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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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ATM is required for the subnuclear localization of phosphorylated (pS367)TRF1. (A) ATM inhibition leads to loss of phosphorylated (pS367)TRF1-containing foci. Indirect IF was performed with anti-pS367 antibody on HeLaI.2.11 cells treated with either DMSO or KU55933. Cell nuclei were stained with DAPI in blue. (B) Quantification of DMSO-treated or KU55933-treated HeLaI.2.11 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for both DMSO-treated and KU55933-treated cells. Standard deviations from three independent experiments are indicated. (C) ATM-deficient cells lack phosphorylated (pS367)TRF1-containing foci. IF was performed with anti-pS367 antibody on IMR90 cells as well as ATM-deficient GM09607 and GM05849 cells. Cell nuclei were stained with DAPI in blue. (D) Quantification of ATM-proficient or ATM-deficient cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for IMR90, GM09607 and GM05849 cells. Standard deviations from three independent experiments are indicated. (E) Introduction of ATM into ATM-deficient cells restores phosphorylated (pS367)TRF1-containing foci. IF was conducted with anti-pS367 antibody on ATM-deficient cells expressing either the vector alone (GM16666) or complemented with ATM (GM16667). Cell nuclei were stained with DAPI in blue. (F) Quantification of GM16666 or GM16667 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of 1500 cells from three independent experiments were scored. Standard deviations from three independent experiments are indicated.
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gks035-F4: ATM is required for the subnuclear localization of phosphorylated (pS367)TRF1. (A) ATM inhibition leads to loss of phosphorylated (pS367)TRF1-containing foci. Indirect IF was performed with anti-pS367 antibody on HeLaI.2.11 cells treated with either DMSO or KU55933. Cell nuclei were stained with DAPI in blue. (B) Quantification of DMSO-treated or KU55933-treated HeLaI.2.11 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for both DMSO-treated and KU55933-treated cells. Standard deviations from three independent experiments are indicated. (C) ATM-deficient cells lack phosphorylated (pS367)TRF1-containing foci. IF was performed with anti-pS367 antibody on IMR90 cells as well as ATM-deficient GM09607 and GM05849 cells. Cell nuclei were stained with DAPI in blue. (D) Quantification of ATM-proficient or ATM-deficient cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for IMR90, GM09607 and GM05849 cells. Standard deviations from three independent experiments are indicated. (E) Introduction of ATM into ATM-deficient cells restores phosphorylated (pS367)TRF1-containing foci. IF was conducted with anti-pS367 antibody on ATM-deficient cells expressing either the vector alone (GM16666) or complemented with ATM (GM16667). Cell nuclei were stained with DAPI in blue. (F) Quantification of GM16666 or GM16667 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of 1500 cells from three independent experiments were scored. Standard deviations from three independent experiments are indicated.

Mentions: To investigate whether phosphorylated (pS367)TRF1-containing foci might be ATM-dependent, indirect IF with anti-pS367 antibody was performed on HeLaI.2.11 cells treated with either DMSO or KU55933. We observed a severe loss of anti-pS367 staining along with the disappearance of distinct nuclear foci of phosphorylated (pS367)TRF1 in KU55933-treated cells as compared to DMSO-treated cells (Figure 4A). Treatment with KU55933 led to a 6-fold reduction in the percentage of cells exhibiting phosphorylated (pS367)TRF1-containing foci (Figure 4B). The absence of distinct anti-pS367 nuclear foci was also evident in ATM-deficient GM09607 and GM05849 cells when compared to ATM-proficient IMR90 cells (Figure 4C and 4D). In addition, we found that introduction of ATM into ATM-deficient cells restored phosphorylated (pS367)TRF1-containing foci (Figure 4E and F). Taken together, these results suggest that the subnuclear localization of phosphorylated (pS367)TRF1 is dependent upon functional ATM.Figure 4.


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

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

ATM is required for the subnuclear localization of phosphorylated (pS367)TRF1. (A) ATM inhibition leads to loss of phosphorylated (pS367)TRF1-containing foci. Indirect IF was performed with anti-pS367 antibody on HeLaI.2.11 cells treated with either DMSO or KU55933. Cell nuclei were stained with DAPI in blue. (B) Quantification of DMSO-treated or KU55933-treated HeLaI.2.11 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for both DMSO-treated and KU55933-treated cells. Standard deviations from three independent experiments are indicated. (C) ATM-deficient cells lack phosphorylated (pS367)TRF1-containing foci. IF was performed with anti-pS367 antibody on IMR90 cells as well as ATM-deficient GM09607 and GM05849 cells. Cell nuclei were stained with DAPI in blue. (D) Quantification of ATM-proficient or ATM-deficient cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for IMR90, GM09607 and GM05849 cells. Standard deviations from three independent experiments are indicated. (E) Introduction of ATM into ATM-deficient cells restores phosphorylated (pS367)TRF1-containing foci. IF was conducted with anti-pS367 antibody on ATM-deficient cells expressing either the vector alone (GM16666) or complemented with ATM (GM16667). Cell nuclei were stained with DAPI in blue. (F) Quantification of GM16666 or GM16667 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of 1500 cells from three independent experiments were scored. Standard deviations from three independent experiments are indicated.
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gks035-F4: ATM is required for the subnuclear localization of phosphorylated (pS367)TRF1. (A) ATM inhibition leads to loss of phosphorylated (pS367)TRF1-containing foci. Indirect IF was performed with anti-pS367 antibody on HeLaI.2.11 cells treated with either DMSO or KU55933. Cell nuclei were stained with DAPI in blue. (B) Quantification of DMSO-treated or KU55933-treated HeLaI.2.11 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for both DMSO-treated and KU55933-treated cells. Standard deviations from three independent experiments are indicated. (C) ATM-deficient cells lack phosphorylated (pS367)TRF1-containing foci. IF was performed with anti-pS367 antibody on IMR90 cells as well as ATM-deficient GM09607 and GM05849 cells. Cell nuclei were stained with DAPI in blue. (D) Quantification of ATM-proficient or ATM-deficient cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of at least 1500 cells from three independent experiments were scored in blind for IMR90, GM09607 and GM05849 cells. Standard deviations from three independent experiments are indicated. (E) Introduction of ATM into ATM-deficient cells restores phosphorylated (pS367)TRF1-containing foci. IF was conducted with anti-pS367 antibody on ATM-deficient cells expressing either the vector alone (GM16666) or complemented with ATM (GM16667). Cell nuclei were stained with DAPI in blue. (F) Quantification of GM16666 or GM16667 cells showing five or more phosphorylated (pS367)TRF1-containing foci. A total of 1500 cells from three independent experiments were scored. Standard deviations from three independent experiments are indicated.
Mentions: To investigate whether phosphorylated (pS367)TRF1-containing foci might be ATM-dependent, indirect IF with anti-pS367 antibody was performed on HeLaI.2.11 cells treated with either DMSO or KU55933. We observed a severe loss of anti-pS367 staining along with the disappearance of distinct nuclear foci of phosphorylated (pS367)TRF1 in KU55933-treated cells as compared to DMSO-treated cells (Figure 4A). Treatment with KU55933 led to a 6-fold reduction in the percentage of cells exhibiting phosphorylated (pS367)TRF1-containing foci (Figure 4B). The absence of distinct anti-pS367 nuclear foci was also evident in ATM-deficient GM09607 and GM05849 cells when compared to ATM-proficient IMR90 cells (Figure 4C and 4D). In addition, we found that introduction of ATM into ATM-deficient cells restored phosphorylated (pS367)TRF1-containing foci (Figure 4E and F). Taken together, these results suggest that the subnuclear localization of phosphorylated (pS367)TRF1 is dependent upon functional ATM.Figure 4.

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