Construction of the POT1 promoter report gene vector, and the effect and underlying mechanism of the POT1 promoter in regulating telomerase and telomere length

Zeng,Liang; Wang,Yue‑Li; Wang,Fa; Cui,Shi‑Quan; Hu,Liang; Huang,Di‑Nan; Hou,Gan

doi:10.3892/ol.2017.7127

Construction of the POT1 promoter report gene vector, and the effect and underlying mechanism of the POT1 promoter in regulating telomerase and telomere length

Authors:
- Liang Zeng
- Yue‑Li Wang
- Fa Wang
- Shi‑Quan Cui
- Liang Hu
- Di‑Nan Huang
- Gan Hou
View Affiliations

Affiliations: Department of Basic Medicine, Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang, Guangdong 524023, P.R. China, Department of Clinical Biochemistry, Guangdong Medical University, Dongguan, Guangdong 523808, P.R. China
Published online on: October 3, 2017 https://doi.org/10.3892/ol.2017.7127
Pages: 7232-7240
Copyright: © Zeng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

By using human genomic DNA as a template to clone protection of telomere 1 (POT1) promoter gene segments and construct the POT1 promoter luciferase report gene vector (pGL3‑Control‑POT1‑promoter), the association between POT1, and the regulation of telomerase and telomere length was investigated. In the present study, two recombinant luciferase report gene vectors were constructed, which included different regions of the POT1 promoter. The plasmids were transformed into DH5α and the positive clones were obtained. The two plasmids termed as pGL3‑Control‑POT1‑promoter‑1 and pGL3‑Control‑POT1‑promoter‑2, were confirmed using restriction enzyme analysis and sequencing. They were separately and transiently transfected into four types of human tumor cells (A549, H460, HepG2 and HeLa). The transcriptional activities of the POT1 promoter were verified using the dual‑luciferase assay. The relative expression of POT1 and human telomerase reverse transcriptase (hTERT), and telomere length were analyzed using quantitative polymerase chain reaction in the four types of non‑transfected tumor cells. Using SPSS software, correlations between POT1 promoter activity, and POT1 expression, hTERT expression and telomere length were analyzed. Two POT1 promoter fragments (POT1‑promoter‑1 and ‑2) were successfully constructed into the pGL3‑Control luciferase report gene vector. POT1‑promoter‑1 exhibited significantly stronger transcription activity compared with POT1‑promoter‑2. The results of the partial correlation and linear regression analyses were similar: POT1 promoter activity was identified to be significantly and positively correlated with POT1 expression and telomere length (partial correlation coefficients, both P<0.05; linear regression, both P<0.01). However, POT1 promoter activity and hTERT expression were significantly negatively correlated (both P<0.05). The results obtained in the present study suggest that the POT1 promoter influences telomere length. Furthermore, these data indicated that POT1 promoter activity and POT1, as well as telomere length, may be a useful biomarker for tumor detection and future patient prognosis.

View Figures

View References

1	Lau LM, Dagg RA, Henson JD, Au AY, Royds JA and Reddel RR: Detection of alternative lengthening of telomeres by telomere quantitative PCR. Nucleic Acids Res. 41:e342013. View Article : Google Scholar : PubMed/NCBI
2	Baumann P, Podell E and Cech TR: Human Pot1 (protection of telomeres) protein: Cytolocalization, gene structure, and alternative splicing. Mol Cell Biol. 22:8079–8087. 2002. View Article : Google Scholar : PubMed/NCBI
3	Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA and Greider CW: Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 91:25–34. 1997. View Article : Google Scholar : PubMed/NCBI
4	van Steensel B, Smogorzewska A and de Lange T: TRF2 protects human telomeres from end-to-end fusions. Cell. 92:401–413. 1998. View Article : Google Scholar : PubMed/NCBI
5	Lee HW, Blasco MA, Gottlieb GJ, Horner JW II, Greider CW and DePinho RA: Essential role of mouse telomerase in highly proliferative organs. Nature. 392:569–574. 1998. View Article : Google Scholar : PubMed/NCBI
6	Nguyen BN, Elmore LW and Holt SE: Mechanism of dominant-negative telomerase function. Cell Cycle. 8:3227–3233. 2009. View Article : Google Scholar : PubMed/NCBI
7	Ozturk S, Sozen B and Demir N: Telomere length and telomerase activity during oocyte maturation and early embryo development in mammalian species. Mol Hum Reprod. 20:15–30. 2014. View Article : Google Scholar : PubMed/NCBI
8	Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q, et al: hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell. 90:785–795. 1997. View Article : Google Scholar : PubMed/NCBI
9	Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, Harley CB and Cech TR: Telomerase catalytic subunit homologs from fission yeast and human. Science. 277:955–959. 1997. View Article : Google Scholar : PubMed/NCBI
10	O'Sullivan JN, Bronner MP, Brentnall TA, Finley JC, Shen WT, Emerson S, Emond MJ, Gollahon KA, Moskovitz AH, Crispin DA, et al: Chromosomal instability in ulcerative colitis is related to telomere shortening. Nat Genet. 32:280–284. 2002. View Article : Google Scholar : PubMed/NCBI
11	Plentz RR, Schlegelberger B, Flemming P, Gebel M, Kreipe H, Manns MP, Rudolph KL and Wilkens L: Telomere shortening correlates with increasing aneuploidy of chromosome 8 in human hepatocellular carcinoma. Hepatology. 42:522–526. 2005. View Article : Google Scholar : PubMed/NCBI
12	Loayza D and De Lange T: POT1 as a terminal transducer of TRF1 telomere length control. Nature. 423:1013–1018. 2003. View Article : Google Scholar : PubMed/NCBI
13	Wang F, Podell ER, Zaug AJ, Yang Y, Baciu P, Cech TR and Lei M: The POT1-TPP1 telomere complex is a telomerase processivity factor. Nature. 445:506–510. 2007. View Article : Google Scholar : PubMed/NCBI
14	Latrick CM and Cech TR: POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation. EMBO J. 29:924–933. 2010. View Article : Google Scholar : PubMed/NCBI
15	Baumann P and Cech TR: Pot1, the putative telomere end-binding protein in fission yeast and humans. Science. 292:1171–1175. 2001. View Article : Google Scholar : PubMed/NCBI
16	Veldman T, Etheridge KT and Counter CM: Loss of hPot1 function leads to telomere instability and a cut-like phenotype. Curr Biol. 14:2264–2270. 2004. View Article : Google Scholar : PubMed/NCBI
17	Armbruster BN, Linardic CM, Veldman T, Bansal NP, Downie DL and Counter CM: Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1. Mol Cell Biol. 24:3552–3561. 2004. View Article : Google Scholar : PubMed/NCBI
18	Colgin LM, Baran K, Baumann P, Cech TR and Reddel RR: Human POT1 facilitates telomere elongation by telomerase. Curr Biol. 13:942–946. 2003. View Article : Google Scholar : PubMed/NCBI
19	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
20	Prather AA, Puterman E, Lin J, O'Donovan A, Krauss J, Tomiyama AJ, Epel ES and Blackburn EH: Shorter leukocyte telomere length in midlife women with poor sleep quality. J Aging Res. 2011:7213902011. View Article : Google Scholar : PubMed/NCBI
21	Zee RY, Michaud SE, Germer S and Ridker PM: Association of shorter mean telomere length with risk of incident myocardial infarction: A prospective, nested case-control approach. Clin Chim Acta. 403:139–141. 2009. View Article : Google Scholar : PubMed/NCBI
22	Lapham K, Kvale MN, Lin J, Connell S, Croen LA, Dispensa BP, Fang L, Hesselson S, Hoffmann TJ, Iribarren C, et al: Automated assay of telomere length measurement and informatics for 100,000 subjects in the genetic epidemiology research on adult health and aging (GERA) cohort. Genetics. 200:1061–1072. 2015. View Article : Google Scholar : PubMed/NCBI
23	Naylor LH: Reporter gene technology: The future looks bright. Biochem Pharmacol. 58:749–757. 1999. View Article : Google Scholar : PubMed/NCBI
24	Miraglia LJ, King FJ and Damoiseaux R: Seeing the light: Luminescent reporter gene assays. Comb Chem High Throughput Screen. 14:648–657. 2011. View Article : Google Scholar : PubMed/NCBI
25	Tiffen JC, Bailey CG, Ng C, Rasko JE and Holst J: Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo. Mol Cancer. 9:2992010. View Article : Google Scholar : PubMed/NCBI
26	Wentzensen IM, Mirabello L, Pfeiffer RM and Savage SA: The association of telomere length and cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev. 20:1238–1250. 2011. View Article : Google Scholar : PubMed/NCBI
27	Zhang CL, Chen XH, Li L, Zhou Y, Wang C and Hou S: The association between telomere length and cancer prognosis: Evidence from a meta-analysis. PLoS One. 10:e01331742015. View Article : Google Scholar : PubMed/NCBI
28	Gilson E and Londoño-Vallejo JA: Telomere length profiles in humans: All ends are not equal. Cell Cycle. 6:2486–2494. 2007. View Article : Google Scholar : PubMed/NCBI
29	Ju Z and Rudolph Lenhard K: Telomere dysfunction and stem cell ageing. Biochimie. 90:24–32. 2008. View Article : Google Scholar : PubMed/NCBI
30	Xin H, Liu D, Wan M, Safari A, Kim H, Sun W, O'Connor MS and Songyang Z: TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase. Nature. 445:559–562. 2007. View Article : Google Scholar : PubMed/NCBI
31	Izgi A, Gunal A, Yalcin S and Gunduz U: Telomere 1 (POT1) gene expression and its association with telomerase activity in colorectal tumor samples with different pathological features. Biomed Pharmacother. 68:841–846. 2014. View Article : Google Scholar : PubMed/NCBI
32	Churikov D and Price CM: Pot1 and cell cycle progression cooperate in telomere length regulation. Nat Struct Mol Biol. 15:79–84. 2008. View Article : Google Scholar : PubMed/NCBI
33	Possemato R, Timmons JC, Bauerlein EL, Wada N, Baldwin A, Masutomi K and Hahn WC: Suppression of hPOT1 in diploid human cells results in an hTERT-dependent alteration of telomere length dynamics. Mol Cancer Res. 6:1582–1593. 2008. View Article : Google Scholar : PubMed/NCBI
34	Butler KS, Hines WC, Heaphy CM and Griffith JK: Coordinate regulation between expression levels of telomere-binding proteins and telomere length in breast carcinomas. Cancer Med. 1:165–175. 2012. View Article : Google Scholar : PubMed/NCBI
35	Zimmermann S, Biniossek ML, Maurer C, Münzer P, Pantic M, Veelken H and Martens UM: Proteomic profiling in distinct cellular compartments of tumor cells reveals p53-dependent upregulation of S100A6 upon induction of telomere dysfunction. Proteomics. 9:5175–5187. 2009. View Article : Google Scholar : PubMed/NCBI