Telomerase Expression in Normal Human Fibroblasts Stabilizes DNA 5-Methylcytosine Transferase I

The finite proliferative potential of normal human fibroblasts can be overcome, a process commonly called immortalization, by the introduction of the catalytic subunit of human telomerase. In contrast to malignant transformation, the pattern of gene expression remains largely unmodified in telomerase-induced immortalization. Here we show evidence that suggests that the maintenance of a “young” pattern of gene expression by telomerization is mediated, at least in part, by a novel function of human telomerase that involves regulation of DNA methyltransferase I gene expression. The finite proliferative potential of normal human fibroblasts can be overcome, a process commonly called immortalization, by the introduction of the catalytic subunit of human telomerase. In contrast to malignant transformation, the pattern of gene expression remains largely unmodified in telomerase-induced immortalization. Here we show evidence that suggests that the maintenance of a “young” pattern of gene expression by telomerization is mediated, at least in part, by a novel function of human telomerase that involves regulation of DNA methyltransferase I gene expression. Primary cultures of normal human diploid cells have a limited proliferative lifespan (1Hayflick L. Moorhead P.S. Exp. Cell Res. 1961; 25: 585-621Crossref PubMed Scopus (5517) Google Scholar, 2Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4284) Google Scholar). After completing a finite number of divisions, the culture enters a state called replicative senescence(definition) that is characterized by a growth arrest refractory to further mitogenic stimulation and by altered gene expression (3Shelton D.N. Chang E. Whittier P.S. Choi D. Funk W.D. Curr. Biol. 1999; 9: 939-945Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). The introduction of the catalytic subunit of human telomerase (hTERT), 1The abbreviations used are: hTERT and hT, human telomerase; huLF, human embryonic lung fibroblast; SNRPN, small nuclear riboprotein-associated peptide N; DNAMeTase, DNA methyltransferase; CMV, cytomegalovirus; TRAP, telomerase repeat amplification protocol; CAT, chloramphenicol acetyltransferase; PD, population doubling. the enzyme that elongates telomere(definition) ends, into pre-senescent cells extends their proliferative lifespan (4Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4114) Google Scholar). This observation is consistent with the hypothesis that telomere attrition resulting from the incomplete replication of chromosome ends by DNA polymerase α triggers cellular senescence (5Harley C.B. Futcher A.B. Greider C.W. Nature. 1990; 345: 458-460Crossref PubMed Scopus (4625) Google Scholar). One of the most interesting aspects of lifespan extension by hTERT is that in addition to preventing irreversible growth arrest, it also prevents the changes in gene expression observed to occur during cellular aging (3Shelton D.N. Chang E. Whittier P.S. Choi D. Funk W.D. Curr. Biol. 1999; 9: 939-945Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). Additionally, there are no signs of transformation or changes in the differentiation state of the telomerized cultures (6Jiang X.R. Jimenez G. Chang E. Frolkis M. Kusler B. Sage M. Beeche M. Bodnar A.G. Wahl G.M. Tlsty T.D. Chiu C.P. Nat. Genet. 1999; 21: 111-114Crossref PubMed Scopus (575) Google Scholar, 7Morales C.P. Holt S.E. Ouellette M. Kaur K.J. Yan Y. Wilson K.S. White M.A. Wright W.E. Shay J.W. Nat. Genet. 1999; 21: 115-118Crossref PubMed Scopus (683) Google Scholar). The pattern of gene expression of a particular cell type is determined mainly by chromatin architecture (the concerted pattern of cis-DNA sequences, DNA methylation, and nucleosomal protein modifications) and a particular set of transcription factors. Faithful heritability of gene expression patterns is critical to the maintenance of a particular differentiated state. Although the effects of hTERT re-expression in normal human fibroblasts have been regarded mostly as rejuvenation, the experimental evidence indicates that reactivation of telomerase most likely freezes the culture in its current state, possibly by fixing the pattern of gene expression. Here we investigated the mechanisms by which telomerase expression in fibroblasts maintains a fixed epigenomic state that prevents age-related changes in gene expression and senescence. Cells and Plasmids—The human foreskin fibroblast cell strains Wi38, HCA2, and ImR90 and their derivatives expressing the catalytic subunit of hTERT were a kind gift of J. Campisi (Lawrence Berkeley National Laboratory, Berkeley, CA). The human embryonic lung fibroblast cell strains HuLF and HuLF-hTERT were established and cultured as described previously (8Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (681) Google Scholar). pCMV-hTERT and pMet-CAT were kindly provided by R. A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA) and M. Szyf (McGill University, Quebec, Canada), respectively. Determination of the 5mC Content—Genomic DNA was isolated using the DNAZol DNA extraction kit (Molecular Research Center, Inc.) according to the manufacturer's directions. RNA was removed by alkaline hydrolysis (0.5 m NaOH, 37 °C for 1 h). The 5mC content in DNA samples was determined by high pressure liquid chromatography analysis of enzymatic hydrolysates of DNA. 10 μg of DNA were digested at 37 °C for 3 h using 2 units of microccocal nuclease (United States Biochemical, Cleveland, OH), 2 μg of spleen phosphodiesterase II (Roche Diagnostics) in 10 mm CaCl2, 20 mm sodium succinate, pH 6.0. The resulting 3′-deoxymonophosphate nucleosides were further hydrolyzed by overnight incubation at 37 °C with 20 units of alkaline phosphatase (Amersham Biosciences). Samples were injected into a Beckman Ultrasphere ODS, 4.6 mm × 25 cm (5-μm par