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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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Phosphorylated (pS367)TRF1 forms distinct non-telomeric foci in a cell cycle regulated manner, enriched in S and G2 phases. (A) FACS analysis of synchronized HeLaI.2.11 cells. y-axis, cell numbers; x-axis, relative DNA content on the basis of staining with propidium iodine. Asyn, asynchronous population; 0–10 h, cells were released for 0–10 h from a double thymidine block. (B) Indirect IF using anti-pS367 antibody was performed on GM847 cells released for 0–16 h from a double thymidine block. Cell nuclei were stained with DAPI shown in blue. (C) Quantification of percentage of GM847 and HeLaI.2.11 cells exhibiting five or more phosphorylated (pS367)TRF1-containing foci. For each of the indicated time-points post-release from a double thymidine block, a total of at least 1500 cells from three independent experiments were scored in blind for both GM847 and HeLaI.2.11 cells. Standard deviations from three independent experiments are indicated.
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gks035-F3: Phosphorylated (pS367)TRF1 forms distinct non-telomeric foci in a cell cycle regulated manner, enriched in S and G2 phases. (A) FACS analysis of synchronized HeLaI.2.11 cells. y-axis, cell numbers; x-axis, relative DNA content on the basis of staining with propidium iodine. Asyn, asynchronous population; 0–10 h, cells were released for 0–10 h from a double thymidine block. (B) Indirect IF using anti-pS367 antibody was performed on GM847 cells released for 0–16 h from a double thymidine block. Cell nuclei were stained with DAPI shown in blue. (C) Quantification of percentage of GM847 and HeLaI.2.11 cells exhibiting five or more phosphorylated (pS367)TRF1-containing foci. For each of the indicated time-points post-release from a double thymidine block, a total of at least 1500 cells from three independent experiments were scored in blind for both GM847 and HeLaI.2.11 cells. Standard deviations from three independent experiments are indicated.

Mentions: To address the cell cycle-dependent nature of anti-pS367 staining, we arrested HeLaI.2.11 and GM847 cells at the G1/S boundary with a double thymidine block and then released them into fresh media for 0, 2, 4, 6, 8, 10 or 16 h. Consistent with our previous findings (34), we found that HeLa cells progressed through S phase 2–6 h post release and enter mitosis 10 h post release (Figure 3A). Analysis of immunofluoresence showed that the bright foci stained by anti-pS367 antibody were predominantly seen in cells fixed 2–8 h post release from a double thymidine block whereas very few cells arrested at G1/S or in G1 (16 h post release from a double thymidine block) displayed such foci (Figure 3B and C). These results suggest that phosphorylated (pS367)TRF1-containing foci are cell cycle regulated, occuring in S and G2 phase.Figure 3.


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

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

Phosphorylated (pS367)TRF1 forms distinct non-telomeric foci in a cell cycle regulated manner, enriched in S and G2 phases. (A) FACS analysis of synchronized HeLaI.2.11 cells. y-axis, cell numbers; x-axis, relative DNA content on the basis of staining with propidium iodine. Asyn, asynchronous population; 0–10 h, cells were released for 0–10 h from a double thymidine block. (B) Indirect IF using anti-pS367 antibody was performed on GM847 cells released for 0–16 h from a double thymidine block. Cell nuclei were stained with DAPI shown in blue. (C) Quantification of percentage of GM847 and HeLaI.2.11 cells exhibiting five or more phosphorylated (pS367)TRF1-containing foci. For each of the indicated time-points post-release from a double thymidine block, a total of at least 1500 cells from three independent experiments were scored in blind for both GM847 and HeLaI.2.11 cells. Standard deviations from three independent experiments are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351164&req=5

gks035-F3: Phosphorylated (pS367)TRF1 forms distinct non-telomeric foci in a cell cycle regulated manner, enriched in S and G2 phases. (A) FACS analysis of synchronized HeLaI.2.11 cells. y-axis, cell numbers; x-axis, relative DNA content on the basis of staining with propidium iodine. Asyn, asynchronous population; 0–10 h, cells were released for 0–10 h from a double thymidine block. (B) Indirect IF using anti-pS367 antibody was performed on GM847 cells released for 0–16 h from a double thymidine block. Cell nuclei were stained with DAPI shown in blue. (C) Quantification of percentage of GM847 and HeLaI.2.11 cells exhibiting five or more phosphorylated (pS367)TRF1-containing foci. For each of the indicated time-points post-release from a double thymidine block, a total of at least 1500 cells from three independent experiments were scored in blind for both GM847 and HeLaI.2.11 cells. Standard deviations from three independent experiments are indicated.
Mentions: To address the cell cycle-dependent nature of anti-pS367 staining, we arrested HeLaI.2.11 and GM847 cells at the G1/S boundary with a double thymidine block and then released them into fresh media for 0, 2, 4, 6, 8, 10 or 16 h. Consistent with our previous findings (34), we found that HeLa cells progressed through S phase 2–6 h post release and enter mitosis 10 h post release (Figure 3A). Analysis of immunofluoresence showed that the bright foci stained by anti-pS367 antibody were predominantly seen in cells fixed 2–8 h post release from a double thymidine block whereas very few cells arrested at G1/S or in G1 (16 h post release from a double thymidine block) displayed such foci (Figure 3B and C). These results suggest that phosphorylated (pS367)TRF1-containing foci are cell cycle regulated, occuring in S and G2 phase.Figure 3.

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