Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2651807&req=5

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.

Mentions: To further investigate the association between telomere maintenance and the epigenetic changes at the subtelomere, and to examine their temporal relationship, we performed bisulphite allelic sequencing on a panel of simian virus 40 (SV40)-transformed cell lines. These cell lines were clonally derived and independently immortalized from the fibroblasts of a single individual; approximately half of SV40-immortalized fibroblast lines maintain telomere length via ALT, and the other half activate telomerase (25). We examined methylation patterns at the same three subtelomeric loci (2p, 4p and 18p) in SV40-transformed immortalized clones (three telomerase-positive and three ALT), and compared these with patterns in pre-immortal SV40-transformed clones with no detectable telomere maintenance mechanism (Figure 3). We found that subtelomeric methylation patterns in pre-immortal and immortalized fibroblasts largely mirrored the patterns we observed in normal PBMC and tumour-derived cell lines, respectively. Pre-immortal fibroblasts demonstrated methylation patterns that were similar to those seen in normal primary PBMC, with the exception of one subtelomere in one cell line (JFCF-6/T.5B at chromosome 2p) which exhibited a considerable loss of methylation (Figure 3). Subtelomeric methylation was significantly increased in telomerase-positive SV40-transformed immortalized cells compared with pre-immortal cells (93 ± 3% versus 80 ± 5%, P < 0.001; Figure 3 and Table S3); ALT-positive immortalized cells showed heterogeneous subtelomeric methylation between clonal lines and between loci, with average percentage methylation ranging from 53% to 91%. The methylation patterns from pre-immortal cells suggest that, in general, changes in subtelomeric methylation do not precede immortalization by a significant extent, but are related to the activation of a telomere maintenance mechanism. However, our observation of reduced subtelomeric methylation at one locus suggests that losses of subtelomeric methylation may occur prior to activation of telomere maintenance; whether or not this cell line would be more likely to use ALT if immortalized cannot be ascertained.Figure 3.


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)

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

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

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.
Mentions: To further investigate the association between telomere maintenance and the epigenetic changes at the subtelomere, and to examine their temporal relationship, we performed bisulphite allelic sequencing on a panel of simian virus 40 (SV40)-transformed cell lines. These cell lines were clonally derived and independently immortalized from the fibroblasts of a single individual; approximately half of SV40-immortalized fibroblast lines maintain telomere length via ALT, and the other half activate telomerase (25). We examined methylation patterns at the same three subtelomeric loci (2p, 4p and 18p) in SV40-transformed immortalized clones (three telomerase-positive and three ALT), and compared these with patterns in pre-immortal SV40-transformed clones with no detectable telomere maintenance mechanism (Figure 3). We found that subtelomeric methylation patterns in pre-immortal and immortalized fibroblasts largely mirrored the patterns we observed in normal PBMC and tumour-derived cell lines, respectively. Pre-immortal fibroblasts demonstrated methylation patterns that were similar to those seen in normal primary PBMC, with the exception of one subtelomere in one cell line (JFCF-6/T.5B at chromosome 2p) which exhibited a considerable loss of methylation (Figure 3). Subtelomeric methylation was significantly increased in telomerase-positive SV40-transformed immortalized cells compared with pre-immortal cells (93 ± 3% versus 80 ± 5%, P < 0.001; Figure 3 and Table S3); ALT-positive immortalized cells showed heterogeneous subtelomeric methylation between clonal lines and between loci, with average percentage methylation ranging from 53% to 91%. The methylation patterns from pre-immortal cells suggest that, in general, changes in subtelomeric methylation do not precede immortalization by a significant extent, but are related to the activation of a telomere maintenance mechanism. However, our observation of reduced subtelomeric methylation at one locus suggests that losses of subtelomeric methylation may occur prior to activation of telomere maintenance; whether or not this cell line would be more likely to use ALT if immortalized cannot be ascertained.Figure 3.

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