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• W2163854316 abstract "Cyclin-dependent kinase inhibitor p16INK4a is implicated in replicative senescence, cell immortalization, and tumor generation. However, the mechanism regulating its overexpression in senescent cells is unknown. We used the enhanced green fluorescent protein reporter system to scan regulatory elements in the upstream region of p16INK4a. The results of 5′-deletion studies indicated that the transcription regulatory elements contributing to overexpression of p16INK4a in senescent cells were located in the region of the p16INK4a promoter from −622 to −280 bp. According to the results of in vitro DNase I footprinting, EMSA, and Southwestern blotting, we found a novel negative regulatory element, the INK4a transcription silence element (ITSE), at −491 to −485 bp of the p16INK4a promoter. A 24-kDa protein that was highly expressed in young cells may inhibit the expression of p16INK4a by interacting with the ITSE. The activity of the p16INK4a promoter increased significantly in young cells when the ITSE was deleted. The GC-rich region of the p16INK4a promoter from −466 to −451 was a positive transcription regulatory element. Deletion of this region showed 91.4% loss of p16INK4a promoter activity in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably. Cyclin-dependent kinase inhibitor p16INK4a is implicated in replicative senescence, cell immortalization, and tumor generation. However, the mechanism regulating its overexpression in senescent cells is unknown. We used the enhanced green fluorescent protein reporter system to scan regulatory elements in the upstream region of p16INK4a. The results of 5′-deletion studies indicated that the transcription regulatory elements contributing to overexpression of p16INK4a in senescent cells were located in the region of the p16INK4a promoter from −622 to −280 bp. According to the results of in vitro DNase I footprinting, EMSA, and Southwestern blotting, we found a novel negative regulatory element, the INK4a transcription silence element (ITSE), at −491 to −485 bp of the p16INK4a promoter. A 24-kDa protein that was highly expressed in young cells may inhibit the expression of p16INK4a by interacting with the ITSE. The activity of the p16INK4a promoter increased significantly in young cells when the ITSE was deleted. The GC-rich region of the p16INK4a promoter from −466 to −451 was a positive transcription regulatory element. Deletion of this region showed 91.4% loss of p16INK4a promoter activity in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably. INK4a transcription silence element population doubling(s) phosphate-buffered solution enhanced green fluorescent protein dithiothreitol secreted alkaline phosphatase Cellular senescence consists of the loss of proliferative potential produced by the accumulation of cell doublings (1Hayflick L. Moorhead P.S. Exp. Cell Res. 1965; 25: 585-621Crossref Scopus (5572) Google Scholar). A number of regulatory proteins have been proposed to transduce senescence-inducing signals or mediate entrance of the cell into the senescence stage. Prominent among these is the p16INK4atumor suppressor protein (2Alcorta D.A. Xiong Y. Phelps D. Hannon G. Beach D. Barrett J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13742-13747Crossref PubMed Scopus (801) Google Scholar, 3Hara E. Smith R. Parry D. Tahara H. Stone S. Peters G. Mol. Cell. Biol. 1996; 16: 859-867Crossref PubMed Scopus (657) Google Scholar). p16INK4a is a negative regulator of the cell cycle. It inhibits CDK4/CDK6-mediated phosphorylation of retinoblastoma gene product (pRB) and causes cell cycle arrest in G1 phase (4Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Crossref PubMed Scopus (3387) Google Scholar). The p16INK4a gene is progressively up-regulated as fibroblasts undergo increasing numbers of cell divisions and approach senescence (2Alcorta D.A. Xiong Y. Phelps D. Hannon G. Beach D. Barrett J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13742-13747Crossref PubMed Scopus (801) Google Scholar, 6Zindy F. Quelle D.E. Roussel M.F. Sherr C.J. Oncogene. 1997; 15: 203-211Crossref PubMed Scopus (467) Google Scholar, 7Huschtscha L.I. Reddel R.R. Carcinogenesis. 1999; 20: 921-926Crossref PubMed Scopus (96) Google Scholar, 8Jarrard D.F. Sarker S. Shi Y. Yeager T.R. Magrane G. Kinoshita H. Nassif N. Meisner L. Newton M.A. Waldman F.M. Reznikoff C.A. Cancer Res. 1999; 59: 2957-2964PubMed Google Scholar). Reexpression of the gene by promoter demethylation with 5-azacytidine (9Chen P.L. Tong T.J. Zhang Z.Y. Chin. J. Geriatr. 2001; 20: 44-46Google Scholar) or transfection into normal young human diploid fibroblast cells to induce overexpression of p16 (10Liu P.H. Tong T.J. Zhang Z.Y. Chin. J. Geriatr. 2001; 20: 128-131Google Scholar) causes a senescent-like changes including suppression of growth rate with cell cycle arrest at G1phase, increase of cell volume, and expression of a neutral senescence-associated β-galactosidase. Neilsen et al. (11Nielsen G.P. Stemmer-Rachamimov A.O. Shaw J. Roy J.E. Koh J. Louis D.N. Lab. Invest. 1999; 79: 1137-1143PubMed Google Scholar) analyzed the immunohistochemical localization of p16INK4ain all human organs and demonstrated that cellular p16INK4aexpression is highly selective. In adults, p16INK4a is widely expressed in many tissues such as proliferative endometrium, breast ductal epithelium, squamous and tubal metaplastic epithelium of the uterine cervix, esophageal squamous epithelium, and salivary glands among others. In infants, p16INK4a staining was limited to thymic Hassall's corpuscles, occasional thymic lymphocytes, and only rare pancreatic epithelial cells. Therefore, restriction of p16INK4a expression in infants to the thymus, the only organ committed to early senescence and increased expression of p16INK4a in adult tissues, may reflect a role of p16INK4a in normal organ senescence. Consistent with the role of p16INK4a in senescence, inactivation of the p16INK4a/Rb pathway results in life span extension or immortalization. Mouse fibroblasts that have bypassed senescence have often lost expression of p16INK4a (12Serrano M. Exp. Cell. Res. 1997; 237: 7-13Crossref PubMed Scopus (279) Google Scholar). In certain human cell types, such as human keratinocytes or mammary epithelial cells, in which telomerase expression alone is insufficient to bypass senescence, the additional inactivation of p16INK4a by genetic or epigenetic mechanisms is required to bypass senescence and render the cells immortal (13Kiyono T. Foster S.A. Koop J.I. McDougall J.K. Galloway D.A. Klingelhutz A.J. Nature. 1998; 396: 84-88Crossref PubMed Scopus (1084) Google Scholar, 14Dickson M.A. Hahn W.C. Ino Y. Ronfard V. Wu J.Y. Weinberg R.A. Louis D.N. Li F.P. Rheinwald J.G. Mol. Cell. Biol. 2000; 20: 1436-1447Crossref PubMed Scopus (799) Google Scholar). Similarly, viral oncoproteins such as SV40 large T antigen, adenoviral E1a protein, or herpesvirus E7 protein inactivate the growth-suppressive functions of Rb and facilitate immortalization (15Jansen-Dürr P. Trends Genet. 1996; 12: 270-275Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 16Yeager T.R. Reddel R. Curr. Opin. Biotech. 1999; 10: 465-469Crossref PubMed Scopus (34) Google Scholar). Despite the widespread alterations of p16INK4a in senescence, immortalization and tumorgenesis, little is known about the transcription regulatory mechanism of this gene. Therefore, we have initiated a search for specific transcription regulatory elements whose function may contribute to up-regulation of p16INK4a in senescent fibroblasts. In this paper, we describe a novel negative transcription regulatory element, the INK4a transcription silence element (ITSE),1 within sequence −491 to −485 bp of the p16INK4a promoter. A 24-kDa protein that was highly expressed in young cells may inhibit the expression of p16INK4a by interacting with ITSE. A GC-abundant element located in the region from −466 to −451 bp was also involved with high expression of p16INK4a in senescent fibroblasts. 2BS cells were previously isolated from female fetal lung fibroblast tissue and have been fully characterized (17Tang Z. Zhang Z. Zheng Y. Corbley M.J. Tong T. Mech. Aging Dev. 1994; 73: 57-67Crossref PubMed Scopus (47) Google Scholar). The 2BS cell line was originally established at the National Institute of Biological Products (Beijing, China). The current expected life span is ∼70 population doublings (PD). 2BS cells were considered to be young at PD 30 or below and to be fully senescent at PD 55 or above. RetroPackTM PT-67 cell line (CLONTECH) is a retrovirus packaging cell line. In conjunction with a retroviral vector, it allows production of infectious, replication-incompetent retrovirus that can be used to introduce a gene of interest into a wide variety of mammalian cell types in vitro or in vivo. 2BS and PT-67 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), which contained 10% fetal bovine serum (Life Technologies), 100 units/ml penicillin, and 100 μg/ml streptomycin. Self-inactivating retrovirus vector pSIR-EGFP was constructed in pSIR (CLONTECH) by inserting an EGFP-SV40poly(A) fragment derived from pEGFP-1 (CLONTECH) into EcoRI andBamHI positions. A p16INK4a promoter fragment containing nucleotides −1 to −3017 relative to the ATG in pGL2-Basic vector was generously provided by Dr. Gorden Peters (Imperial Cancer Research Fund Laboratories, London, UK). A series of 5′ truncations were generated with different endonucleases listed in Fig.1. Various fragments were isolated from restriction digests and were ligated between the XhoI andHindIII sites in pSIR-EGFP. Transfections of plasmids containing p16 promoter 5′-deletions into PT67 retroviral packaging cells were performed with LipofectinTM (Life Technologies). Drug selection (500 μg of G418/ml) was started 2 days after transfection and continued for 10–14 days. Virus-producing cell clones were mixed and cultured to generate retroviral supernatants in Dulbecco's modified Eagle's medium, which were gathered by passing through a 0.45-μm pore size filter and stored at −80 °C until use. Young 2BS cells (PDL 22) plated 1 day earlier at ∼105cells/9-cm2 well were transduced by refeeding them for 10–12 h with retroviral supernatant plus polybrene (4 μg/ml; Sigma). The treated cells were subcultured the next day into 50-cm2flasks (Costar). Drug selection (200 μg of G418/ml) was started 2 days after transfection and continued for 14 days. Cell clones were mixed and cultured to their finite life span under the selection with 50 μg of G418/ml. Monolayer cell cultures were trypsinized and fixed with cold calcium- and magnesium-free phosphate-buffered solution (pH 7.2) containing 3.5% paraformaldehyde for 30 min on ice. Cells were washed three times in sterile PBS, and resuspended in 1 ml of sterile PBS for flow cytometry analysis. The cells were flow cytometrically analyzed (FACStar; Becton-Dickinson, San Jose, CA) with excitation at 488 nm from an argon laser operating at a constant power output of 200 W. The enhanced green fluorescence protein (EGFP) fluorescence signals were collected with a total accumulation of 10,000 cells/run. Young (PD 25) and senescent 2BS cells (PD 62) were grown to 80% confluence before being washed three times with PBS and harvested by scraping with a rubber policeman. The cells were pelleted, washed twice with cold PBS, and resuspended in five packed cell volumes of 10 mm HEPES (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, and 0.5 mm DTT (Buffer A). Following a 10 min incubation on ice, the cells were pelleted and resuspended in two packed cell volumes of buffer A. The cells were then lysed by Dounce homogenization and centrifuged at 25,000 × g at 4 °C for 20 min. The pellet was resuspended in 1 ml of 20 mm HEPES (pH 7.9), 25% glycerol, 0.42 m NaCl, 1.5 mmMgCl2, 0.2 mm EDTA, 0.5 mm DTT, and 0.5 mm phenylmethlylsulfonyl fluoride. The resuspended cells were stirred gently for 30 min at 4 °C and centrifuged at 25,000 × g for 30 min at 4 °C. The supernatant was extensively dialyzed against 20 mm HEPES (pH 7.9), 20% glycerol, 0.1 m KCl, 0.2 mm EDTA, 0.5 mm DTT, and 0.5 mm PMSF. The protein concentration of the dialyzed material was determined by Lowry assay. DNase I footprinting reactions were performed as described in the protocol of SureTrack Footprinting Kit (Amersham Pharmacia Biotech). Briefly, young and senescent 2BS nuclear extracts were combined with binding buffer (20% glycerol, 20 mm Na-HEPES, pH 7.9, 100 mm KCl, 2 mm EDTA, 2 mm DTT, 4 mm Tris-HCl, pH 7.9), 1 μg of poly(dI-dC), and 10,000 cpm of the end-radiolabeled probe in a final volume of 50 μl at room temperature for 30 min. Fresh dilutions of DNase I were made in 4 mmCaCl2, 10 mm MgCl2, and an appropriate volume was added to each reaction. After 1 min, 150 μl of stop buffer (0.2 m NaCl, 20 mm EDTA, 1% SDS, and 250 mg/ml yeast RNA) was added, and reactions were subsequently extracted with phenol/chloroform, DNA-precipitated in ethanol, vacuum-dried, and resuspended in formamide buffer. Samples were resolved on 6% sequencing gel. The gel was dried under vacuum and exposed to Eastman Kodak Co. X-Omat AR film for 48–72 h. G + A chemical sequence marker was electrophoresed with the sample to identify the size and location of the protected DNase I footprints. Nuclear extracts from young and senescent 2BS cells were preincubated for 10 min at room temperature in a buffer containing 2 μg of poly(dI-dC)·poly(dI-dC), 4% glycerol, 1.5 mm MgCl2, 0.5 mmEDTA, 0.5 mm DTT, 50 mm NaCl, and 5 mm Tris-HCl, pH 7.5. Probe (50,000 cpm) was added and incubated at room temperature for 20 min. In competition experiments unlabeled oligonucleotides were added just prior to the addition of the probe. Reactions were analyzed on 4% polyacrylamide (30:1 acrylamide/bisacrylamide) gels in 0.5× TBE. The gels were electrophoresed at 300 V at 4 °C for ∼3 h. After electrophoresis, the gel was dried and autoradiographed with an intensifier screen. 120 μg of young and senescent 2BS nuclear extracts were electrophoretically separated on a 7% SDS-polyacrylamide gel. Proteins from the gel were subsequently electroblotted onto nitrocellulose membranes. The blot was incubated in binding buffer (25 mm HEPES (pH 7.9), 3 mmMgCl2, 4 mm KCl, 1 mmdithiothreitol) at 4 °C for 5 min. The filter was then blocked for 30 min in a 5% blotto solution. The blot was rinsed three times with binding buffer containing 0.25% blotto before the radiolabeled probe was added to the buffer. The blot was incubated with the probe for 16 h at 4 °C, washed five times with binding buffer, and then exposed to Kodak X-Omat AR film for 24–72 h. Site-directed mutagenesis of the −870 bp p16INK4a promoter was prepared by QuickChangeTM site-directed mutagenesis methods. We designed a synthetic double-stranded oligonucleotide (5′-GGAAGGTTGGATCCCGGAGGAAGGAAACG-3′) to create a new BamHI site at position −482. Then two double-stranded oligonucleotide 5′-CTTTCCCTATGAGATCTAACACCCCGATTC-3′ and 5′-GGCGGGGGCAGATCTCTTTTTAACAGAG-3′ were used to create a newBglII site at position −522 and −451 of the promoter, respectively. BglII and BamHI produce compatible overhangs. When digested with these two endonucleases and ligated with T4-ligase, the fragment between BglII and BamHI was deleted. The nucleotide sequences of the deleted mutants were confirmed by a DNA sequencer, model 3700 (PerkinElmer Life Sciences). The wide type −870 bp p16INK4a promoter and two deletion mutants 5′-MUT (−522 to −482 region deletion) and 3′-MUT (−482 to −451 region deletion) were inserted into XhoI andHindIII sites in pSEAP-Basic vector. Transfection of the young (PD26) and senescent (PD62) 2BS cells were performed using FuGENE 6 (Roche Molecular Biochemicals). Transfected cells were harvested 48 h post-transfection, and SEAP activity was assayed using the Great EscAPe SEAPTM chemiluminescence detection kit (CLONTECH). SEAP light output was measured in a luminometer and normalized against the transfection pSV-β-galactosidase control vector. Measurement of β-galactosidase was performed using a β-galactosidase enzyme assay system (Promega). We used the EGFP reporter system to scan regulatory elements in the upstream region of p16INK4agene. On the base of self-inactivating retrovirus vector pSIR-EGFP, we constructed a series of plasmids in which various lengths of the p16INK4a promoter were placed upstream of the EGFP. Transfecting the recombinant vectors into retrovirus packaging cells, we got virus supernatants. The young human fibroblasts were infected with virus. Then the infected cells were cultured to their finite life span. EGFP activity was measured by fluorescence-activated cell sorting. The data in Fig. 2 showed that very low expression of EGFP was observed in young and senescent cells infected by pSIR-EGFP−280, which contained −280 bp upstream from ATG of the p16 promoter. The activities of the −622, −870, −1400, −2070, and −3017 bp of the p16 promoter increased in senescent cells 5–7 times relative to that in young cells. Deletion from −3017 to −622 had little effect on promoter activity. A marked loss of promoter activity was observed when the region between −622 and −280 was excised (pSIR-EGFP−280). Together, these results indicated that the main transcription-regulatory elements contributing to overexpression of p16 in senescent cells were located in the region from −622 to −280 of the promoter. We analyzed the DNA-protein interactions within the functionally important segment of the p16INK4a promoter as a means to identify putative cis-acting elements that mediate promoter function. In vitro DNase I footprinting was performed with nuclear extracts from young and senescent 2BS cells and single end-labeled probe spanning −622 to −280. As seen in Fig.3, three footprints were detected in this region. Two regions (region A, from −507 to −493, and region C, extended from −466 to −450) could be combined with nuclear proteins extracted from both young and senescent 2BS cells. Region B that extended from −491 to −485 was mainly detected in the presence of extract from young 2BS cells. The positions and sequences of footprinted regions A, B, and C are shown in Fig.4. Regions A and B were separated only by one nucleotide. Region C was rich in GC sequence and contained the conventional consensus Sp1 binding site, 5′-GGGCGG-3′.Figure 4The partial sequence of p16INK4apromoter. Regions A, B, and C are three nuclear protein binding regions identified in footprinting experiments. 5′ and 3′ oligonucleotide probes were used for competition in EMSA experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine the nuclear factors that bind to the footprinted region A, B, and C, we performed EMSA with young and senescent fibroblasts extracts. A 32P-end-labeled segment from −622 to −374 of the p16 promoter was used for EMSA, and two double-stranded oligonucleotide probes (5′-oligo, whose sequence extended from −525 to −481 covering the footprinted region A and B, and 3′-oligo, whose sequence extended from −480 to −447 covering the footprinted region C; Fig. 4) were used for competition. Six retarded bands were detected as shown in Fig. 5. Band e, which was completely competed by 5′-oligo, could only be detected in the presence of extract from young 2BS cells. Bands a, b, c, and f could be competed by 3′-oligo probe. Among them, bands b and c could only be detected in the presence of extract from senescent 2BS cells. We next studied the molecular weight of DNA-binding proteins that could combine with the region from −622 to −280 of the p16 promoter with Southwestern blotting. As showed in Fig.6, a 24-kDa binding protein was observed only in young cells, and 15.5-kDa binding protein could be detected in both kinds of cells. In order to investigate the contribution of the protected regions acquired from the DNase I footprinting assay toward the promoter activity, we deleted the region from −522 to −482 (named 5′-MUT) and the region −482 to −451 (called 3′-MUT) from −870 bp of the p16 promoter, respectively. After inserting these two kinds of mutated promoters and the wild type −870 bp p16 promoter into the multiple clone sites of pSEAP-Basic vector, we generated three mutational reporter gene expression vectors, pSEAP−870, pSEAP-5′-MUT, and pSEAP-3′-MUT. Recombinant vectors were transfected into young and senescent 2BS cells. pSEAP-5′-MUT with the −522 to −482 deletion showed that promoter activity increased by 72% in young cells, while no significant change occurred in senescent cells. Deletion of the region from −482 to −451 showed a 91.4% loss of activity of p16INK4a promoter in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably (Figs.7 and8).Figure 8SEAP activity in senescent 2BS cells.The pSEAP-5′-MUT construct with a −522 to −482 deletion showed no significant change of promoter activity. The pSEAP-3′-MUT construct with a −482 to −451 deletion showed a 91.2% loss of activity. The mean activity and S.D. values were relative to wide type promoter activity, which was arbitrarily set at 100% (**, p < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The vector pSIR used for 5′-deletion study is a self-inactivating retrovirus vector (19Schnell T. Foley P. Wirth M. Munch J. Uberla K. Hum. Gene Ther. 2000; 11: 439-447Crossref PubMed Scopus (108) Google Scholar). It contains a 176-bp deletion in the 3′-long terminal repeat that removes the enhancer sequence. Following reverse transcription, the 3′-long terminal repeat is copied and replaces 5′-long terminal repeat resulting in inactivation of the 5′-long terminal repeat promoter. It can be used for transcription regulatory research. The results of the 5′-deletion assay indicated that the activities of different length (−622 to −3017 bp) of the p16INK4a promoter were enhanced by 5–7-fold during the fibroblast aging process, which was much lower than the increment level of p16INK4a mRNA and protein in senescent cells. In many cultured cell lines, such as fibroblasts, keratinocytes, and urothelial cells, p16INK4a accumulates with increasing numbers of population doublings (2Alcorta D.A. Xiong Y. Phelps D. Hannon G. Beach D. Barrett J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13742-13747Crossref PubMed Scopus (801) Google Scholar, 5Adrian G.S. Seto E. Fischbach K.S. Rivera E.V. Adrian E.K. Herbert D.C. Walter C.A. Weaker F.J. Bowman B.H. J. Gerontol. A Biol. Sci. Med. Sci. 1996; 51: B66-B75Crossref PubMed Scopus (48) Google Scholar, 6Zindy F. Quelle D.E. Roussel M.F. Sherr C.J. Oncogene. 1997; 15: 203-211Crossref PubMed Scopus (467) Google Scholar, 7Huschtscha L.I. Reddel R.R. Carcinogenesis. 1999; 20: 921-926Crossref PubMed Scopus (96) Google Scholar, 8Jarrard D.F. Sarker S. Shi Y. Yeager T.R. Magrane G. Kinoshita H. Nassif N. Meisner L. Newton M.A. Waldman F.M. Reznikoff C.A. Cancer Res. 1999; 59: 2957-2964PubMed Google Scholar). Interestingly, the levels of p16INK4a continue to rise after the cells have effectively ceased to divide (2Alcorta D.A. Xiong Y. Phelps D. Hannon G. Beach D. Barrett J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13742-13747Crossref PubMed Scopus (801) Google Scholar). Since p16 RNA is extremely stable (3Hara E. Smith R. Parry D. Tahara H. Stone S. Peters G. Mol. Cell. Biol. 1996; 16: 859-867Crossref PubMed Scopus (657) Google Scholar), many researchers hypothesize that accumulation of p16INK4a mRNA and proteins with each PD may lead to overexpression of this gene in senescent cells (3Hara E. Smith R. Parry D. Tahara H. Stone S. Peters G. Mol. Cell. Biol. 1996; 16: 859-867Crossref PubMed Scopus (657) Google Scholar, 20Stein C.H. Drullinger L.F. Soulard A. Dulic V. Mol. Cell. Biol. 1999; 19: 2109-2117Crossref PubMed Scopus (587) Google Scholar). However, the significant elevation of p16INK4a begins from the late stage of senescence, but not from early passage, gradually. There are no significant changes in p16INK4a RNA stability between early passage and late passage fibroblast cells (3Hara E. Smith R. Parry D. Tahara H. Stone S. Peters G. Mol. Cell. Biol. 1996; 16: 859-867Crossref PubMed Scopus (657) Google Scholar). So we hypothesized that there are some negative regulatory mechanisms in young fibroblasts that inhibit the transcription of p16INK4a by negative feedback to prevent the accumulation of p16INK4a in young cells. In senescent cells, the negative regulatory mechanisms are turned off, which will lead to elevation of p16 promoter activity. On the other hand, the loss of negative regulation will initiate the accumulation of p16INK4a. Therefore, we suppose that the loss of negative regulatory mechanisms plays a primary role in overexpression of p16INK4a in senescent cells. We located the main regulatory elements contributing to the overexpression of p16INK4a in the region of the promoter from −622 to −280 through 5′-deletion assays. Then we studied the DNA-protein interactions with this fragment in vitro. The result of DNase I footprinting showed that the fragment spanning −491 to −485 relative to the ATG was specifically protected from DNase I digestion by nuclear extracts from young 2BS cells. As mentioned above, the activity of the −622 bp promoter increased by 5-fold in the fibroblast aging process. We postulate that the region (−491 to −485) may be a silencer. In order to confirm this theory, we performed EMSA and site-directed deletion assays. The fragment (−622 to −374) was end-labeled and incubated with nuclear extracts from young and senescent cells, respectively. The formation of DNA-protein complexes that could be competed by a 100-fold molar excess of the synthetic 5′-oligo oligonucleotides completely was only detected in young cells (Fig. 5, band e). The fragment (−522 to −482) was deleted from −870 bp of the p16 promoter. The deletion resulted in a drastic increase in promoter activity to about 170.2% compared with the wild type promoter (set arbitrarily at 100%) in young cells and no significant change in senescent cells. The results demonstrated that the region (−491 to −485) was a negative regulatory element, and it mediated transcriptional inhibition of p16INK4a by interacting with a protein factor highly expressed in young cells. This functional element did not contain consensus sequence of cis-acting transcription factors that have been reported. It was a novel silencer, ITSE. The sequence of ITSE is 5′-GAAGGTT-3′. A transcription inhibition factor that was highly expressed in young cells may inhibit the expression of p16 by interacting with ITSE. Up to now, it is known that the Id family of helix-loop-helix proteins can inhibit the transcription of p16INK4a. The loss of Id function has been linked to replicative senescence (21Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1397) Google Scholar). The mouse embryo fibroblasts derived from Id1-deficient mice express high levels of p16INK4a (22Lyden D. Young A.Z. Zagzag D. Yan W. Gerald W. O'Reilly R. Bader B.L. Hynes R.O. Zhuang Y. Manova K. Benezra R. Nature. 1999; 401: 670-677Crossref PubMed Scopus (781) Google Scholar). Id1 interacts with Ets2 and blocks its transcriptional activation effect on p16INK4a (23Ohtani N Zebedee Z. Huot T.H. Stinson J.A. Sugimoto M. Ohashi Y. Sharrocks A.D. Peters G. Hara E. Nature. 2001; 409: 1067-1070Crossref PubMed Scopus (537) Google Scholar). Unlike basic helix-loop-helix proteins, Ids lack a DNA-binding activity due to the absence of a basic domain (24Takai N. Miyazaki T. Fujisawa K. Nasu K. Miyakawa I. Cancer Lett. 2001; 165: 185-193Crossref PubMed Scopus (66) Google Scholar). The protein binding to ITSE did not belong to the Id family of transcription factors. We performed Southwestern blot to detect the molecular weight of the proteins that can bind to the fragment (−622 to −280) of the p16INK4apromoter. A 24-kDa protein could only be detected in young 2BS cells. We postulated that this protein was the transcription inhibition factor binding to and acting through ITSE, but it needs further confirmation. The GC-abundant region C (−466 to −450; Fig. 3) was another footprinted region. It could be protected by nuclear protein extracted from both young and senescent fibroblasts. The electrophoretic mobility shift assays with fragment −622 to −374 as the labeled probe and a 100-fold molar excess of the 3′-oligo unlabeled oligonucleotide as competitor indicated that the gel mobility-retarded DNA-protein complexes a, b, c, and f were competed by the 3′-oligo oligonucleotide. Moreover, complexes b and c were present in nuclear extracts from the senescent 2BS cells. This suggested that some new transcription factors bound to this region in senescent cells. The result of deletion mutation demonstrated that region C was a positive regulatory element related to the increase of expression of p16INK4a in senescent cells. Deletion of this region showed 91.4% loss of activity of the p16INK4a promoter in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably. The sequence of region C was 5′-ACGGGGCGGGGGCGGA-3′. It contained two conventional consensus Sp family protein (25Courey A.J. Tjian R. Cell. 1998; 55: 887-898Abstract Full Text PDF Scopus (1079) Google Scholar) binding sites, 5′-GGGCGG-3′. This GC-box element is an important and widely distributed promoter element (26Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (987) Google Scholar). It is required for the appropriate expression of many ubiquitous, tissue-specific and viral genes. In addition, it occurs frequently in the regulatory region of genes that are under a specific mode of control such as cell cycle regulation, tumor genesis, hormonal activation, and developmental and senescent patterning. The transcription factors that can bind to and act though the GC-boxes are the Sp family of proteins (27Courey A.J. Tjian R. Cell. 1989; 594: 827-836Abstract Full Text PDF Scopus (391) Google Scholar). This family consists of four proteins designated Sp1, Sp2, Sp3, and Sp4. All four human Sp family members have similar structures. The DNA binding domain close to the C terminus contains three zinc fingers. The zinc finger structure is highly conserved in Sp1, Sp3, and Sp4 but not in Sp2. Consistently, Sp1, Sp3, and Sp4 recognize the classical GC-box element with identical affinity (28Pavletich N.P. Pabo C.O. Science. 1991; 252: 809-817Crossref PubMed Scopus (1760) Google Scholar, 29Fairall L. Schwabe J.W. Chapman L. Finch J.T. Rhodes D. Nature. 1993; 366: 483-487Crossref PubMed Scopus (299) Google Scholar), while Sp2 does not bind to the GC-box but to a GT-rich element (30Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4261Crossref PubMed Scopus (490) Google Scholar). Both Sp1 and Sp4 are known to be strong transcriptional activators. Sp4 expression appears to be restricted to a few tissues. High levels of Sp4 are predominantly found in the brain (31Supp D.M. Witte D.P. Branford W.W. Smith E.P. Potter S.S. Dev. Biol. 1996; 176: 284-299Crossref PubMed Scopus (151) Google Scholar). The transcriptional role of Sp3 is complicated. 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In our study, we found when the GC-box element located in −466 to −450 was deleted, the decrease of p16 promoter activity in senescent cells was much higher than in young cells, which suggested that the GC-box-binding proteins contributed to the increase of p16INK4a expression in senescent cells. We are very grateful to Dr. Danial L. Kilpatrick (University of Massachusetts Medical School, Worcester, MA) for careful reading of the manuscript and Dr. Gorden Peters for the kind gift of the p16 promoter-Luc vector." @default.
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