Protein requirements for sister telomere association in human cells

Abstract

Previous studies in human cells indicate that sister telomeres have distinct requirements for their separation at mitosis. In cells depleted for tankyrase 1, a telomeric poly(ADP-ribose) polymerase, sister chromatid arms and centromeres separate normally, but telomeres remain associated and cells arrest in mitosis. Here, we use biochemical and genetic approaches to identify proteins that might mediate the persistent association at sister telomeres. We use immunoprecipitation analysis to show that the telomeric proteins, TRF1 (an acceptor of PARsylation by tankyrase 1) and TIN2 (a TRF1 binding partner) each bind to the SA1 ortholog of the cohesin Scc3 subunit. Sucrose gradient sedimentation shows that TRF1 cosediments with the SA1-cohesin complex. Depletion of the SA1 cohesin subunit or the telomeric proteins (TRF1 and TIN2) restores the normal resolution of sister telomeres in mitosis in tankyrase 1-depleted cells. Moreover, depletion of TRF1 and TIN2 or SA1 abrogates the requirement for tankyrase 1 in mitotic progression. Our studies indicate that sister telomere association in human cells is mediated by a novel association between a cohesin subunit and components of telomeric chromatin.

Figure 1

Sister telomeres remain associated in tankyrase 1 siRNA cells. (A–F) Chromosome-specific FISH analysis of HeLaI.2.11 cells collected by mitotic shake-off at 48 h after treatment with control (GFP) or tankyrase 1 siRNA. Cells were fixed directly in methanol-acetic acid without hypotonic swelling and hybridized to a telomere probe 16pter (green) (A, B) or a centromere probe 6cen (red) (D, E). DNA was stained with DAPI (blue). (C, F) Histograms showing the percentage of mitotic cells with unseparated telomeres (C) or separated centromeres (F); at least 100 mitotic cells were scored for each sample. (G–I) Chromosome spread analysis of HelaI.2.11 cells collected after 48 h of treatment with control (GFP) or tankyrase 1 siRNA, swollen in hypotonic buffer and fixed in paraformaldehyde. Cells were treated with colcemide 90 min before harvesting. Chromosome arms were visualized by staining with antibodies to the condensin subunit Smc2. (I) Histogram showing % mitotic cells with single sisters; at least 200 spreads were scored for each sample. (J–L) Histograms showing analysis of nocodazole-arrested cells. HeLaI.2.11 cells were treated with control (GFP) or tankyrase 1 siRNA for 16 h and then incubated without (−) or with (+) nocodazole for an additional 12 h, harvested and processed for telomere FISH (J), centromere FISH (K), and chromosome spreads (L) as described above. Approximately 100 mitotic cells or more were scored for each sample.

Figure 2

TRF1 binds to the SA1 cohesin complex. (A) Endogenous TRF1 is co-immunoprecipitated by SA1 not SA2. HeLaI.2.11 cells were lysed and immunoprecipitated (IP) with anti-Flag as a control (C), anti-SA1 (BL143G), or anti-SA2. Proteins were fractionated on SDS–PAGE and analyzed by immunoblotting with antibodies against SA1, SA2, Scc1, Smc3, TRF1, TRF2, or TIN2. (B, C) Exogenous TRF1 not TRF2 is co-immunoprecipitated by SA1 not SA2. 293T cells were transfected with FlagSA1 or FlagSA2 and (B) MycTRF1 or (C) MycTRF2. Cell lysates were immunoprecipitated with anti-Flag beads and analyzed by immunoblotting with antibodies against Flag, Myc, or Smc3. (D) TRF1 is not co-immunoprecipitated by SA1Δ72. 293T cells were transfected with FlagSA1 or FlagSA1Δ72 and MycTRF1. Cell lysates were immunoprecipitated with anti-Flag beads and analyzed by immunoblotting with antibodies against Flag, Myc, or Smc3. (E) The N-terminal 72 amino acids of SA1 is sufficient for binding to TRF1. 293T cells were transfected with GFP or GFPSA1N72 and MycTRF1. Cell lysates were immunoprecipitated with anti-GFP antibody and analyzed by immunoblotting with antibodies against GFP, Myc, or Smc3. (A–E) Input indicates 4% of extract. (F) TRF1 forms a complex with SA1–cohesin. 293T cells were transfected with FlagSA1 and MycTRF1. Cell extracts (ext; 1% of total) were immunoprecipitated with anti-Flag beads. The immunocomplex was eluted (E; 2.5% of total) from the beads (P, pellet; 2.5% of total) with Flag peptide and separated by sucrose density gradient sedimentation. Fractions (numbered 1–37) were analyzed by immunoblotting with antibodies against Flag, Smc3, Scc1, SA2, or TRF1. The sedimentation positions of aldolase (7.5S) and catalase (11.3S) are indicated.

Figure 3

TIN2 binds to SA1. (A–C) TIN2N not TIN2C is co-immunoprecipitated by SA1 or SA1Δ72, not SA2. 293T cells were transfected with GFPTIN2N or GFPTIN2C and (A) FlagSA1, (B) FlagSA1Δ72, or (C) FlagSA2. Cell lysates were immunoprecipitated with anti-Flag beads. Proteins were fractionated on SDS–PAGE and analyzed by immunoblotting with antibodies against Flag, GFP, or Smc3. Input indicates 4% of extract. (D) Schematic representation of the interactions between SA1 and TRF1 and TIN2.

Figure 4

Depletion of SA1 or TIN2 (and TRF1) rescues the mitotic arrest phenotype induced by tankyrase 1 siRNA. (A–C) Immunoblot analysis of extracts from HeLaI.2.11 cells transfected for 48 h with tankyrase 1 siRNA and a 2nd siRNA: (A) control (GFP), SA1.a, or SA2.a siRNA (B) control (scramble) or TRF1.b siRNA, and (C) control (scramble) or TIN2.a siRNA. (D–F) Histograms showing the percentage of cells in mitosis following 48 h of treatment with (+) or without (−) tankyrase 1 siRNA and the indicated 2nd siRNA. Approximately 1000 cells were scored for each sample by immunofluorescence analysis of cells stained with anti-α-tubulin antibody and DAPI. Control siRNAs; (D) GFP (E, F) scramble. (G, H) Immunoblot analysis of cell extracts from HeLaI.2.11 cells transfected with (G) TRF1.b siRNA or (H) TIN2.a siRNA. (I) TRF1 is degraded by the proteasome in TIN2 siRNA cells. Immunoblot analysis of cell extracts from HeLaI.2.11 cells transfected with TIN2.a siRNA for 48 h. Before harvesting, cells were treated with (+) or without (−) proteasome inhibitor MG132 (12.5 μM) for the indicated times.

Figure 5

Depletion of SA1 or TIN2 rescues the persistent telomere associations in tankyrase 1-depleted cells. (A) Chromosome-specific FISH analysis of HeLaI.2.11 cells collected by mitotic shake-off at 48 h after treatment with (a) control (GFP), (b) SA1.a, (c) SA2.a, (d) TRF1.b, or (e) TIN2.a siRNA without (a–e) or with (f–j) tankyrase 1 siRNA. Cells were fixed directly in methanol-acetic acid without hypotonic swelling and hybridized to a telomere probe 16pter (green). DNA was stained with DAPI (blue). (B) Histogram showing percentage of mitotic cells with unseparated telomeres; at least 100 mitotic cells were scored for each sample.

Figure 6

Depletion of SA1 or TIN2 restores normal centromere cohesion in tankyrase 1-depleted cells. (A) Chromosome-specific FISH analysis of HeLaI.2.11 cells collected by mitotic shake-off at 48 h after treatment with (a) control (GFP), (b) SA1.a, or (c) TIN2.a siRNA without (a–c) or with (d–f) tankyrase 1 siRNA. Cells were fixed directly in methanol-acetic acid without hypotonic swelling and hybridized to a centromere probe 6cen (red). DNA was stained with DAPI (blue). (B) Histogram showing percentage of mitotic cells with separated centromeres; at least 100 mitotic cells were scored for each sample. (C) Chromosome spread analysis of HelaI.2.11 cells collected after 48 h of treatment with (a) control (GFP), (b) SA1.a, or (c) TIN2.a siRNA without (a–c) or with (d–f) tankyrase 1 siRNA. Cells were swollen in hypotonic buffer and fixed in paraformaldehyde. Cells were treated with colcemide for 90 min before harvesting. Chromosome arms were visualized by staining with antibodies to the condensin subunit Smc2. (D) Histogram showing % mitotic cells with separated sisters; at least 200 mitotic cells were scored for each sample.