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.
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📋 Methods
Cloning, Protein Expression and Purification TRFH point mutants of TRF1 and TRF2 were expressed in E. coli . Size exclusion chromatography was performed using a HiLoad 16/600 Superdex 200 pg column. For PALM imaging assays, native and mutant human shelterin components were cloned into the pEGFP-C1 vector containing an N-terminal mEos2. TRF2 ΔBΔM was fused to BFP for multicolor PALM imaging. For telomere rescue assays, a DmrB tag (Clontech) was fused to the N-terminus of TRF2-N53D and TRF2-Y60F. The complete list of plasmids used is shown in Table S1 .
Cell Culture
Human cells were cultured at 37°C. Transfections were performed using Lipofectamine 2000 at 50–70% confluence. 24–48 hours post-transfection, the cells were fixed with 4% paraformaldehyde. For cell-cycle measurements, HeLa cells were synchronized with a double thymidine block. Cell cycle distribution of the cells was quantified by flow cytometry. siRNA and shRNA mediated depletion of telomere associated proteins were determined by real-time PCR and fluorescence imaging.
Imaging and Data Analysis
Images for TIF analysis were collected using confocal microscopy. FRAP measurements were performed on a Zeiss LSM 710 microscope. Telomeres were photobleached using 0.8 mW focused 488 nm beam and fluorescence recovery at telomeres was measured every 3 s under 0.4 μW excitation. FISH samples were prepared as described ( Lackner et al., 2011 ). STORM imaging was done in PBS buffer supplemented with methylethylamine (MEA) and protocatechuic acid/protocatechuate-3,4-dioxygenase (PCA/PCD) system. Superresolution images were recorded using a cycle containing one frame for photoconversion at 405 nm excitation followed by 5 frames of imaging under 532 nm excitation (150 ms per frame). Camera acquisition and laser excitation were synchronized through custom software written in LabView. Images were analyzed using software provided by B. Huang (UCSF). The resolution of 2D PALM imaging is defined as SEM of a typical mEos2 spot. Detailed procedures for cloning, purification, light microscopy and data analysis are described in Extended Experimental Procedures.
Show full methods section
Cloning, Protein Expression and Purification TRFH point mutants of TRF1 and TRF2 were expressed in E. coli . Size exclusion chromatography was performed using a HiLoad 16/600 Superdex 200 pg column. For PALM imaging assays, native and mutant human shelterin components were cloned into the pEGFP-C1 vector containing an N-terminal mEos2. TRF2 ΔBΔM was fused to BFP for multicolor PALM imaging. For telomere rescue assays, a DmrB tag (Clontech) was fused to the N-terminus of TRF2-N53D and TRF2-Y60F. The complete list of plasmids used is shown in Table S1 .
Cell Culture
Human cells were cultured at 37°C. Transfections were performed using Lipofectamine 2000 at 50–70% confluence. 24–48 hours post-transfection, the cells were fixed with 4% paraformaldehyde. For cell-cycle measurements, HeLa cells were synchronized with a double thymidine block. Cell cycle distribution of the cells was quantified by flow cytometry. siRNA and shRNA mediated depletion of telomere associated proteins were determined by real-time PCR and fluorescence imaging.
Imaging and Data Analysis
Images for TIF analysis were collected using confocal microscopy. FRAP measurements were performed on a Zeiss LSM 710 microscope. Telomeres were photobleached using 0.8 mW focused 488 nm beam and fluorescence recovery at telomeres was measured every 3 s under 0.4 μW excitation. FISH samples were prepared as described ( Lackner et al., 2011 ). STORM imaging was done in PBS buffer supplemented with methylethylamine (MEA) and protocatechuic acid/protocatechuate-3,4-dioxygenase (PCA/PCD) system. Superresolution images were recorded using a cycle containing one frame for photoconversion at 405 nm excitation followed by 5 frames of imaging under 532 nm excitation (150 ms per frame). Camera acquisition and laser excitation were synchronized through custom software written in LabView. Images were analyzed using software provided by B. Huang (UCSF). The resolution of 2D PALM imaging is defined as SEM of a typical mEos2 spot. Detailed procedures for cloning, purification, light microscopy and data analysis are described in Extended Experimental Procedures.
Supplementary Material 1 2 3 4 5 6 7 8 9
📊 Figures
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 homod...
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....
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...
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 lo...
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 telo...
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...
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 re...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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