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Human Molecular Genetics, 2003, Vol. 12, No. 11 1329-1336
DOI: 10.1093/hmg/ddg139
© 2003 Oxford University Press

Telomere length and the expression of natural telomeric genes in human fibroblasts

Yi Ning1,*, Jing-fan Xu1, Yu Li2, Liz Chavez3, Harold C. Riethman4, Peter M. Lansdorp3 and Nan-ping Weng2

1Department of Pathology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA, 2Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA, 3Terry-Fox Laboratory, BC Cancer Research Centre, Vancouver, British Columbia, Canada V5Z 1L3 and 4The Wistar Institute, Philadelphia, PA 19104, USA

Received December 20, 2002; Revised February 20, 2003; Accepted March 30, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Progressive telomere shortening occurs with division of normal human cells, and eventually leads to replicative senescence. The mechanism by which the shortened telomeres cause growth arrest is largely unknown. Transcriptional silencing of genes adjacent to telomeres, also called telomere position effect, has been hypothesized as a possible mechanism of telomere-mediated senescence. However, there is no report regarding telomere position effect on natural telomeric genes in human cells. To address whether the expression of natural telomeric genes is regulated by telomere length, we combined quantitative RT–PCR with quantitative fluorescence in situ hybridization to comparatively analyze the expression of 34 telomeric genes and telomere length of their 24 corresponding chromosome ends in young and senescent human fibroblasts. We have demonstrated here that telomere length alone is not sufficient to determine the expression status of natural telomeric genes. An extended analysis of a tandem of eight telomeric genes on a single chromosome end revealed a discontinuous pattern of changed expression during telomere shortening and some of the changes are senescence-specific rather than non-dividing-related. These results suggest that the expression of natural telomeric genes may be influenced by alteration of local heterochromatin structure.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human diploid fibroblasts in culture exhibit a finite life span. Following a limited number of cell divisions, they enter a state of growth arrest referred to as replicative senescence. Substantial experimental evidence has demonstrated that the total cellular life span is measured by the number of cell generations and the progressive loss of the telomere repeat tracts at the chromosome ends is an important timing mechanism in replicative senescence (1). However, it remains unknown how cells sense their telomere shortening and why the shortened telomeres can result in the cessation of cell division.

Transcriptional silencing of the genes adjacent to telomeres has been hypothesized as one of the possible mechanisms of telomere-mediated senescence (2). The silencing phenomenon, also called telomere position effect (TPE), is best characterized in yeast, Saccharomyces cerevisiae, where the reversible silencing of a gene near a telomere appears to depend on telomere length as well as its distance to the telomere (3). Studies of the silencing phenomenon in mammalian cells have yielded inconclusive results (4–8). A recent report by Baur et al. (7) demonstrated the presence of TPE in human HeLa cells when introducing a luciferase reporter gene adjacent to telomere repeats on a truncated telomere and the strength of the silencing effect correlated with telomere length. However, these experiments involve genes that are artificially positioned upstream of a telomere and no intact natural subtelomeric region is present on the truncated telomere. It remains to determine whether and how TPE operates on natural telomeric genes that are located tens to hundreds of kilobases away from the telomeres in human cells.

The terminus of human chromosomes consists of conserved simple tandem repeats (T2AG3)n as well as various types of polymorphic subtelomeric repeats. They are followed by unique sequences where natural telomeric genes reside (9,10). Owing to this unusual sequence composition and organization, cloning and characterizing the telomeric region represents a major challenge. With the development of a specialized yeast artificial chromosome (YAC) cloning strategy called half-YACs, Riethman et al. have recently isolated chromosome ends and connected them to the draft human genome sequence (11). Since each half-YAC clone contains a large segment of telomeric DNA flanked by the cloning vector sequence at one end and the human telomere repeat sequence capable of complementing yeast telomere function at the other end, the genes mapped to the half-YACs represent the most telomeric genes with a known distance to the ends of specific chromosomes.

Taking the advantage of these recent advances, we attempted to determine whether the expression of natural telomeric genes is regulated by telomere length in human fibroblasts. In this study, we analyzed 34 telomeric genes and the telomere length of their 24 corresponding chromosome ends in young and senescent human fibroblasts, and demonstrated that telomere length alone is not sufficient to determine the expression status of telomeric genes. We also analyzed a tandem of eight telomeric genes on a single chromosome end, compared their expression level in senescent versus quiescent and young cells, and propose here that telomere shortening may influence gene expression through the alteration of local heterochromatin structure involving subtelomeric sequences.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Telomeric gene expression in young and senescent cells
We cultured human diploid fibroblasts HCA2 (12) derived from neonatal foreskin and carried them to the senescent stage by serial passage in 10% serum. The maximum lifespan of these cells was ~75 population doublings (PD). We selected 34 single-copy telomeric genes/ESTs that are mapped to 24 half-YACs, corresponding to 24 chromosome ends (Table 1), and analyzed their expression in young (PD18) and senescent (PD75) HCA2 cells by quantitative RT–PCR approach. The RT–PCR experiments for each gene were repeated three to four times. The amplified products from each experiment were then quantified by an Angilent 2100 Bioanalyzer system, a chip-based nucleic acid separation system that provides high fidelity of sizing and quantitation. The expression level of each gene was normalized to GAPDH. To further validate this approach, we used a well-studied gene p21(Waf1/Cip1/Sdi1) as a control. This is a target gene for the p53 transcription factor and has been shown to have a 10–20-fold increase of expression in senescent cells (13,14). Indeed, we observed an average 15-fold increase of expression in senescent versus young cells from three quantitative RT–PCR experiments. The normalized expression levels of the 34 telomeric genes/ESTs in young and senescent cells, together with their name, chromosome location and distance to reference telomere, are summarized in Table 1. An intensity ratio change between young and senescent cells over 1.5-fold in either direction was considered as increased or decreased expression. We found that nine genes/ESTs expressed at higher levels in young cells, seven genes/ESTs expressed at higher levels in senescent cells, and 18 genes/ESTs did not show differential expression between young and senescent cells. Although there was a substantial loss of mean terminal restriction fragment length from PD18 to PD75 in HCA2 cells (data not shown), we did not observe an obvious bias of changed expression of these natural telomeric genes in the senescent cells that possess overall shortened telomeres relative to the young cells (Table 1).


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  		Table 1. Changes of telomeric gene expression and telomere length between young and senescent fibroblasts
 
Telomere dynamics and telomeric gene expression
Since telomere length exhibits a considerable heterogeneity on different chromosome ends (15,16) and the analysis of telomere length using terminal restriction fragment length assay did not provide information on telomere length at individual chromosome end, we chose the quantitative fluorescence in situ hybridization (Q-FISH) technique to examine individual telomere length of 24 chromosome ends where the 34 genes/ESTs were located. This analysis confirmed the accumulation of short telomeres prior to replicative senescence (Fig. 1) and demonstrated a variable range of telomere length among individual chromosome ends (0.4–1.8 kb) in near-senescent cells. It also revealed variations in the net loss of telomeres (1.2–2.9 kb, or 21–50 bp loss per mean population doubling) on different chromosome ends (Table 1). To investigate the potential relationship of telomere length and telomeric gene expression, we compared the changes of telomeric gene expression with either the telomere length of near senescent cells (cells at PD65 with sufficient metaphase for analysis) or the average rate of telomere loss per cell division. No direct correlation was found between the changes of telomeric gene expression and telomere length dynamics (length of telomere in cells at PD65 as well as net loss of telomere from PD18 to PD65) of the corresponding chromosome ends (Fig. 2A and B). These results indicate that telomere length alone is not sufficient to determine the expression status of natural telomeric genes.



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  		Figure 1. Quantitative-FISH analysis of individual telomeres. Representative images of HCA2 cells at PD18 (A) and PD65 (B) demonstrate variable telomere lengths and heterogeneous accumulation of short telomeres prior to replicative senescence.

 


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  		Figure 2. Relationship of telomeric gene expression and telomere length dynamics. (A) Telomere length of near senescent (PD65) HCA2 cells as function of changes of telomeric gene expression in near senescent versus young cells; (B) Rate of telomere loss per cell division at individual ends as function of changes of telomeric gene expression in near senescent versus young cells.

 
Neighboring telomeric genes expression on a single chromosome end
Studies of genes on truncated yeast telomere showed a continuous domain of transcriptional silencing with its strength inversely proportional to the distance from the telomere (17). To examine whether there is a similar pattern for natural telomeric genes in human cells, we included eight chromosome ends with two or three neighboring genes in our analysis. As shown in Table 1, a unidirectional expression of neighboring genes was observed on chromosomes 1q (both had decreased expression in senescent cells), 16q (both had increased expression in senescent cells), and 11p (all three had no significant changes in young versus senescent cells), whereas neighboring genes on chromosomes 10p, 10q, 12p, 13q and 17p exhibited one changed and one or two unchanged expression. These findings prompted us to extend our analysis and further explore the expression pattern of the neighboring telomeric genes. We analyzed a tandem of eight genes ranging from ~170 to 640 kb from the telomere of the long arm of chromosome 16. The order, distance from reference telomere and transcriptional orientation of these eight genes are shown in Figure 3A. This analysis revealed a discontinuous pattern of changed expression: two proximal and two distal genes exhibited an increased expression in senescent cells and four genes in between showed no differential expression (Fig. 3B). In addition, we analyzed the expression of these genes when cells were at PD55 (middle passage). Remarkably, there were no significant differences in expression between PD18 and PD55 cells (Fig. 3B), suggesting that the changes of expression occurred as a late event, rather than a gradual one, during the senescence process. Thus, the shortened telomeres in senescent cells may influence telomeric gene expression in a more complex way. Telomeres are known to regulate chromatin structure in yeast (18), and new evidence from the study of human cells also supports a role for telomere structure, rather than length, in replicative senescence (19). A recent report by Koering et al. (8) provided additional evidence that the control of human TPE may not be the telomere length per se but accompanied changes in chromatin structure. It is therefore possible that TPE on natural chromosome ends depends on whether or not there is an alteration of heterochromatin structure that appears to occur towards the end of cellular lifespan, and such alteration is specific to particular domains rather than a continuous zone of transcriptional repression.



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  		Figure 3. Expression of a tandem of eight genes on the long arm of chromosome 16 terminal region. (A) Name of the genes and their transcriptional orientation and distance to reference telomere. The six additional genes and GenBank accession numbers are: CPNE7 (NM_014427), CDK10 (NM_003674), FANCA (NM_000135), KIAA1049 (NM_014972), TUBB4 (NM_006086), and AFG3L1 (XM_047427). (B) Quantitation of expression of these genes in young (PD18), middle-passage (PD55) and senescent (PD75) HCA2 cells. Relative differences in expression are presented as mean ratio of PD55/PD18 and PD75/PD18 with standard error.

 
Unlike the artificially inserted genes with an immediate link to the telomere repeats, natural telomeric genes and telomere repeats are separated by various length of different types of subtelomeric repeats (10,20). In fact, the role of subtelomeric sequences in TPE has been suggested in a recent study by Pryde and Louis (21). They reported a variable degree of TPE in only about half of the natural telomeres in yeast and correlated this variability to the different types of subtelomeric sequences. In the human population the subtelomeric region is highly polymorphic, and the length variation may be up to hundreds of kilobases on different alleles. Therefore, telomere length-mediated transcriptional regulation of natural telomeric genes in human cells is likely to operate through the telomeric heterochromatin structure, involving long stretches of subtelomeric sequences, and a variable range of natural telomeric gene expression is likely to be present among the population.

Differential expression of telomeric genes in senescent versus quiescent cells
Replicative senescence, triggered by telomere shortening, is defined as a state of irreversible growth arrest. In contrast, quiescence is a reversible growth state that can be induced in dividing young cells upon serum starvation or contact inhibition. In order to determine whether the differential expression of telomeric genes observed in this analysis is a senescence-specific or non-dividing state-related phenomenon, we chose the four 16q telomeric genes with increased expression in senescent cells and compared their expression level with those of the quiescent cells. Quantitative RT–PCR was performed using cDNAs from young (PD18), senescent (PD75), and quiescent (PD20 with serum starvation) cells. After normalizing to GAPDH, the relative expression ratio of GAS11, MGC3101, CDK10 and CPNE7 in senescent or quiescent versus young cells were analyzed and are shown in Figure 4. Unlike what was observed in senescent cells, the expression level of MGC3101 and CPNE7 was lower in quiescent cells and similar to that of the young cells. These results indicate that the increased expression of MGC3101 and CPNE7 is a senescence-triggered change rather than a general growth arrest-induced phenomenon. In contrast, GAS11 and CDK10 showed elevated expression in senescent as well as quiescent cells, suggesting their involvement in cell growth regulation in both senescence and quiescence.



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  		Figure 4. Comparison of selected telomeric gene expression in senescent and quiescent cells. Relative differences in expression of the four 16q telomeric genes are presented as mean ratio of senescent/young and quiescent/young with standard error. The mean value was derived from three independent measurements.

 
Despite efforts to understand the differences between quiescence and senescence at the molecular level, only a few markers can distinguish these two states. Histochemically detectable beta-galactosidase is the most commonly used senescent cell specific marker (22). Early population doubling level cDNA, also known as pigment epithelium-derived factor (EPC1/PEDF), was found to have higher level expression in quiescent versus senescent cells (23), and was also differentially expressed in quiescent versus senescent HCA2 cells (data not shown). The finding of differential expression of MGC3101 and CPNE7 in senescent versus quiescent cells demonstrates the existence of senescence-specific change of telomeric gene expression for the first time, and provides novel markers that can distinguish senescence from quiescence.

This study has led to the identification of a total of 18 telomeric genes with differential expression in young versus senescent cells. While the function of most of these differentially expressed genes are unknown, CDK10, previously referred to as PISSLRE, has been reported as one of the CDC2-related kinases that play a role in regulating the G2/M phase of cell cycle (24), and GAS11 has been implicated in cell growth arrest (25). Furthermore, it is worth noting that four telomeric genes on the long arm of chromosome 16, where a frequent loss of heterozygosity has been observed in breast cancer (26,27), exhibited increased expression in senescent cells. Viewing that replicative senescence as one of the protective mechanisms against tumor formation, it is conceivable that senescence-associated genes may play significant roles in tumor suppression. Further studies will be necessary to elucidate the biological function of telomeric genes that are differentially expressed in senescent cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells and cell culture
Human diploid fibroblasts HCA2 were obtained from Dr O. Pereira-Smith (University of Texas, San Antonio, TX, USA) and cultured in minimum essential medium supplemented with 10% fetal calf serum. The number of population doublings was determined at each subculture. Cells were subcultured 1 : 4 in early passage and 1 : 2 in late passage with weekly medium change until they failed to double in cell number in a greater than 2 week period. The HCA2 cells have been used in other studies (12) and have a known maximum lifespan of ~75 PD. Young cells refer to early passage of exponentially growing cells, and senescent cells are those in their permanent growth arrest state with characteristic large flattened senescent cell morphology and elevated expression of p21. Quiescent cells were obtained by growing young cells in 0.2% serum for 7 days, as described (28). The quiescent status was confirmed by assay for the expression of EPC1/PEDF, known to have elevated expression level in quiescent cells (25).

Quantitative RT–PCR analysis of telomeric gene expression
The genes/ESTs analyzed in this study were mapped to the half-YACs, representing the most telomeric genes from 24 chromosome ends. Their name and GenBank accession number are listed in Table 1 and in the Figure 3 legend. Total RNA from the HCA2 cells at PD 18, 55, 75 and quiescent stage was isolated and used for first-strand cDNA synthesis. RT–PCR primers were designed based on GenBank sequence for each specific gene (Table 2). The PCR conditions were optimized for each gene, and 28–35 cycles of PCR were carried out depending on the products abundance of a specific gene. Same cycle numbers were used for a specific gene in both young and senescent cells. The RT–PCR experiments for each gene were repeated three to four times, and the expression level of each gene was normalized to GAPDH. The RT–PCR products from each experiment were quantified using an Agilent 2100 Bioanalyzer (a chip-based nucleic acid separation system that separates nucleic acid fragments in micro-fabricated channels and automates detection). Relative differences in expression are presented as mean ratio of PD55/PD18 and PD75/PD18 with standard error. The comparison of selected telomeric gene expression in senescent versus quiescent cells are presented as mean ratio of senescent/young and quiescent/young with standard error.


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  		Table 2. Primer sequences for the analysis of telomeric gene expression
 
Telomere length measurement by Q-FISH
The telomere length of individual chromosome ends of HCA2 cells was measured by Q-FISH technique as described (17,18). The analysis was performed on chromosomes from 10–15 metaphase spreads when the cells at PD18 and PD65 (prior to senescent stage with sufficient metaphase cells). The measurement was based on telomere fluorescence units (TFUs), with each unit corresponding to 1 kb of (T2AG3)n repeats. The telomere length and the net loss of specific chromosome ends were determined to correlate to the expression level of telomeric genes analyzed in this study.


   ACKNOWLEDGEMENTS
 
We thank Dr Wang Zhi for technical assistance. This work was supported by a grant (AG21208) to Y.N. from the National Institute on Aging and a grant to P.M.L. from the Canadian Institute of Health Research.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 4107061282; Fax: +1 4107068414; Email: yning{at}som.umaryland.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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