TRF1 phosphorylation on T271 modulates telomerase-dependent telomere length maintenance as well as the formation of ALT-associated PML bodies

Abstract

TRF1, a component of the shelterin complex, plays a key role in both telomerase-dependent telomere maintenance and alternative lengthening of telomeres, the latter also known as ALT. Characteristics of ALT cells include C-circles and ALT-associated PML bodies, referred to as APBs. The function of TRF1 is tightly regulated by post-translational modification including phosphorylation, however TRF1 phosphorylation sites have yet to be fully characterized. Here we report a novel TRF1 phosphorylation site threonine 271. We show that a nonphosphorylatable mutation of T271A impairs TRF1 binding to telomeric DNA in vivo and renders TRF1 defective in inhibiting telomerase-dependent telomere elongation. On the other hand, TRF1 carrying a phosphomimic mutation of T271D is competent in not only binding to telomeric DNA but also inhibiting telomerase-mediated telomere lengthening. These results suggest that TRF1 phosphorylation on T271 negatively regulates telomerase-mediated telomere maintenance. We find that in telomerase-negative ALT cells, TRF1 carrying either a T271A or T271D mutation is able to promote C-circle production but fails to support APB formation. These results suggest that TRF1 phosphorylation on T271 is necessary for APB formation but dispensable for C-circle production. These results further imply that APB formation can be mechanistically separated from C-circle production.

Figure 1. T271 of TRF1 is phosphorylated in vivo.

(a) LC/MS/MS analysis was performed on Flag-tagged TRF1 immunoprecipitated from HT1080 cells. The spectrum of the peptide identified to contain phosphorylated threonine at position 271 is shown with the relative abundance plotted against the monoisotopic mass (m/z). The m/z peaks from the y-type ions are indicated. Mass spectrometry analysis of Flag-tagged TRF1 was done through service provided by WEMB Biochem. Inc. Toronto, Canada. (b) Affinity-purified anti-pT271 antibody specifically recognizes TRF1 peptide containing phosphorylated T271 (pT271-peptide). An increasing amount of peptide either carrying unmodified T271 (pT271-peptide) or phosphorylated T271 (pT271-peptide) was spotted on a nitrocellulose membrane, followed by immunoblotting with affinity-purified anti-pT271 antibody or crude serum. The amount of peptide spotted from left to right is 0.5 μg, 1.5 μg and 3 μg. (c) Peptide competition assays. Affinity-purified anti-pT371 antibody was incubated with 5 μg of either unmodified (T271-peptide) or phosphorylated (pT271-peptide) prior to immunoblotting. The blot with crude serum was used to indicate the presence of pT271-peptide on the membrane. The amount of pT271-peptide spotted from left to right is the same as described in (b). (d) Western analysis of HeLaII whole cell lysate with anti-pT271 antibody. (e) Western analysis of HeLII cells depleted for endogenous TRF1. Immunoblotting was performed with anti-pT271 and anti-TRF1 antibody. The γ-tubulin blot was used as a loading control in this experiment and all following westerns shown in this article. (f) Western analysis. HeLa nuclear extract (75 μg) was treated in the presence or absence of Lambda protein phosphatase (800 units, NEB). Immunoblotting was performed with anti-pT271 and anti-γ-tubulin antibodies.

Figure 2. TRF1 carrying a nonphosphorylatable mutation of T271A is defective in inhibiting telomerase-dependent telomere lengthening.

(a) Western analysis of HeLaII cells expressing either the vector pRS alone or shRNA against TRF1 (shTRF1). Immunoblotting was performed with anti-TRF1 and anti-γ-tubulin antibodies. (b) Western analysis of pRS- and shTRF1-expressing HeLaII cells expressing the vector pWZL-NMyc vector alone or various TRF1 alleles as indicated above the lanes. Immunoblotting was performed with anti-Myc and anti-γ-tubulin antibodies. (c) Genomic blots of telomeric restriction fragments from TRF1-depleted HeLaII cells (shTRF1) expressing either the vector pWZL alone or various TRF1 alleles as indicated above the lanes. HeLaII cells expressing both pRS and pWZL were used as a control. PD are indicated above the lanes whereas DNA molecular weight markers are shown on both left and right of the blots. About 3 μg of RsaI/HinfI-digested genomic DNA from each sample was used for gel electrophoresis. (d) Average telomere length of indicated cell lines was ploted against PD. Standard deviations from three independent experiments are indicated. (e) Quantification of average rates of telomere elongation of indicated cell lines. Standard deviations from three independent experiments are indicated. (f) Growth curve of HeLaII cells expressing various constructs as indicated from one of the three independent experiments. The number of PDs was plotted against days in culture.

Figure 3. TRF1 carrying a nonphosphorylatable mutation of T271A is defective in binding to telomeric DNA in vivo.

(a) Western analysis of TRF1-depleted HeLaII cells expressing the pWZL vector alone or varous TRF1 alleles as indicated. Immunoblotting was performed with anti-Myc and anti-γ-tubulin antibodies. (b) Dot blots of anti-Myc ChIPs from TRF1-depleted HeLaII cells expressing various constructs as indicated on the left. (c) Quantification of anti-Myc ChIPs from (b). Standard deviations from three independent experiments are indicated. (d) Cycloheximide chase experiments. HT1080 cells expressing Myc-TRF1, Myc-TRF1-T271A or Myc-TRF1-T271D were treated with 100 μg/ml cycloheximide for the indicated times, followed by immunoblotting of the lysates with anti-Myc and anti-γ-tubulin antibodies. (e) Quantification of Myc-tagged wild type, Myc-tagged TRF1-T271 and Myc-tagged TRF1-T271D from (d). The signals from the western blots were quantified with densitometry. The level of Myc-tagged wild type TRF1 and various mutant TRF1 proteins is represented in arbitrary units after their signals were first normalized relative to those of γ-tubulin and then normalized relative to Myc-tagged wild type TRF1. Standard deviations from three independent experiments are indicated.

Figure 4. TRF1 phosphorylation on T271 is dispensable for C-circle production in ALT cells.

(a) Western analysis of GM847 cells stably expressing the vector pRS alone or shRNA against TRF1 (shTRF1). Immunoblotting was performed with anti-TRF1 and anti-γ-tubulin antibodies. (b) Western analysis of pRS- and shTRF1-expressing GM847 cells expressing the vector alone (pWZL) or various TRF1 alleles as indicated above the lanes. Immunoblotting was performed with anti-Myc and anti-γ-tubulin antibodies. (c) Analysis of C-circle formation. C.C. stands for C-circles. (d,e) Quantification of the level of C-circles from (c). The C-circle signals were quantified with ImageQuant. The level of C-circles is represented in arbitrary units. In (d) the signal in the shTRF1 lane was normalized relative to that in the pRS lane whereas in (e) the signals in lanes representing Myc-tagged wild type TRF1 and various mutant TRF1 proteins were normalized relative to that in the vector alone lane. Standard deviations from three independent experiments are indicated.

Figure 5. TRF1 phosphorylation on T271 is needed to support APB formation in ALT cells.

(a) Dual Indirect immunofluorescence with anti-TRF2 and anti-PML antibodies on GM847 cells expressing various constructs as indicated. Cell nuclei are stained with DAPI in blue in this and subsequent figures. (b) Dual Indirect immunofluorescence with anti-hRap1 and anti-PML antibodies on GM847 cells expressing various constructs as indicated. (c) Quantification of the percentage of cells with TRF2 at APBs from (a). A total of 1000 cells from each independent experiments were scored for each cell line in blind. Standard deviations from three independent experiments are indicated. (d) Quantification of the percentage of cells with hRap1 at APBs from (b). A total of 500 cells from each independent experiments were scored for each cell line in blind. Standard deviations from three independent experiments are indicated. (e) Quantification of the average number of APB foci per APB+ cell. A total of at least 250 cells from captured images from three independent experiments were scored in blind. Standard deviations from three independent experiments are indicated. (f) Quantification of the percentage of GM847 cells expressing various constructs as indicated staining positive for cyclin A. Scoring was done as described in (c). Standard deviations from three independent experiments are indicated. (g) Western analysis of GM847 cells expressing various constructs as indicated. Immunoblotting was performed with anti-TRF2 and anti-γ-tubulin antibodies. (h) Western analysis of GM847 cells expressing various constructs as indicated. Immunoblotting was performed with anti-hRap1 and anti-γ-tubulin antibodies.