Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin

Abstract

Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, but not by DNA methylation, histone deacetylation, or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres accumulate DDR signals and become more accessible to telomere-associated proteins. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery.

Figure 1. Human telomeres form tight globular structures

(A) The human shelterin complex consists of six proteins: TRF1, TRF2, POT1, TPP1, TIN2 and RAP1. TRF1 and TRF2 subunits specifically bind to dsTEL tracts through their C-terminal MYB domains and homodimerize via their N-terminal TRFH domains. (B) Conventional (left) and PALM (right) images of a HeLa cell expressing mEos2-TRF2. TRF2 proteins are localized to nucleus (yellow boundary) and show punctate spots that represent telomeres. Telomeric chromatin appears smaller than a diffraction-limited spot (insert). (C) 3D representation of a telomeric chromatin constructed from individual spots (magenta dots). (D) The average volume and the number of localized spots detected per telomere in regular HeLa and HeLa 1.2.11 cells. Error bars represent SEM. N_telomeres is >150 for each case. (E) The volume of telomeric structures in mEos2-TRF2 expressing cells positively correlates with the number of detected TRF2 molecules per telomeres (N_telomeres >150). Color bar shows the number of telomeres (mean ± SEM). (F) The average volume of telomeres in G1/S phase is similar to that of unsynchronized cells. (G) The average number of TRF2 molecules detected per telomere remains similar in different stages of the cell cycle. (H) The volume of telomeres in mEos2-TRF2 expressing cells remains unaffected by inhibition of histone deacetylation, DNA methylation and Suv knockdown. See also Figures S1 and S2, Movies S1 and S2.

Figure 2. Removal of shelterin subunits leads to decompaction of telomeres

(A) A schematic represents the looping of dsTEL tracts by TIN2, which bridges dsTEL-bound TRF1 and TRF2. (B) Representative FISH-STORM images of telomeres in WT and TRF1, TRF2 and TIN2 depleted cells. (C) The average volume of telomeres in WT and shelterin-depleted cells measured by FISH-STORM (N >150 per condition). Error bars represent SEM. (D) In PALM imaging, the average number of mEos2 spots localized to each telomere correlated with the average volume of the telomere under native and shelterin knockdown (KD) conditions (dotted line, Pearson’s r = 0.89). Error bars represent SEM. See also Figures S3 and S4.

Figure 3. TRF1 and TRF2 dimerization is essential for the compaction of telomeres

(A) A schematic depicting the crosslinking of distal dsTEL tracts through TRF1 or TRF2 homodimerization. (B) Overlay of the atomic structures of the TRFH domains of TRF1 (magenta) and TRF2 (cyan-green) dimers. Single–point mutations were introduced to helix 1 to disrupt dimerization. (C) Gel filtration profiles show that WT TRF1 and TRF2 elute as homodimers, whereas TRFH point mutations elute as monomers. (D) Representative telomeric structures for TRF1 and TRF2 dimerization mutants. (E) The distribution of telomere volume in cells expressing the TRFH mutants (N_telomeres >150). The expression of the TRFH mutants increases the average volume of telomeres (mean ± SEM). Percentages represent the data in pink shaded regions. See also Figure S5.

Figure 4. Decompaction of telomeres correlates with the number of TIF spots per cell

(A) Telomeres were detected using Alexa-488-labeled antibodies against TRF1 or TRF2 (green) and DNA repair sites were probed by immunolabeling of 53BP1 with Alexa-647 (red). The overlay reveals the location of TIF spots (yellow). (B) The knockdown of shelterin components and the expression of TRFH dimerization mutants induce localization of DDR signals at telomeres. TIF spots (yellow) were determined by colocalization of the fluorescence signals of TRF1 or TRF2 (green) and 53BP1 (red). (C) The number of TIF spots per cell as a function of the volume of telomeres under various shelterin perturbation conditions. The number of TIF spots per cell correlates strongly (dotted line, Pearson’s r = 0.82) with telomere volume. See also Figures S4 and S5.

Figure 5. Shelterin-mediated telomere compaction reduces the accessibility of telomere associated proteins

(A) A model for higher-order remodeling of telomeres by shelterin. Shelterin remodels dsTEL tracts into a globular nucleoprotein mesh, which reduces the accessibility of the DDR signals and other telomere-associated proteins to telomeric sites. The removal of shelterin leads to more than a ten-fold decompaction of telomeric chromatin and exposes the protected sites to the DDR signals. (B) Images of selected time points from FRAP experiments in live HeLa cells expressing GFP POT1 in the absence and presence of TRF1 siRNA treatment. Circular regions covering a single telomere (red circles) were photobleached with a focused laser beam. Images were acquired before bleaching and at 3-s intervals after bleaching, starting at 0 s. (C) The recovery of GFP-POT1 fluorescence at individual telomeres is faster in TRF1 depleted cells than untreated cells. Values represent mean ± SEM from 15 cells. (D) Quantitative analysis of the recovery of TRF2 fluorescence at individual telomeres showed that TRFH dimerization mutants recover faster than WT TRF2. Values represent mean ± SEM from 15 cells. See also Figure S6, Movies S3 and S4.

Figure 6. Telomere decompaction upon TRF2 removal is uncoupled from the ATM-pathway

(A) If the accumulation of DDR signals precedes telomere decompaction upon shelterin depletion, inactivation of the DDR pathway would prevent telomere decompaction in shelterin-depleted cells. (B) The overlay images of telomeres detected by mEos2-TRF2 (green) and the DNA repair sites probed by immunolabeling of 53BP1 with Alexa-647 (red). TIF spots (yellow) were determined by colocalization of the fluorescence signals of TRF1 or TRF2 (green) and 53BP1 (red). TRF2^MYB expression leads to a large increase in TIF spots in ATM active cells, but TIF spots are significantly reduced in ATM knockdown cells. (C) Comparison of the number of TIF spots per cell in WT TRF2 (yellow shade) and TRF2^MYB expressing (blue shade) cells. Error bars show SEM. N_telomeres is >50 for each case. (D) Comparison of the telomere volume in WT TRF2 (yellow shade) and TRF2^MYB expressing (blue shade) cells. TRF2^MYB expression leads to telomere decompaction even in cells depleted of DDR proteins. Error bars show SEM. N_telomeres is >150 for each case. See also Figure S6

Figure 7. Recompaction of telomeric chromatin reduces TIFs

(A) If DDR accumulation and DNA decompaction occur simultaneously upon the removal of shelterin from telomeres, recompaction of telomeric chromatin using an orthogonal method (blue arrow) would not reduce DDR accumulation (red arrow). (B) A DmrB tag is fused to the N-terminus of TRF2-N53D. Monomer to dimer transition of TRF2 is controlled by the BB dimerizer and BB washout ligand. (C) (Top) Representative telomeric structures and (Bottom) TIF spots (yellow; green: TRF2 and red: 53BP1) as monomer to dimer transition of TRF2 is reversibly controlled in HeLa cells. (D) Telomeric chromatin in DmrB-tagged TRF2-N53D expressing cells is reversibly compacted by dimerization and decompacted by monomerization of TRF2-N53D (blue shaded region). The number of TIF spots per cells correlates strongly (dotted line, Pearson’s r = 0.91) with changes in telomere volume in these cells (mean ± SEM). Values for cells expressing WT TRF2 and TRF2-N53D without the DmrB tag are shown for comparison (green shaded region). (E) Overexpression of TRF1 and TRF2 in TIN2-depleted cells leads to telomere compaction and reduction in the number of TIFs per cell. See also Figure S7