A role for heterochromatin protein 1γ at human telomeres

Abstract

Human telomere function is mediated by shelterin, a six-subunit complex that is required for telomere replication, protection, and cohesion. TIN2, the central component of shelterin, has binding sites to three subunits: TRF1, TRF2, and TPP1. Here we identify a fourth partner, heterochromatin protein 1γ (HP1γ), that binds to a conserved canonical HP1-binding motif, PXVXL, in the C-terminal domain of TIN2. We show that HP1γ localizes to telomeres in S phase, where it is required to establish/maintain cohesion. We further demonstrate that the HP1-binding site in TIN2 is required for sister telomere cohesion and can impact telomere length maintenance by telomerase. Remarkably, the PTVML HP1-binding site is embedded in the recently identified cluster of mutations in TIN2 that gives rise to dyskeratosis congenita (DC), an inherited bone marrow failure syndrome caused by defects in telomere maintenance. We show that DC-associated mutations in TIN2 abrogate binding to HP1γ and that DC patient cells are defective in sister telomere cohesion. Our data indicate a novel requirement for HP1γ in the establishment/maintenance of cohesion at human telomeres and, furthermore, may provide insight into the mechanism of pathogenesis in TIN2-mediated DC.

Figure 1.

HP1 binds to the PTVML site in the DC-associated TIN2 mutation cluster. (A) Schematic representation of TIN2 and two-hybrid interaction with HP1. TPP1-, TRF2-, and TRF1-binding domains (BDs) and the double point mutation in TIN2 (RTDML) are indicated. (B) Schematic representation of HP1 and two-hybrid interaction with TIN2. The chromodomain (CD), hinge (H), and chromoshadow domain (CSD) in HP1 are indicated. (A,B) Two-hybrid interactions were scored according to the number of minutes required for the color change: 20 min (++), 45 min (+), and 150–180 min (+/−). (C) Alignment of the TIN2 domain containing the PTVML HP1-binding site. Identical amino acids are indicated in red. (D) HP1 binds to TIN2 in human cells. HT1080 stable cell lines expressing HP1γ or vector (V) were transfected with vector or FlagTIN2 (WT or RD). Cell lysates were immunoprecipitated with anti-myc beads and analyzed by immunoblotting with anti-myc or anti-TIN2 701 antibody. (E) The DC-associated mutation cluster in TIN2. DC mutations are indicated by dots and asterisks; asterisks indicate mutations shown to give rise to shortened telomeres.

Figure 2.

An intact HP1-binding site in TIN2 is required for sister telomere cohesion. (A–F) FISH analysis of HTC75 stable cell lines expressing Vector (A,D), TIN2.WT (B,E), or TIN2.RD (C,F) following transfection with GFP siRNA (A–C) or TIN2 siRNA (D–F) with a 16ptelo probe. DNA was stained with DAPI (blue). Bar, 5 μm. Histograms based on measurements from two independent experiments (Table 1) showing the distance between FISH signals are on the right, with the average (Avg) distance ± SEM indicated. (G) Graphical representation of the average distance between sister telomeres ± SEM. (H) Immunoblot analysis of extracts from stable HTC75 lines expressing Vector, TIN2.WT, or TIN2.RD transfected with GFP siRNA or TIN2 siRNA and probed with anti-TIN2 701 or anti-α-tubulin antibody. Asterisk (*) indicates a nonspecific band.

Figure 3.

HP1 is required to establish/maintain sister telomere cohesion in S phase. (A) Immunoblot analysis of extracts from HeLaI.2.11 cells transfected for 48 h with GFP, HP1α, HP1β, or HP1γ siRNA. (B) Schematic representation of the protocol to analyze siRNA-treated synchronized cells. (C–E) FACS (C) and FISH (D,E) analysis 4 h after release from the second thymidine block. Cells were hybridized with a telomere 16ptelo (green) (D) or centromere 6cen (red) (E) probe. The cen locus is trisomic. DNA was stained with DAPI (blue). Bar, 5 μm. (F) Graphical representation of the frequency of doublets from two independent experiments: 16pter and 6cen probes (Experiment 1) and 4pter and 10cen probes (Experiment 2) (see Table 2).

Figure 4.

HP1γ colocalizes with TIN2 at telomeres in S phase. (A,C–E) Immunofluorescence analysis of HeLaI.2.11 cells 4 h after release from a double thymidine block. Cells were extracted with Triton prior to fixation and dually stained with antibodies against HP1γ (green) (A,C–E), TIN2 (red) (A), TRF1 (red) (C), and RAP1 (red) (D), or processed for FISH with a PNA-TTAGGG repeat probe (red) (E). Merge is yellow. Bar, 5 μm. (B) Graphical representation of the frequency of cells containing colocalizing HP1γ–TIN2 foci across the cell cycle. Cells were synchronized by a double thymidine block and released for 0 h (G1/S), 2 h (early S), 4 h (mid S), 6 h (late S), 8 h (G2), 10 h (M), and 12 h (G1). Mean ± SD of three independent experiments (n, ∼100 cells each).

Figure 5.

TIN2 mutations in the HP1-binding site interfere with telomere length maintenance. (A) Immunoblot analysis of extracts from stable HTC75 cell lines expressing Vector (V), TIN2.WT, TIN2.RD, TIN2-C, or TIN2-C.RD at population doubling 4 (PD 4) or 120 (PD 120) probed with anti-TIN2 701 or anti-α-tubulin antibody. (B) Analysis of telomere restriction fragments isolated from the stable HTC75 cell lines at the indicated population doubling, fractionated on agarose gel, denatured, and probed with a ³²P-labeled CCCTAA probe. (C) Graphical representation of telomere length changes in B. (D–F) FISH analysis of HTC75 stable cell lines expressing Vector (D), TIN2-C (E), or TIN2-C.RD (F) with a 16ptelo probe. DNA was stained with DAPI (blue). Bar, 5 μm. Histograms based on measurements from two independent experiments (Table 1) showing the distance between FISH signals are on the right, with the average (Avg) distance ± SEM indicated. (G) Graphical representation of the average distance between sister telomeres ± SEM.

Figure 6.

DC-associated TIN2 mutations interfere with HP1 binding. (A) Two-hybrid analysis of the interaction between DC-associated TIN2 mutations and HP1γ and TRF1. Two-hybrid interactions were scored according to the number of minutes required for the color change: 20 min (++), 45 min (+), and 150–180 min (+/−). (B) Coimmunoprecipitation analysis of the interaction between DC-associated TIN2 mutations and HP1γ. HT1080 stable cell lines expressing HP1γ or vector (V) were transfected with the indicated FlagTIN2 plasmids. Cell lysates were immunoprecipitated with anti-myc beads and analyzed by immunoblotting with anti-myc or anti-TIN2 701 antibody.

Figure 7.

DC patient cells harboring TIN2 mutations suffer loss in sister telomere cohesion. (A) DC mutations in patient cell lines are indicated. (B–E) Loss in sister telomere cohesion in DC patient fibroblasts harboring the TIN2p.Q269X mutation. FISH analysis with a 16ptelo (B,C) or 10cen (D,E) probe. DNA was stained with DAPI (blue). Bar, 5 μm. Histograms showing the distance between FISH signals (Table 3) are on the right, with the average (Avg) distance ± SEM indicated. (F) Graphical representation of the average distance ± SEM. (G–K) Loss in sister telomere cohesion in DC patient LCLs harboring the following mutations: TIN2p.K280Rfs36X, TIN2p.R282H, and DKC1p.A2V. (G–J) FISH analysis with a 16ptelo probe. DNA was stained with DAPI (blue). Bar, 5 μm. Histograms showing the distance between FISH signals (Table 3) are shown on the right, with the average (Avg) distance ± SEM indicated. (K) Graphical representation of the average distance ± SEM.

Figure 8.

Schematic diagram showing how HP1 might influence cohesion at telomeres. (A) HP1 could act as a bridge in telomeric heterochromatin by binding to the heterochromatin mark H3K9Me3 via its CD and to TIN2-SA1 cohesin via its CSD. (B) HP1 associates with telomeres in S phase to aid in establishment of cohesion. Once cohesion is established, chromosome ends from both sisters could undergo coordinated processing, including C-strand resection, telomerase-dependent elongation, and C-strand synthesis.