Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1

Abstract

The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.

FIG. 1.

hPot1 binds telomeres and promotes telomere elongation. (A) 293 cells transiently transfected with Flag-tagged hPot1 or hTRF1 were treated with or without formaldehyde (X-link), lysed, and subjected to immunoprecipitation with the indicated antibodies. After purification, coimmunoprecipitated DNA was analyzed by Southern dot blot analysis with the indicated probes. Genomic DNA was used as a control for probe integrity. (B and C) 293 cells were stably infected with a control virus (vector), Flag-tagged hPot1_ΔOB (f-hPot1_ΔOB), or Flag- and HA-tagged hPot1 (f-hPot1-HA). The resulting cells were assayed for ectopic hPot1 expression by immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies (B) and for changes in telomere length by Southern blot analysis with a telomeric probe and with genomic DNA isolated at the indicated pd (right panel) (C). 293 cells infected with vector or Flag-tagged hPot1 (f-hPot1) alone were similarly assayed (left panel).

FIG. 2.

Fusion of hPot1 to hTERT relocalizes catalytically active hTERT to telomeres. (A and B) Flag-tagged hPot1, hPot1-hTERT, and hPot1-hTERT₊₁₂₈ were stably introduced into U2OS cells. (A) Flag-hPot1 proteins were immunoprecipitated with an anti-Flag antibody and detected by immunoblotting with the same antibody. (B) hPot1-containing proteins stably expressed in U2OS cells were detected by immunofluorescence with an anti-Flag antibody (red) and compared with localization of transfected hTRF2-YFP (green), which was detected by virtue of its fluorescence. (C and D) HA5 cells stably infected with a control retrovirus (vector) or one encoding Flag-tagged hTERT, hPot1-hTERT, or hPot1-hTERT₊₁₂₈ in either pBabepuro or (for lower expression) pBabehygro (indicated by asterisks) were confirmed to express the appropriate protein by immunoblotting with an anti-Flag antibody (tubulin served as a loading control) (C) and were assayed for telomerase activity with threefold serial dilutions of cellular lysates, starting with 0.2 μg (internal control [IC] results and average TRAP activity levels [normalized to those of wild-type hTERT] are indicated at the bottom of the panel) (D). HT, heat treatment to inactivate telomerase.

FIG. 3.

Elongation of telomeres and life span extension by hPot1-hTERT and hPot1-hTERT₊₁₂₈ in mortal telomerase-negative cells. HA5 cells stably infected with a control retrovirus (vector) or one encoding Flag-tagged hTERT, hTERT₊₁₂₈, hPot1-hTERT, or hPot1-hTERT₊₁₂₈ were monitored for changes in telomere length by Southern blot analysis with a telomeric probe and with genomic DNA isolated at the indicated pd (A) and were grown in cultures to monitor cell life span (B). Average telomere length values [Avg length] are shown below panel A. hPot1-hTERT, □; hPot1-hTERT₊₁₂₈, ○; hTERT₊₁₂₈, •; vector, ▴; hTERT, ▪.

FIG. 4.

Mutations in the hPot1 DNA-binding region diminish the ability of hPot1 to interact with a telomeric G-strand oligonucleotide in vitro. (A) Pictorial representation of hPot1_ΔOB and hPot1_3A mutants (amino acid positions are indicated by numbers). Sequence alignment of putative telomere end-binding proteins from divergent eukaryotes (accession numbers: Macaca fascicularis [AB066545], Homo sapiens [NM_015450], Mus musculus [NM_133931], Euplotes crassus [M96818], and Caenorhabditis elegans [T22006]) through the use of a manually modified ClustalW alignment shows three identically conserved amino acids (asterisks) that were replaced with alanines in hPot1_3A. Conserved residues are show in gray-shaded boxes; identical residues are shown with white characters in black boxes. (B) Association of in vitro ³⁵S-labeled hPot1 proteins with a ³²P-labeled G-strand oligonucleotide. IP, immunoprecipitation.

FIG. 5.

Rescue of hTERT₊₁₂₈ by fusion with hPot1 mutants. HA5 cells stably infected with a vector control, hTERT, hPot1-hTERT, hPot1_3A-hTERT, hPot1_ΔOB-hTERT, hTERT₊₁₂₈, hPot1-hTERT₊₁₂₈, hPot1_3A-hTERT₊₁₂₈, or hPot1_ΔOB-hTERT₊₁₂₈ were confirmed by immunoblot analysis with an anti-Flag antibody to express the appropriate fusion proteins (actin serves as a loading control) (A) and assayed for telomerase activity with a ninefold dilution of cellular extracts, starting at a total of 0.2 μg (B). The internal control (IC) results and average values of TRAP activity (normalized to that of wild-type hTERT) are shown at the bottom of the panel. (C and D) Cell life span (mutants: hPot1_3A-hTERT [•], hPot1_3A-hTERT₊₁₂₈ [○], hPot1_ΔOB-hTERT [▴], hPot1_ΔOB-hTERT₊₁₂₈ [▵]; controls: hPot1-hTERT [▪], hPot1-hTERT₊₁₂₈ [□], [hTERT₊₁₂₈ [⋄]) (C) and telomere length (as measured by Southern blotting analysis with a telomeric probe) (D) were monitored over time.

FIG. 6.

Rescue of in vivo functions of hTERT₊₁₂₈ requires direct fusion with hPot1. HA5 cells were stably coinfected with either hTERT or hTERT₊₁₂₈ in combination with control vector, hPot1, or hPot1_ΔOB and were confirmed to express the appropriate proteins by immunoblotting with an anti-Flag antibody following immunoprecipitation (A), monitored for life span (hTERT with vector, ▪; hTERT with hPot1, •; hTERT with hPot1_ΔOB, ▴; hTERT₊₁₂₈ with vector, □; hTERT₊₁₂₈ with hPot1, ○; hTERT₊₁₂₈ with hPot1_ΔOB, ▵) (B), and assayed for telomere length by Southern blot analysis with a telomeric probe and with genomic DNA isolated at the indicated pd (C).