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

Ng LJ, Cropley JE, Pickett HA, Reddel RR, Suter CM - Nucleic Acids Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Victor Chang Cardiac Research Institute, Darlinghurst 2010, Australia.

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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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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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.

Mentions: Heterochromatic states and dense cytosine methylation are tightly linked to transcriptional repression. Although it has been assumed that telomeres are transcriptionally silent, telomeric transcription has recently been described in mammalian cells (27,28). The telomeric repeat-containing transcript (TERRA) is transcribed from the C-rich strand using promoters situated in the subtelomeric region. Because ALT and telomerase cells exhibit different methylation profiles at the subtelomere, we investigated the levels of TERRA transcripts in ALT and telomerase-positive cells by RNA slot-blot. Total RNA from each of the cell lines described above, as well as from primary human fibroblasts derived from five individuals, was hybridized to probes directed against the sense (UUAGGG) and antisense (CCCUAA) TERRA sequence; signals were normalized to the signal for GAPDH. We observed no significant signal in any cell line with the CCCUAA-directed probe (data not shown). The TERRA:GAPDH ratio was higher in ALT cell lines than in any other sample (Figure 4A); telomerase-positive cell lines gave a much lower TERRA:GAPDH ratio. ALT cells tend to have longer telomeres than telomerase-positive cells or normal cells and this could account for their higher TERRA signal. Therefore to control for telomere length we measured the amount of telomeric DNA in each sample (Figure 4B). When corrected for the total amount of telomeric DNA, tumour-derived ALT cells still exhibited high TERRA levels compared to tumour-derived telomerase-positive cells (Figure 4C). The series of fibroblasts, ranging from normal through to immortalized, allowed us to compare TERRA levels in matching cell types and determine its normal abundance. In this series, the most striking feature is a dramatic reduction in TERRA levels in the telomerase-positive immortalized cells (Figure 4C); normal fibroblasts, SV40 transformed, non-immortal fibroblasts and immortalized ALT cells all show comparable levels of TERRA. Taken together, our data indicate that TERRA transcription is dampened in telomerase-positive cells, but permitted in ALT cells.Figure 4.


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

Ng LJ, Cropley JE, Pickett HA, Reddel RR, Suter CM - Nucleic Acids Res. (2009)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651807&req=5

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.
Mentions: Heterochromatic states and dense cytosine methylation are tightly linked to transcriptional repression. Although it has been assumed that telomeres are transcriptionally silent, telomeric transcription has recently been described in mammalian cells (27,28). The telomeric repeat-containing transcript (TERRA) is transcribed from the C-rich strand using promoters situated in the subtelomeric region. Because ALT and telomerase cells exhibit different methylation profiles at the subtelomere, we investigated the levels of TERRA transcripts in ALT and telomerase-positive cells by RNA slot-blot. Total RNA from each of the cell lines described above, as well as from primary human fibroblasts derived from five individuals, was hybridized to probes directed against the sense (UUAGGG) and antisense (CCCUAA) TERRA sequence; signals were normalized to the signal for GAPDH. We observed no significant signal in any cell line with the CCCUAA-directed probe (data not shown). The TERRA:GAPDH ratio was higher in ALT cell lines than in any other sample (Figure 4A); telomerase-positive cell lines gave a much lower TERRA:GAPDH ratio. ALT cells tend to have longer telomeres than telomerase-positive cells or normal cells and this could account for their higher TERRA signal. Therefore to control for telomere length we measured the amount of telomeric DNA in each sample (Figure 4B). When corrected for the total amount of telomeric DNA, tumour-derived ALT cells still exhibited high TERRA levels compared to tumour-derived telomerase-positive cells (Figure 4C). The series of fibroblasts, ranging from normal through to immortalized, allowed us to compare TERRA levels in matching cell types and determine its normal abundance. In this series, the most striking feature is a dramatic reduction in TERRA levels in the telomerase-positive immortalized cells (Figure 4C); normal fibroblasts, SV40 transformed, non-immortal fibroblasts and immortalized ALT cells all show comparable levels of TERRA. Taken together, our data indicate that TERRA transcription is dampened in telomerase-positive cells, but permitted in ALT cells.Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Victor Chang Cardiac Research Institute, Darlinghurst 2010, Australia.

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.

Show MeSH
Related in: MedlinePlus