A and C did, in fact, bind to Sp1 specifically (Fig. 7aGo), and they also competed with each other in binding to Sp1. Element B also gave rise to two specific binding complexes in EMSA (Fig. 7bGo), which was, however, not competed by a c-Myb motif, showing that B is probably a novel element. Using nuclear extracts derived from several different cell lines it was found that these two specific binding complexes are present in all of the samples tested, showing that the nuclear protein(s) corresponding to element B is a widespread and general transcription factor(s). Furthermore, an oligo spanning -221/-201 nt showed some homology to an activating protein-4 consensus element, but did not yield specific binding in the EMSA (data not shown).



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  		Figure 7. EMSA characterization of three elements contributing to PTTG promoter activity. In the EMSA, the free probe is run out of the gel matrix to maximize resolution of the binding complex; thus, unbound free probe is not depicted. a, EMSA of elements A and C. In each reaction about 5 µg NIH-3T3 cell nuclear extract was added as well as the indicated radiolabeled probe, EMSA was performed as indicated in Materials and Methods. Lane 1, Element A as radiolabeled probe; lanes 2–5, cold competitor, as indicated, was added at a 200-fold excess concentration besides the hot probe; lane 11, element C as radiolabeled probe; lanes 12–15, cold competitor, as indicated, was added at a 200-fold excess concentration besides the hot probe. The specific binding complex is indicated with an arrow. b, EMSA of element B. NIH-3T3 cell nuclear extracts were used in lanes 1–4. Lane 1, Element B as radiolabeled probe; lanes 2–4, cold competitor, as indicated, was added at a 200-fold excess concentration besides the hot probe. Lanes 11–16, different nuclear extracts, as indicated, were added. Specific binding complexes are indicated as arrows.

 
As element A contributes more than elements B and C to promoter activity, three mutants were generated within this fragment, replacing every six bases, and mutant promoter activities were compared with those of the wild-type construct. EMSA results of the corresponding mutant oligos were also compared with those of the wild-type oligo, and the results are shown in Fig. 8Go. As depicted, mutating -311/-06 nt did not alter promoter activity, nor did the corresponding mutant oligo lose its ability to compete with wild-type oligo in the EMSA. On the other hand, mutating -305/-300 nt and -299/-294 nt reduced promoter activity by 60% and 65%, respectively, and both corresponding mutant oligos lost their abilities to compete with wild-type oligo in the EMSA. These results confirmed that -305 to -293 nt was the critical region within -313 to -293 nt for the murine PTTG promoter activity.



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  		Figure 8. Loss of transcription factor binding to element A results in significant loss of PTTG promoter activity. About 5 µg AtT20 cell nuclear extract were used in each reaction. a, EMSA of element A and its mutant oligos. Three mutant oligos were designed to replace the contiguous six bases in element A as shown and were used in competition assay to test their transcription factor binding ability. Lane 1, Element A as radiolabeled probe; lanes 2–4, cold competitors as indicated were added at a 200-fold excess concentration besides the hot probe. b, Promoter activity analysis of pGL3-MP-D4 and three mutants with mutations at contiguous six bases in element A as shown in a. Transfection and luciferase assays were performed as indicated in Materials and Methods.

 
Database searching also revealed a potential c-myb oncogene binding site, at -243 to -235 nt. We therefore performed EMSA using -249 to -229 nt region as a probe and found that it produced two specific binding complexes; nevertheless, these specific bindings could not be competed by c-myb oligo despite high homology with the c-myb consensus binding site, indicating that the corresponding transcription factor(s) are probably novel factors rather than c-myb.

Sp1/Sp3 are major transcription factors binding to elements A and C
As shown in Fig. 7aGo, elements A and C can compete with each other, and both elements produced three specific binding complexes in the EMSA. As element A seems to be more important for promoter activity, element A was used for further study. When using this -313/-293 element as a probe for the EMSA, three specific binding complexes were produced (Fig. 9Go). Database searching indicated the presence of a potential Sp1 binding site. We performed EMSA competition and supershift assays to test whether this is indeed the case. The results presented in Fig. 9Go show that all three specific binding complexes were competed by a known Sp1 oligo; one complex was supershifted by an Sp1 antibody (lane 5); the other two complexes were supershifted by an Sp3 antibody (lane 6), indicating that these two complexes involve interaction with Sp3 protein.



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  		Figure 9. Sp1/Sp3 binds to element A specifically. EMSA and supershift assay of element A and its binding proteins; about 5 µg AtT20 cell nuclear extract were used in each reaction. Lane 1, Element A as radiolabeled probe; lanes 2–4, cold competitors, as indicated, were added at a 200-fold excess concentration besides the hot probe; lanes 5 and 6, the same as lane 1, except that an Sp1 antibody or an Sp3 antibody was added to the mixture, respectively. Supershifted complexes are arrowed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although rat and human PTTG cDNAs have been cloned, detailed knowledge of the murine PTTG cDNA and gene is essential for in vivo functional study using transgenic and knockout techniques. Thus, we specifically screened a 129 Sv murine genomic library and characterized the murine PTTG gene structure and its promoter. Cloning of murine PTTG cDNA shows that PTTG is a highly conserved protein that shares 88% and 66% homology with rat and human PTTG, respectively. The soft agar assay also indicates that murine PTTG is capable of transforming NIH-3T3 cells (Wang Z., and S. Melmed, unpublished data). Computational analysis revealed that PTTG is rich in alkaline, acidic, and proline residues within conserved regions, suggesting that these regions might be important for PTTG function. In this study, primary, approximately 1-kb murine PTTG transcript was most abundant in the testis and was also detected in thymus, spleen, ovary, and intact embryo (days 10–15), but not in lung, liver, heart, brain, and kidney, arguing that in normal tissues the primary PTTG transcript has a restricted expression pattern. Furthermore, alternative murine PTTG transcripts are also weakly detected. An alternative transcript was cloned and found to yield a murine PTTG variant protein (Wang, Z., and S. Melmed, unpublished data).

Expression of murine PTTG promoter-driven EGFP in the testis demonstrated the functionality of the cloned promoter in vivo. Very low level EGFP activity was observed in other tissues. DNA elements within the mPTTG gene exons or introns (which are absent in EGFP cDNA) may be required to mediate transcription in vivo; alternatively, other DNA elements outside the cloned promoter may be required to mediate transcription in vivo. The xpression level of EGFP in nontesticular tissues may also not exceed the threshold required for green fluorescence emission. The results showed that cloned mPTTG is highly active in the testis in vivo, which is also the most abundant PTTG-expressing normal tissue. It is thus likely that a testis-specific enhancer is included in the cloned promoter and studying the PTTG promoter in different testis cell lines should elucidate the mechanism of testis-preferential expression of PTTG.

the identification of two transcription factor Sp1/Sp3-binding sites as important elements for murine PTTG promoter activity is of interest. Sp1 is an ubiquitous transcription factor that binds to a GC-rich region and plays important roles in regulation of basal and/or inducible gene transcription, Sp1 has also been implicated in the maintenance of methylation-free CpG islands and the formation of active chromatin structures (11, 12, 13, 14). Specifically, Sp1 is an important transcription factor affecting the regulation of several genes involved in cellular growth and differentiation. Sp1 binding elements are found in oncogene promoter regions such as Her2 (15), Abl (16), and Src (17); Sp1 also interacts with the Rb tumor suppressor gene (18, 19) through the Rb control element to regulate several cellular proliferation-related genes (20, 21), including c-fos, c-myc, c-jun, transforming growth factor-ß1, and neu (22, 23, 24, 25, 26). Previous observations have suggested that Sp1 C-terminal phosphorylation by casein kinase II decreased DNA-binding activity (27), whereas dephosphorylation enhanced Sp1 DNA-binding activity (28).

On the other hand, although less characterized than Sp1, Sp3 is also a member of the Sp1 transcription factor family (29) and plays an inhibitory or stimulatory role in trans-activation depending on the promoter used. The results obtained here suggest that the ratio of Sp1 to Sp3 regulates mPTTG promoter activity.

Identification of a novel motif resembling a c-Myb element is also of interest. Its corresponding transcription factor(s) is present in several different cell lines tested, indicating that it may be a widespread transcription factor. As Sp1 also interacts with a variety of other transcription factors, and this novel element is in close proximity to two neighboring Sp1 elements, it is likely that its binding factor(s) may interact with Sp1/Sp3 to regulate basal PTTG promoter activity.

Murine PTTG promoter activities were higher in the transformed than in the nontransformed cell lines tested. Although Sp1/Sp3 are generally considered to be constitutively expressed, nuclear Sp1 levels and phosphorylation status correlate with tumor cell mitotic rates, cellular proliferation, and cell cycle regulators (30, 31, 32). Considering that tumor cells generally have enhanced transcriptional machinery, modification and/or interaction of Sp1/Sp3 and the novel general transcription factor(s) in transformed cells or cell-specific factors in transformed cells may be responsible for the higher mPTTG promoter activity observed here.


   Footnotes
 
1 This work was supported by NIH Grant CA-75979 and the Doris Factor Molecular Endocrinology Laboratory. Back

Received July 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hesketh R 1995 The oncogene and tumor suppressor gene factsbook. Academic Press, San Diego
  2. Pei L, Melmed S 1997 Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Mol Endocrinol 11:433–441[Abstract/Free Full Text]
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