Telomerase activity is associated with an increase in DNA methylation at the proximal subtelomere and a reduction in telomeric transcription - PubMed (original) (raw)

Telomerase activity is associated with an increase in DNA methylation at the proximal subtelomere and a reduction in telomeric transcription

Laura J Ng et al. Nucleic Acids Res. 2009 Mar.

Abstract

Tumours and immortalized cells avoid telomere attrition by using either the ribonucleoprotein enzyme telomerase or a recombination-based alternative lengthening of telomeres (ALT) mechanism. Available evidence from mice suggests that the epigenetic state of the telomere may influence the mechanism of telomere maintenance, but this has not been directly tested in human cancer. Here we investigated cytosine methylation directly adjacent to the telomere as a marker of the telomere's epigenetic state in a panel of human cell lines. We find that while ALT cells show highly heterogeneous patterns of subtelomeric methylation, subtelomeric regions in telomerase-positive cells invariably show denser methylation than normal cells, being almost completely methylated. When compared to matched normal and ALT cells, telomerase-positive cells also exhibit reduced levels of the telomeric repeat-containing-RNA (TERRA), whose transcription originates in the subtelomere. Our results are consistent with the notion that TERRA may inhibit telomerase: the heavy cytosine methylation we observe in telomerase-positive cells may reflect selection for TERRA silencing in order to facilitate telomerase activity at the telomere. These data suggest that the epigenetic differences between telomerase-positive and ALT cells may underlie the mechanism of telomere maintenance in human tumorigenesis and highlight the broad reaching consequences of epigenetic dysregulation in cancer.

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Figures

Figure 1.

Figure 1.

Allelic methylation patterns at human subtelomeric regions in normal cells. (A) Subtelomeric regions of chromosomes 2p and 18p analysed by bisulphite sequencing. CpGs are represented by small vertical lines and PCR primers by arrows. (B) Bisulphite sequencing maps from normal human PBMC are shown below the corresponding region in (A). Within each map, horizontal lines show the methylation pattern of individual alleles: black squares represent methylated CpGs and white squares unmethylated CpGs. The average percent methylation for each individual is shown at the bottom right of each map. The gap in some alleles is due to a G/A polymorphism/mutation that results in loss of a CpG. C, cord blood; A, adult.

Figure 2.

Figure 2.

Subtelomeric methylation is heterogeneous in tumour-derived ALT cells and increased in telomerase-positive cells. Bisulphite sequencing maps for subtelomeres at chromosomes 2p, 4p and 18p are shown; those from tumour-derived ALT cells at top and those from tumour-derived telomerase-positive cells at bottom. Within each map, horizontal lines show the methylation pattern of individual alleles: black squares represent methylated CpGs and white squares unmethylated CpGs. The average percent methylation for each individual is shown at the bottom right of each map. The gap in some alleles is due to a G/A polymorphism/mutation that results in loss of a CpG.

Figure 3.

Figure 3.

Subtelomeric methylation in SV40-transformed immortalized cell lines mirrors that of tumour-derived cells. Bisulphite sequencing maps for subtelomeres at chromosomes 2p, 4p and 18p are shown; those from pre-immortal cells at top, immortalized ALT cells at middle, and immortalized telomerase-positive cells at bottom. Within each map, horizontal lines show the methylation pattern of individual alleles: black squares represent methylated CpGs and white squares unmethylated CpGs. The average percent methylation for each individual is shown at the bottom right of each map. The gap in some alleles is due to a G/A polymorphism/mutation that results in loss of a CpG.

Figure 4.

Figure 4.

Telomeric transcription is low in telomerase-positive cells compared with matched ALT cells. (A) Quantitation of TERRA transcripts in the normal fibroblasts and cell lines indicated. The intensity of each TERRA sense strand signal was measured by densitometry and normalized to the GAPDH signal. (B) Quantitation of telomeric DNA from the same cells. The intensity of telomeric signal was measured by densitometry and normalized to the Alu repeat signal. Error bars represent standard deviation between three separate experiments. (C) TERRA levels corrected for the amount of telomeric DNA, as shown in (B). In each case, levels for each cell line are expressed relative to the first normal fibroblast sample, JFCF-6.

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