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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 nuclear foci that are not associated with telomere chromatin. (A) Indirect IF using anti-pS367 antibody was performed on human primary IMR90 cells as well as transformed HeLaI.2.11 and GM847 cells. Cell nuclei were stained with DAPI shown in blue. (B) Indirect IF using anti-pS367 antibody was performed on TRF1-depleted HeLaII cells expressing the vector alone, wild-type TRF1, TRF1-S367A or TRF1-S367D. Wild-type TRF1, TRF1-S367A or TRF1-S367D were engineered to be resistant to shTRF1. Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are visible in early prophase but become diffuse in the rest of mitosis. Indirect IF using anti-pS367 antibody was performed on GM847 cells. Cell nuclei were stained with DAPI shown in blue. (D) Phosphorylated (pS367)TRF1 is not associated with telomeric DNA. IF–FISH analysis was performed on fixed GM847 cells with anti-pS367 antibody (green) in conjunction with TRITC-conjugated telomeric DNA-containing PNA probe (red). Cell nuclei were stained with DAPI shown in blue. (E) Phosphorylated (pS367)TRF1 is predominantly free of chromatin. Differential salt (KCl) extraction of chromatin was performed on HeLaI.2.11 cells. Immunoblotting was carried out with anti-pS367, anti-TRF1 or anti-H3K9m3 antibody. The anti-H3K9m3 blot was used as control to assess the extraction of chromatin.
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gks035-F2: Phosphorylated (pS367)TRF1 forms distinct nuclear foci that are not associated with telomere chromatin. (A) Indirect IF using anti-pS367 antibody was performed on human primary IMR90 cells as well as transformed HeLaI.2.11 and GM847 cells. Cell nuclei were stained with DAPI shown in blue. (B) Indirect IF using anti-pS367 antibody was performed on TRF1-depleted HeLaII cells expressing the vector alone, wild-type TRF1, TRF1-S367A or TRF1-S367D. Wild-type TRF1, TRF1-S367A or TRF1-S367D were engineered to be resistant to shTRF1. Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are visible in early prophase but become diffuse in the rest of mitosis. Indirect IF using anti-pS367 antibody was performed on GM847 cells. Cell nuclei were stained with DAPI shown in blue. (D) Phosphorylated (pS367)TRF1 is not associated with telomeric DNA. IF–FISH analysis was performed on fixed GM847 cells with anti-pS367 antibody (green) in conjunction with TRITC-conjugated telomeric DNA-containing PNA probe (red). Cell nuclei were stained with DAPI shown in blue. (E) Phosphorylated (pS367)TRF1 is predominantly free of chromatin. Differential salt (KCl) extraction of chromatin was performed on HeLaI.2.11 cells. Immunoblotting was carried out with anti-pS367, anti-TRF1 or anti-H3K9m3 antibody. The anti-H3K9m3 blot was used as control to assess the extraction of chromatin.

Mentions: We have shown that TRF1 is phosphorylated at S367 in vivo and we decided to examine the nuclear localization of phosphorylated (pS367)TRF1. Using the phospho-specific anti-pS367 antibody, we performed indirect IF on both primary and transformed cell lines. We found that in interphase cells, anti-pS367 staining formed distinct nuclear foci in both primary and transformed cell lines (Figure 2A) and that these foci were very heterogenous in size, a feature distinct from telomere staining. We found that depletion of TRF1 led to a severe reduction in anti-pS367 staining and the formation of anti-pS367-containing foci (Figure 2B). On the other hand, anti-pS367-containing foci were fully restored in TRF1-depleted cells expressing wild-type TRF1 but not in TRF1-depleted cells expressing TRF1 carrying either a non-phosphorylatable mutation (S367A) or a phosphomimic mutation (S367D) (Figure 2B), suggesting that these foci represent phosphorylated (pS367)TRF1. The lack of rescue of anti-pS367-containing foci by TRF1 mutants demonstrates that phospho-specific anti-pS367 antibody does not recognize TRF1 carrying a single amino acid substitution of S367. Phosphorylated (pS367)TRF1-containing foci were also visible in early prophase cells (Figure 2C); however, anti-pS367 staining became very diffuse in the rest of mitosis from metaphase to telophase (Figure 2C). These results suggest that the formation of phosphorylated (pS367)TRF1-containing foci may be cell cycle regulated.Figure 2.


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 nuclear foci that are not associated with telomere chromatin. (A) Indirect IF using anti-pS367 antibody was performed on human primary IMR90 cells as well as transformed HeLaI.2.11 and GM847 cells. Cell nuclei were stained with DAPI shown in blue. (B) Indirect IF using anti-pS367 antibody was performed on TRF1-depleted HeLaII cells expressing the vector alone, wild-type TRF1, TRF1-S367A or TRF1-S367D. Wild-type TRF1, TRF1-S367A or TRF1-S367D were engineered to be resistant to shTRF1. Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are visible in early prophase but become diffuse in the rest of mitosis. Indirect IF using anti-pS367 antibody was performed on GM847 cells. Cell nuclei were stained with DAPI shown in blue. (D) Phosphorylated (pS367)TRF1 is not associated with telomeric DNA. IF–FISH analysis was performed on fixed GM847 cells with anti-pS367 antibody (green) in conjunction with TRITC-conjugated telomeric DNA-containing PNA probe (red). Cell nuclei were stained with DAPI shown in blue. (E) Phosphorylated (pS367)TRF1 is predominantly free of chromatin. Differential salt (KCl) extraction of chromatin was performed on HeLaI.2.11 cells. Immunoblotting was carried out with anti-pS367, anti-TRF1 or anti-H3K9m3 antibody. The anti-H3K9m3 blot was used as control to assess the extraction of chromatin.
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gks035-F2: Phosphorylated (pS367)TRF1 forms distinct nuclear foci that are not associated with telomere chromatin. (A) Indirect IF using anti-pS367 antibody was performed on human primary IMR90 cells as well as transformed HeLaI.2.11 and GM847 cells. Cell nuclei were stained with DAPI shown in blue. (B) Indirect IF using anti-pS367 antibody was performed on TRF1-depleted HeLaII cells expressing the vector alone, wild-type TRF1, TRF1-S367A or TRF1-S367D. Wild-type TRF1, TRF1-S367A or TRF1-S367D were engineered to be resistant to shTRF1. Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are visible in early prophase but become diffuse in the rest of mitosis. Indirect IF using anti-pS367 antibody was performed on GM847 cells. Cell nuclei were stained with DAPI shown in blue. (D) Phosphorylated (pS367)TRF1 is not associated with telomeric DNA. IF–FISH analysis was performed on fixed GM847 cells with anti-pS367 antibody (green) in conjunction with TRITC-conjugated telomeric DNA-containing PNA probe (red). Cell nuclei were stained with DAPI shown in blue. (E) Phosphorylated (pS367)TRF1 is predominantly free of chromatin. Differential salt (KCl) extraction of chromatin was performed on HeLaI.2.11 cells. Immunoblotting was carried out with anti-pS367, anti-TRF1 or anti-H3K9m3 antibody. The anti-H3K9m3 blot was used as control to assess the extraction of chromatin.
Mentions: We have shown that TRF1 is phosphorylated at S367 in vivo and we decided to examine the nuclear localization of phosphorylated (pS367)TRF1. Using the phospho-specific anti-pS367 antibody, we performed indirect IF on both primary and transformed cell lines. We found that in interphase cells, anti-pS367 staining formed distinct nuclear foci in both primary and transformed cell lines (Figure 2A) and that these foci were very heterogenous in size, a feature distinct from telomere staining. We found that depletion of TRF1 led to a severe reduction in anti-pS367 staining and the formation of anti-pS367-containing foci (Figure 2B). On the other hand, anti-pS367-containing foci were fully restored in TRF1-depleted cells expressing wild-type TRF1 but not in TRF1-depleted cells expressing TRF1 carrying either a non-phosphorylatable mutation (S367A) or a phosphomimic mutation (S367D) (Figure 2B), suggesting that these foci represent phosphorylated (pS367)TRF1. The lack of rescue of anti-pS367-containing foci by TRF1 mutants demonstrates that phospho-specific anti-pS367 antibody does not recognize TRF1 carrying a single amino acid substitution of S367. Phosphorylated (pS367)TRF1-containing foci were also visible in early prophase cells (Figure 2C); however, anti-pS367 staining became very diffuse in the rest of mitosis from metaphase to telophase (Figure 2C). These results suggest that the formation of phosphorylated (pS367)TRF1-containing foci may be cell cycle regulated.Figure 2.

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