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ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance.

McKerlie M, Lin S, Zhu XD - Nucleic Acids Res. (2012)

Bottom Line: In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers.Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis.Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON L8S4K1, Canada.

ABSTRACT
Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

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Overexpression of TRF1-S367D promotes the formation of telomere doublets in TRF1-depleted HeLaII cells. (A) Images of metaphase chromosomes depicting telomere abnormalities. Metaphase chromosomes were stained with DAPI and false-colored in red. Telomeric DNA was detected by FISH using a FITC-conjugated (CCCTAA)3-containing PNA probe in green. Open arrows represent telomere doublets whereas asterisks indicate telomere loss. (B) Quantification of telomere doublets from TRF1-depleted cells expressing indicated constructs. For each cell line, a total of 3150–3750 chromosomes from 60 to 69 metaphase cells were scored in a blind manner for the presence of telomere doublets in B and D and telomere loss in C. Standard deviations derived from three independent experiments are indicated. (C) Quantification of telomere loss from TRF1-depleted cells expressing indicated constructs. Telomere loss refers to chromatids without a detectable telomere signal. The total number of chromatid ends without a detectable telomere signal was divided by the total number of chromatid ends scored, giving rise to the percentage of telomere loss per chromatid end. (D) Quantification of telomere doublets from aphidicolin-treated cells. TRF1-depleted cells expressing indicated constructs were treated with aphidicolin (0.2 µM) for 16 h prior to FISH analysis.
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gks035-F8: Overexpression of TRF1-S367D promotes the formation of telomere doublets in TRF1-depleted HeLaII cells. (A) Images of metaphase chromosomes depicting telomere abnormalities. Metaphase chromosomes were stained with DAPI and false-colored in red. Telomeric DNA was detected by FISH using a FITC-conjugated (CCCTAA)3-containing PNA probe in green. Open arrows represent telomere doublets whereas asterisks indicate telomere loss. (B) Quantification of telomere doublets from TRF1-depleted cells expressing indicated constructs. For each cell line, a total of 3150–3750 chromosomes from 60 to 69 metaphase cells were scored in a blind manner for the presence of telomere doublets in B and D and telomere loss in C. Standard deviations derived from three independent experiments are indicated. (C) Quantification of telomere loss from TRF1-depleted cells expressing indicated constructs. Telomere loss refers to chromatids without a detectable telomere signal. The total number of chromatid ends without a detectable telomere signal was divided by the total number of chromatid ends scored, giving rise to the percentage of telomere loss per chromatid end. (D) Quantification of telomere doublets from aphidicolin-treated cells. TRF1-depleted cells expressing indicated constructs were treated with aphidicolin (0.2 µM) for 16 h prior to FISH analysis.

Mentions: TRF1 has been shown to be important for telomere replication (6,9), the defect of which can give rise to fragile telomeres (6,9), also known as telomere doublets. Previously we have shown that depletion of TRF1 induces the formation of telomere doublets and telomere loss, both of which can be suppressed by wild-type TRF1 (12). We asked whether S367 phosphorylation might affect the ability of TRF1 to suppress these abnormalities. FISH analysis was performed on metaphase cells derived from HeLaII cells stably expressing shTRF1/pWZL, shTRF1/TRF1, shTRF1/S367A or shTRF1/S367D. Consistent with previous findings (12), we found that overexpression of wild-type TRF1 was able to suppress the formation of both telomere doublets and telomere loss in TRF1-depleted cells (Figure 8A–C). Introduction of TRF1-S367A into TRF1-depleted cells was also able to suppress these telomere abnormalities (Figure 8A–C). On the other hand, we found that overexpression of TRF1-S367D failed to result in any reduction in telomere doublets or telomere loss in TRF1-depleted HeLaII cells (Figure 8B and C). In fact, we detected a further increase in the formation of telomere doublets as a result of TRF1-S367D expression in TRF1-depleted HeLaII cells (Figure 8B). Aphidicolin, an inhibitor of DNA replication, has been shown to induce telomere doublets (6,9). We observed a substantial increase in telomere doublets in TRF1-depleted cells upon treatment with aphidicolin (Figure 8D), consistent with a previous report that the effect of aphidicolin was additive with the loss of TRF1 (9). Such a increase was also observed in TRF1-S367D-expressing cells (Figure 8D). Taken together, these results suggest that the phosphomimic mutation of S367D impairs TRF1 function in telomere replication.Figure 8.


ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance.

McKerlie M, Lin S, Zhu XD - Nucleic Acids Res. (2012)

Overexpression of TRF1-S367D promotes the formation of telomere doublets in TRF1-depleted HeLaII cells. (A) Images of metaphase chromosomes depicting telomere abnormalities. Metaphase chromosomes were stained with DAPI and false-colored in red. Telomeric DNA was detected by FISH using a FITC-conjugated (CCCTAA)3-containing PNA probe in green. Open arrows represent telomere doublets whereas asterisks indicate telomere loss. (B) Quantification of telomere doublets from TRF1-depleted cells expressing indicated constructs. For each cell line, a total of 3150–3750 chromosomes from 60 to 69 metaphase cells were scored in a blind manner for the presence of telomere doublets in B and D and telomere loss in C. Standard deviations derived from three independent experiments are indicated. (C) Quantification of telomere loss from TRF1-depleted cells expressing indicated constructs. Telomere loss refers to chromatids without a detectable telomere signal. The total number of chromatid ends without a detectable telomere signal was divided by the total number of chromatid ends scored, giving rise to the percentage of telomere loss per chromatid end. (D) Quantification of telomere doublets from aphidicolin-treated cells. TRF1-depleted cells expressing indicated constructs were treated with aphidicolin (0.2 µM) for 16 h prior to FISH analysis.
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gks035-F8: Overexpression of TRF1-S367D promotes the formation of telomere doublets in TRF1-depleted HeLaII cells. (A) Images of metaphase chromosomes depicting telomere abnormalities. Metaphase chromosomes were stained with DAPI and false-colored in red. Telomeric DNA was detected by FISH using a FITC-conjugated (CCCTAA)3-containing PNA probe in green. Open arrows represent telomere doublets whereas asterisks indicate telomere loss. (B) Quantification of telomere doublets from TRF1-depleted cells expressing indicated constructs. For each cell line, a total of 3150–3750 chromosomes from 60 to 69 metaphase cells were scored in a blind manner for the presence of telomere doublets in B and D and telomere loss in C. Standard deviations derived from three independent experiments are indicated. (C) Quantification of telomere loss from TRF1-depleted cells expressing indicated constructs. Telomere loss refers to chromatids without a detectable telomere signal. The total number of chromatid ends without a detectable telomere signal was divided by the total number of chromatid ends scored, giving rise to the percentage of telomere loss per chromatid end. (D) Quantification of telomere doublets from aphidicolin-treated cells. TRF1-depleted cells expressing indicated constructs were treated with aphidicolin (0.2 µM) for 16 h prior to FISH analysis.
Mentions: TRF1 has been shown to be important for telomere replication (6,9), the defect of which can give rise to fragile telomeres (6,9), also known as telomere doublets. Previously we have shown that depletion of TRF1 induces the formation of telomere doublets and telomere loss, both of which can be suppressed by wild-type TRF1 (12). We asked whether S367 phosphorylation might affect the ability of TRF1 to suppress these abnormalities. FISH analysis was performed on metaphase cells derived from HeLaII cells stably expressing shTRF1/pWZL, shTRF1/TRF1, shTRF1/S367A or shTRF1/S367D. Consistent with previous findings (12), we found that overexpression of wild-type TRF1 was able to suppress the formation of both telomere doublets and telomere loss in TRF1-depleted cells (Figure 8A–C). Introduction of TRF1-S367A into TRF1-depleted cells was also able to suppress these telomere abnormalities (Figure 8A–C). On the other hand, we found that overexpression of TRF1-S367D failed to result in any reduction in telomere doublets or telomere loss in TRF1-depleted HeLaII cells (Figure 8B and C). In fact, we detected a further increase in the formation of telomere doublets as a result of TRF1-S367D expression in TRF1-depleted HeLaII cells (Figure 8B). Aphidicolin, an inhibitor of DNA replication, has been shown to induce telomere doublets (6,9). We observed a substantial increase in telomere doublets in TRF1-depleted cells upon treatment with aphidicolin (Figure 8D), consistent with a previous report that the effect of aphidicolin was additive with the loss of TRF1 (9). Such a increase was also observed in TRF1-S367D-expressing cells (Figure 8D). Taken together, these results suggest that the phosphomimic mutation of S367D impairs TRF1 function in telomere replication.Figure 8.

Bottom Line: In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers.Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis.Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON L8S4K1, Canada.

ABSTRACT
Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

Show MeSH
Related in: MedlinePlus