The Pot1a-associated proteins Tpt1 and Pat1 coordinate telomere protection and length regulation in Tetrahymena

Abstract

We have identified two new telomere proteins, Tpt1 and Pat1, from the ciliate Tetrahymena thermophila. Although Tetrahymena telomerase is well characterized, only one telomere protein had previously been identified. This was the G-overhang binding-protein Pot1a. Tpt1 and Pat1 were isolated as Pot1a binding partners and shown to localize to telomeres. As Tpt1 and Pat1 were both found to be essential, conditional cell lines were generated to explore their function. Tpt1 depletion caused a rapid growth arrest and telomere elongation in the absence of cell division. The phenotype was similar to that seen after Pot1a depletion suggesting that Tpt1 and Pot1a function together to regulate telomere length and prevent telomere deprotection. In contrast, Pat1 depletion had a modest effect on cell growth but caused progressive telomere shortening similar to that observed upon TERT depletion. Thus Pat1 appears to be needed for telomerase to maintain the chromosome terminus. Analysis of Pot1a-Tpt1-Pat1 complex formation using purified proteins indicated that Tpt1 interacts directly with Pot1a while Pat1 interacts with Tpt1. Our results indicate that Tpt1 is the Tetrahymena equivalent of mammalian TPP1, Schizosaccharomyces pombe Tpz1, and Oxytricha nova TEBPβ.

FIGURE 1:

Generation of cell lines with Tpt1 or Pat1 gene replacements. (A–C) Gene-targeting constructs used to make modified Tpt1 or Pat1 cell lines. In each case, the gene-targeting construct was recombined into the native gene locus (panel II). Exons are in gray. Southern blot probes are shown as black bars. X, XbaI; H, HindIII; N, NdeI restriction sites. (A) Constructs used to make TAP-Tpt1 (I) and Tpt1 KD cells (III). (B) Pat-HA (I) and TAP-Pat1 (III) cells. (C) Pat1 KD cells (I). (D and E) Southern blots showing Pat1 or Tpt1 gene replacement. Bands from endogenous, knockout, or HA- or TAP- tagged genes are marked. (D) (I) Pat1 KD or Pat1-HA (HA), HindIII digest. (II) Pat1 KD or TAP-Pat1, XbaI digest. (E) WT, KD, or TAP-Tpt1 (TAP), NdeI digest.

FIGURE 2:

Tpt1 and Pat1 bind Pot1a and associate with telomeres. (A and B) Tpt1 and Pat1 copurify with Pot1a. (A) Ni-Sepharose precipitation (PPT) of nuclear extracts (NE) from WT or conditional TAP-Tpt1 (TAP) cells (panel I) or TAP-Pat1 (TAP) cells (panel II). Western blot with Pot1a antibody. Lanes 7 and 8, extract was treated with MNase before pull down. (B) Immunoprecipitation (IP) of nuclear extracts (NE) from Pat1-HA cells with no antibody (NA) or Pot1a antibody (Pot1). Western blot with HA antibody. *, Cross-reacting bands. (C) Tpt1 and Pat1 are present at rDNA telomeres. (I) Schematic of rDNA telomere. Arrows indicate positions of PCR primers used to amplify subtelomeric (Tel) and internal (Int) regions. (II) Multiplex PCR of ChIP products from WT or conditional TAP-Tpt1 or TAP-Pat1 cells grown ± cadmium. Lanes 1–5, fourfold dilutions of input DNA; lane 6, protein A beads, no antibody (NA); lane 7, Protein A beads + Pot1a antibody (Pot1); lane 8, IgG Sepharose to precipitate Tap-tagged Tpt1 or Pat1 (TAP). (III) Quantification of multiplex PCR to show fold enrichment of telomeric DNA. Minimum of three experiments.

FIGURE 3:

Tpt1 depletion causes a cell-cycle checkpoint and telomere elongation. (A) Southern blot showing length of rDNA telomere in WT and Tpt1 KD cells. (B) Growth curves for TAP-Tpt1 conditional cells grown with (+Cd) or without (–Cd) cadmium. Cd was readded to a portion of the –Cd culture after 24 h. (C) Phase contrast (PC) images of conditional TAP-Tpt1 cells grown with (+Cd) or without (–Cd) cadmium for 24 h. Nuclei were stained with DAPI. (D) Southern blot showing length of rDNA telomeres in TAP- Tpt1 cells grown ± Cd for 0–96 h. (E) Localization of Pot1a in TAP-Pat1 cells grown ± Cd for 24 h. Cells were stained with Pot1a antibody and DAPI.

FIGURE 4:

Pat1 depletion results in shorter telomeres. (A) Southern blot showing length of rDNA telomere in WT and Pat1 KD cells. (B) Phase contrast (PC) images of conditional TAP-Pat1 cells grown with (+Cd) or without (–Cd) cadmium for 6 d. Nuclei were stained with DAPI. (C) Southern blot showing length of rDNA telomeres in conditional TAP-Pat1 cells grown ± Cd for 1–9 d.

FIGURE 5:

Telomere shortening is reversible and similar to that seen after TERT depletion. (A) Localization of Pat1 in conditional TAP-Pat1 cells grown ± Cd for 3 d. Cells were stained with Pat1 antibody and DAPI. (B) RT-PCR showing level of TAP-Pat1 expression. (I) mRNA was isolated from TAP-Pat1 cells grown ± Cd for 3, 6, or 9 d. (II) as for (I) but without reverse transcriptase. (C) Southern blot showing the length of the rDNA telomere in TAP-Pat1 cells after growth without (–Cd) cadmium for 9 d followed by addition of cadmium for 6, 24, or 48 h. (D) Southern blots showing rDNA telomere length in conditional TAP-Pat1 cells (TAP-Pat1) and conditional TERT knockout cells (TERT-KO) after growth without cadmium for 2–16 d.

FIGURE 6:

Lack of interaction between Pat1 or Tpt1 and telomerase. (A) Telomerase activity in extracts made from WT cells and TAP-TERT or TAP-Pat1 conditional cells grown ± cadmium. RC, recovery control for telomerase product isolation. (B) Telomerase activity precipitated with IgG beads and extracts from WT cells or cells expressing TAP-tagged TERT (TT), TAP-Pat1 (Pat1), or TAP-Tpt1 (Tpt1) (lanes 5–8). Input activity is shown in lanes 1–4.

FIGURE 7:

Configuration of the Pot1a-Tpt1-Pat1 complex. (A) Protein–protein interactions within the complex. (I) His-SUMO–tagged Tpt1 was incubated with Pot1a and Pat1, complexes were isolated on Ni-Sepharose, and copurified proteins were detected by Western blotting. Lane 1 shows the input protein. (II) As for (I) but with SUMO-Pat1 and Pot1a. (B) Mobility shift gels to examine Pot1 and Tpt1 binding to telomeric DNA. (I) Addition of increasing amounts of Pot1a (∼0.25–50 nM) to 15 pM TelG₂₀ oligonucleotide TG₄(T₂G4)₂T₂G. (II) Addition of increasing concentrations of Tpt1 to 15 pM TelG₂₀ ± ∼25 nM Pot1a. Lanes 1–7, 50–1000 nM Tpt1; lane 8, Pot1a alone; lanes 9–11, 50–200 nM Tpt1 + Pot1a. (C) Model showing organization of the Pot1a telomeric complex.