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J. Biol. Chem., Vol. 276, Issue 52, 48655-48661, December 28, 2001

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Characterization of Regulatory Elements on the Promoter Region of p16^INK4a That Contribute to Overexpression of p16 in Senescent Fibroblasts^*

Wei Wang, Junfeng Wu, Zongyu Zhang, and Tanjun Tong

From the Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xueyuan Rd., Beijing 100083, People's Republic of China

Received for publication, August 28, 2001, and in revised form, September 24, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cyclin-dependent kinase inhibitor p16^INK4a 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 p16^INK4a. The results of 5'-deletion studies indicated that the transcription regulatory elements contributing to overexpression of p16^INK4a in senescent cells were located in the region of the p16^INK4a 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 p16^INK4a promoter. A 24-kDa protein that was highly expressed in young cells may inhibit the expression of p16^INK4a by interacting with the ITSE. The activity of the p16^INK4a promoter increased significantly in young cells when the ITSE was deleted. The GC-rich region of the p16^INK4a promoter from 466 to 451 was a positive transcription regulatory element. Deletion of this region showed 91.4% loss of p16^INK4a promoter activity in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cellular senescence consists of the loss of proliferative potential produced by the accumulation of cell doublings (1). 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 p16^INK4a tumor suppressor protein (2, 3). p16^INK4a 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 (4). The p16^INK4a gene is progressively up-regulated as fibroblasts undergo increasing numbers of cell divisions and approach senescence (2, 6-8). Reexpression of the gene by promoter demethylation with 5-azacytidine (9) or transfection into normal young human diploid fibroblast cells to induce overexpression of p16 (10) causes a senescent-like changes including suppression of growth rate with cell cycle arrest at G₁ phase, increase of cell volume, and expression of a neutral senescence-associated -galactosidase. Neilsen et al. (11) analyzed the immunohistochemical localization of p16^INK4a in all human organs and demonstrated that cellular p16^INK4a expression is highly selective. In adults, p16^INK4a 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, p16^INK4a staining was limited to thymic Hassall's corpuscles, occasional thymic lymphocytes, and only rare pancreatic epithelial cells. Therefore, restriction of p16^INK4a expression in infants to the thymus, the only organ committed to early senescence and increased expression of p16^INK4a in adult tissues, may reflect a role of p16^INK4a in normal organ senescence.

Consistent with the role of p16^INK4a in senescence, inactivation of the p16^INK4a/Rb pathway results in life span extension or immortalization. Mouse fibroblasts that have bypassed senescence have often lost expression of p16^INK4a (12). 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 p16^INK4a by genetic or epigenetic mechanisms is required to bypass senescence and render the cells immortal (13, 14). 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 (15, 16).

Despite the widespread alterations of p16^INK4a 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 p16^INK4a 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 p16^INK4a promoter. A 24-kDa protein that was highly expressed in young cells may inhibit the expression of p16^INK4a by interacting with ITSE. A GC-abundant element located in the region from 466 to 451 bp was also involved with high expression of p16^INK4a in senescent fibroblasts.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- 2BS cells were previously isolated from female fetal lung fibroblast tissue and have been fully characterized (17). 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.

RetroPack^TM 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.

Plasmid Construction-- 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 and BamHI positions. A p16^INK4a 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 and HindIII sites in pSIR-EGFP.

View larger version (5K): [in this window] [in a new window]   Fig. 1.   Restriction map of the p16INK4a promoter. The restriction endonuclease sites were used in 5' promoter deletion analyses.

Retrovirus Packaging and Transduction Assay-- Transfections of plasmids containing p16 promoter 5'-deletions into PT67 retroviral packaging cells were performed with Lipofectin^TM (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 ~10⁵ cells/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-cm² flasks (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.

Extraction of Nuclear Protein-- 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 mM MgCl₂, 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 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 mM CaCl₂, 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.

Electrophoretic Mobility Shift Assay-- 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 mM EDTA, 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.

Southwestern Blot-- 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 mM MgCl₂, 4 mM KCl, 1 mM dithiothreitol) 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.

Deletion Mutation Assay-- Site-directed mutagenesis of the 870 bp p16^INK4a promoter was prepared by QuickChange^TM 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 new BglII 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 p16^INK4a promoter and two deletion mutants 5'-MUT (522 to 482 region deletion) and 3'-MUT (482 to 451 region deletion) were inserted into XhoI and HindIII 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 SEAP^TM chemiluminescence detection kit (CLONTECH). SEAP light output was measured in a luminometer and normalized against the transfection pSV--galactosidase control vector. Measurement of -galactosidase was performed using a -galactosidase enzyme assay system (Promega).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Definition of Functional Segments of p16^INK4a Promoter That Contribute to Overexpression of p16 in Senescent Fibroblasts by Functional Deletion Analysis-- We used the EGFP reporter system to scan regulatory elements in the upstream region of p16^INK4a gene. On the base of self-inactivating retrovirus vector pSIR-EGFP, we constructed a series of plasmids in which various lengths of the p16^INK4a 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-EGFP280, 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-EGFP280). 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.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Deletion analysis of the human p16INK4a promoter activities in young and senescent 2BS cells. The left panel is a schematic representation of the constructs used in transcription activity assays. The right panel displays the relative promoter activity of the different constructs. The promoter relative activity was calculated relative to the 870-bp promoter activity in senescent cells, which was arbitrarily set at 100%.

Identification of Functional Elements in the p16^INK4a Promoter-- We analyzed the DNA-protein interactions within the functionally important segment of the p16^INK4a 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'.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A, DNase I footprinting of the region from 622 to 280 bp of the p16 promoter. The fragment was 3'-end-labeled (coding strand) and incubated with nuclear extracts of young and senescent cells. Complexes were digested with 2 units of DNase I in the presence of 80 µg of nuclear extracts; 3.5 units of DNase I in the presence of 120 µg of nuclear extracts, and 0.2 units DNase I in the presence of 40 µg of bovine serum albumin (control). Aliquots of the probes were chemically treated as for Maxam-Gilbert sequencing of G + A residues to give markers. A, B, and C indicate three DNase I protection regions. B, standardized corrected, integrated optical density values for binding site blocks to total DNA in lane. The optical densities of three DNase I protection regions A, B, and C (Dsite) were integrated for each lane. The optical density of region D (Dstd) was integrated to represent the total DNA concentration in a lane. Protein binding equilibria were analyzed by calculating optical density ratio (Dsite/Dstd) (18).

View larger version (46K): [in this window] [in a new window]   Fig. 4.   The partial sequence of p16INK4a promoter. 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.

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.

View larger version (54K): [in this window] [in a new window]   Fig. 5.   Electrophoretic mobility shift assay of the region from 622 to 374 of the p16 promoter. EMSA was performed using nuclear extracts obtained from either young or senescent 2BS cells. The fragment was radiolabeled and incubated with 4 µg of nuclear protein or 4 µg of BSA for control (lane 1). Competition was performed in the presence of a 100-fold molar excess of the synthetic nonradioactive oligonucleotides according to the footprinting result. 5'-oligonucleotides were the sequence of 525 to 481 of the p16 promoter (lanes 4 and 5). 3'-Oligonucleotides were the sequence of 480 to 447 of the p16 promoter (lanes 6 and 7). a-f, six shifted binding bands.

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.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Southwestern blotting of the fragment from 622 to 280 of the p16 promoter. End-labeled probe was incubated with nuclear extracts of young and senescent 2BS cells, which were separated by SDS-PAGE and transferred to nitrocellulose membrane. A 24-kDa binding protein was observed only in young cells, and a 15.5-kDa binding protein could be detected in both 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, pSEAP870, 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 p16^INK4a promoter in senescent cells, and the promoter activity decreased by 41.2% in young cells comparably (Figs. 7 and 8).

View larger version (12K): [in this window] [in a new window]   Fig. 7.   SEAP activity in young 2BS cells. The pSEAP-5'-MUT construct with a 522 to 482 deletion showed a 72% increase of promoter activity. The pSEAP-3'-MUT construct with a 482 to 451 deletion showed a 41.2% loss of activity. The mean activity and S.D. values were relative to wide type promoter activity, which was arbitrarily set at 100% (*, p < 0.05; **, p < 0.01).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   SEAP 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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The vector pSIR used for 5'-deletion study is a self-inactivating retrovirus vector (19). 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 p16^INK4a promoter were enhanced by 5-7-fold during the fibroblast aging process, which was much lower than the increment level of p16^INK4a mRNA and protein in senescent cells. In many cultured cell lines, such as fibroblasts, keratinocytes, and urothelial cells, p16^INK4a accumulates with increasing numbers of population doublings (2, 5-8). Interestingly, the levels of p16^INK4a continue to rise after the cells have effectively ceased to divide (2). Since p16 RNA is extremely stable (3), many researchers hypothesize that accumulation of p16^INK4a mRNA and proteins with each PD may lead to overexpression of this gene in senescent cells (3, 20). However, the significant elevation of p16^INK4a begins from the late stage of senescence, but not from early passage, gradually. There are no significant changes in p16^INK4a RNA stability between early passage and late passage fibroblast cells (3). So we hypothesized that there are some negative regulatory mechanisms in young fibroblasts that inhibit the transcription of p16^INK4a by negative feedback to prevent the accumulation of p16^INK4a 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 p16^INK4a. Therefore, we suppose that the loss of negative regulatory mechanisms plays a primary role in overexpression of p16^INK4a in senescent cells.

We located the main regulatory elements contributing to the overexpression of p16^INK4a 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 p16^INK4a 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 p16^INK4a. The loss of Id function has been linked to replicative senescence (21). The mouse embryo fibroblasts derived from Id1-deficient mice express high levels of p16^INK4a (22). Id1 interacts with Ets2 and blocks its transcriptional activation effect on p16^INK4a (23). Unlike basic helix-loop-helix proteins, Ids lack a DNA-binding activity due to the absence of a basic domain (24). 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 p16^INK4a promoter. 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 p16^INK4a in senescent cells. Deletion of this region showed 91.4% loss of activity of the p16^INK4a 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 (25) binding sites, 5'-GGGCGG-3'. This GC-box element is an important and widely distributed promoter element (26). 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 (27). 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 (28, 29), while Sp2 does not bind to the GC-box but to a GT-rich element (30).

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 (31). The transcriptional role of Sp3 is complicated. In some experiments, Sp3 was shown to act as a transcriptional activator similar to Sp1 (32, 33). In other experiments, Sp3 acted as a transcriptional repressor of Sp1-mediated activation (34, 35). The Sp family of proteins are involved in regulating the activities of senescence-regulated genes such as p21^WAF1 (36) and Werner helicase genes (37). Fukami-Kobayashi et al. (38) investigated the binding of nuclear protein factors to the cyclin D1 gene promoter domain in young and senescent normal human fibroblasts to clarify the molecular mechanisms of cyclin D1 expression during in vitro cellular aging. They found that the binding of Sp1 to its promoter element occurred only in senescent cells, which may promote the increase of cyclin D1 expression during cellular aging. In humans, serum transferrin levels decrease during the aging process. A decrease in Sp1-like binding activity was shown to cause decreased transcription of the human transferrin gene (5). 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 p16^INK4a expression in senescent cells.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported by special funds for Major State Basic Research Program of China (Grant G2000057001) and the National Natural Science Foundation of China (Grant 39930170).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.

To whom correspondence should be addressed. Tel.: 8610-62091454; Fax: 8610-62015582; E-mail: ttjzzy@public.gb.com.cn.

Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M108278200

	ABBREVIATIONS

The abbreviations used are: ITSE, INK4a transcription silence element; PD, population doubling(s); PBS, phosphate-buffered solution; EGFP, enhanced green fluorescent protein; DTT, dithiothreitol; SEAP, secreted alkaline phosphatase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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				X. F. Lu, X. G. Jiang, Y. B. Lu, J. H. Bai, and Z. B. Mao Characterization of a Novel Positive Transcription Regulatory Element That Differentially Regulates the Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Gene in Senescent Cells J. Biol. Chem., June 17, 2005; 280(24): 22606 - 22615. [Abstract] [Full Text] [PDF]

				H. Youn, Y. Koo, I. Ji, and T. H. Ji An Upstream Initiator-Like Element Suppresses Transcription of the Rat Luteinizing Hormone Receptor Gene Mol. Endocrinol., May 1, 2005; 19(5): 1318 - 1328. [Abstract] [Full Text] [PDF]

				L. Zhao, T. Tong, and Z. Zhang Expression of the Leo1-like domain of replicative senescence down-regulated Leo1-like (RDL) protein promotes senescence of 2BS fibroblasts FASEB J, April 1, 2005; 19(6): 521 - 532. [Abstract] [Full Text] [PDF]

				W. Zheng, H. Wang, L. Xue, Z. Zhang, and T. Tong Regulation of Cellular Senescence and p16INK4a Expression by Id1 and E47 Proteins in Human Diploid Fibroblast J. Biol. Chem., July 23, 2004; 279(30): 31524 - 31532. [Abstract] [Full Text] [PDF]

				I. B. Roninson Tumor Cell Senescence in Cancer Treatment Cancer Res., June 1, 2003; 63(11): 2705 - 2715. [Abstract] [Full Text] [PDF]

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