Characterization of Regulatory Elements on the Promoter Region of p16INK4a That Contribute to Overexpression of p16 in Senescent Fibroblasts

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 were with buffer glycerol, 20 mm 100 mm KCl, 2 mm EDTA, 2 mm DTT, 4 mm 7.9), 1 μg of and 10,000 of the in a of 50 at for 30 min. of DNase I were in 4 10 mm MgCl2, and an was to 1 of buffer m NaCl, 20 mm EDTA, and was and reactions were with in and resuspended in were on The was under and to for A sequence was with the to the size and of the DNase I from young and senescent 2BS cells were for 10 min at in a buffer containing 2 μg of glycerol, 1.5 mm MgCl2, 0.5 0.5 mm DTT, 50 mm NaCl, and mm was and at for 20 min. In were to the of the were analyzed on in The were at at 4 °C for the was and with an μg of young and senescent 2BS were on a from the were The was in buffer mm HEPES (pH 7.9), mmMgCl2, 4 mm KCl, 1 at 4 °C for min. The filter was then for 30 min in a The was three times with buffer containing before the was to the The was with the for h at 4 washed five times with and then to for of the bp p16INK4a promoter was by We a to a at two and were used to a at and −451 of the and with these two endonucleases and ligated with the fragment between and was deleted. The of the were by a The wide bp p16INK4a promoter and two to region and to −451 region were into XhoI andHindIII sites in of the young and senescent 2BS cells were performed cells were harvested h and activity was the output was in a and against the transfection of was performed a system We used the reporter system to scan regulatory elements in the upstream region of the of retrovirus vector we constructed a series of plasmids in which of the p16INK4a promoter were upstream of the the into retrovirus packaging cells, we The young human fibroblasts were with the cells were cultured to their finite life activity was by cell The in 2 showed that expression of was in young and senescent cells by which contained −280 bp upstream from ATG of the p16 promoter. The of the and −3017 bp of the p16 promoter increased in senescent cells times relative to that in young cells. Deletion from −3017 to −622 little on promoter A loss of promoter activity was when the region between −622 and −280 was these results indicated that the elements contributing to overexpression of p16 in senescent cells were located in the region from −622 to −280 of the promoter. We analyzed the within the of the p16INK4a promoter as a to elements that mediate promoter In vitro DNase I footprinting was performed with from young and senescent 2BS cells and −622 to in three were in this from to and region from −466 to be with proteins from young and senescent 2BS cells. that from −491 to −485 was in the of from young 2BS cells. The and of and in A and were only by was in sequence and contained the sequence of and three protein in footprinting 5′ and were used for in the that to the region and we performed with young and senescent fibroblasts A from −622 to of the p16 promoter was used for EMSA, and two whose sequence from to the region A and and whose sequence from to the region were used for were as in which was by only be in the of from young 2BS cells. and be by and only be in the of from senescent 2BS cells. We next the of proteins that with the region from −622 to −280 of the p16 promoter with Southwestern showed in a 24-kDa protein was only in young cells, and protein be in of cells. In to the of the from the DNase I footprinting the promoter we the region from to and the region to −451 from bp of the p16 inserting these two of and the bp p16 promoter into the sites of vector, we generated three reporter gene expression and were into young and senescent 2BS cells. with the to showed that promoter activity increased by in young cells, in senescent cells. Deletion of the region from 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 activity in senescent 2BS with a to showed of promoter The with a to −451 showed a loss of The activity and were relative to wide promoter which was at The vector pSIR used for 5′-deletion is a retrovirus vector T. P. M. J. 2000; PubMed Scopus Google Scholar). It a in the that the Following the is and in inactivation of the promoter. It can be used for transcription regulatory The results of the 5′-deletion indicated that the of different to −3017 of the p16INK4a promoter were enhanced by the fibroblast which was the of p16INK4a and protein in senescent cells. In many cultured cell such as and cells, p16INK4a 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, E. C.A. J. A Biol. Sci. Sci. 1996; PubMed Scopus 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). the of p16INK4a to after the cells have to (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). p16 is E. Smith R. Parry D. Tahara H. Stone S. Peters G. Mol. Cell. Biol. 1996; 16: 859-867Crossref PubMed Scopus (657) Google many that accumulation of p16INK4a and proteins with PD may to overexpression of this gene in senescent cells E. Smith R. Parry D. Tahara H. Stone S. Peters G. Mol. Cell. Biol. 1996; 16: 859-867Crossref PubMed Scopus (657) Google Scholar, A. V. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). However, the of p16INK4a from the of senescence, from early changes in p16INK4a between early and fibroblast cells E. Smith R. Parry D. Tahara H. Stone S. Peters G. Mol. Cell. Biol. 1996; 16: 859-867Crossref PubMed Scopus (657) Google Scholar). we that negative regulatory mechanisms in young fibroblasts that inhibit the transcription of p16INK4a by negative to the accumulation of p16INK4a in young cells. In senescent cells, the negative regulatory mechanisms which to of p16 promoter the the loss of negative the accumulation of p16INK4a. Therefore, we that the loss of negative regulatory mechanisms a role in overexpression of p16INK4a in senescent cells. We located the regulatory elements contributing to the overexpression of p16INK4a in the region of the promoter from −622 to −280 through 5′-deletion we the with this fragment in The of DNase I footprinting showed that the fragment −491 to −485 relative to the ATG was from DNase I by from young 2BS cells. the activity of the −622 bp promoter increased by in the fibroblast We that the region to may be a In to this we performed and The fragment to was and with from young and senescent cells, The of that be by a of the was only in young cells The fragment to was from bp of the p16 promoter. The in a increase in promoter activity to about with the promoter at in young cells and in senescent cells. The results demonstrated that the region to was a negative regulatory element, and it of p16INK4a by interacting with a protein highly expressed in young cells. element sequence of transcription that have been It was a novel ITSE. The sequence of ITSE is A transcription that was highly expressed in young cells may inhibit the expression of p16 by interacting with ITSE. to it is known that the of proteins can inhibit the transcription of p16INK4a. The loss of function been to replicative senescence Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). The fibroblasts derived from high of p16INK4a D. Young D. W. W. R. Y. R. Nature. 1999; PubMed Scopus Google Scholar). with and its on p16INK4a Z. M. Y. Peters G. E. Nature. 2001; PubMed Scopus Google Scholar). a activity to the of a N. T. Cancer 2001; PubMed Scopus Google Scholar). The protein to ITSE to the of transcription We performed Southwestern to the of the proteins that can to the fragment to of the A 24-kDa protein only be in young 2BS cells. We that this protein was the transcription to and through it The GC-abundant region to was It be by protein from young and senescent fibroblasts. The with fragment −622 to as the and a of the as indicated that the and were by the and were in from the senescent 2BS cells. that transcription to this region in senescent cells. The of demonstrated that region was a positive regulatory element 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 was It contained two protein A.J. R. Cell. 1998; Full Text PDF Scopus Google Scholar) element is an and widely promoter element G. 1999; PubMed Scopus Google Scholar). It is required for the expression of many and viral In it in the regulatory region of that under a specific of such as cell cycle tumor and and senescent The transcription that can to and the the of proteins A.J. R. Cell. Full Text PDF Scopus Google Scholar). consists of proteins and human have The to the three The is highly in and in and the element with PubMed Scopus Google Scholar, L. L. D. Nature. 1993; 366: PubMed Scopus Google to the to a element A. Mol. Cell. Biol. 12: PubMed Scopus Google Scholar). and known to be expression to be to a of found in the Smith Dev. Biol. 1996; PubMed Scopus Google Scholar). The role of is In was to as a to L. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. Res. 1997; 25: PubMed Scopus Google Scholar). In as a of Res. 1997; 25: PubMed Scopus Google Scholar, J. M. G. J. 1996; 15: PubMed Scopus Google Scholar). The of proteins involved in regulating the of such as L. A. G.P. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and Y. A. M. J. M. Y. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). et al. J. Y. Exp. Cell Res. 1998; PubMed Scopus Google Scholar) the of protein to the gene promoter in young and senescent normal human fibroblasts to the mechanisms of expression in vitro cellular found that the of to its promoter element only in senescent cells, which may the increase of expression cellular In serum the A in activity was to decreased transcription of the human gene E. C.A. J. A Biol. Sci. Sci. 1996; PubMed Scopus Google Scholar). In we found when the element located in −466 to was the of p16 promoter activity in senescent cells was in young cells, which that the proteins to the increase of p16INK4a expression in senescent cells. We to Dr. L. of for of the and Dr. Gorden Peters for the of the p16