Putative telomere-recruiting domain in the catalytic subunit of human telomerase

Abstract

Telomerase, the enzyme that elongates telomeres, is essential to maintain telomere length and to immortalize most cancer cells. However, little is known about the regulation of this enzyme in higher eukaryotes. We previously described a domain in the hTERT telomerase catalytic subunit that is essential for telomere elongation and cell immortalization in vivo but dispensable for catalytic activity in vitro. Here, we show that fusions of hTERT containing different mutations in this domain to the telomere binding protein hTRF2 redirected the mutated hTERT to telomeres and rescued its in vivo functions. We suggest that this domain posttranscriptionally regulates telomerase function by targeting the enzyme to telomeres.

FIG. 1.

Fusion of hTERT to hTRF2 relocalizes hTERT to telomeres. (A) Scale diagram of the protein structure of hTRF2-hTERT fusion. The positions of epitope tags and substitution mutations generated in hTRF2 and hTERT are indicated. (B) Stable expression of hTRF2 and hTRF2 fusion proteins in LM217 cells. Lysates isolated from LM217 cells expressing the described constructs were immunoblotted with anti-flag (α-flag) and anti-myc antibodies to detect ectopic hTERT and hTRF2, respectively. Actin served as a loading control. (C) Fusion of hTRF2 targets hTERT to telomeres. Telomeric localization of hTRF2, hTRF2-hTERT, and hTRF2-hTERT₊₁₂₈ proteins visualized by indirect immunofluorescence with an anti-myc antibody and colocalization with transfected GFP-hTRF1 in LM217 cells. Cell nuclei were stained with DAPI. (D) Diffuse nuclear distribution of hTERT. An example of LM217 cells transiently expressing YFP-hTERT is shown as a phase-contrast image (right) to visualize the cells and as a fluorescent image (left) to visualize the YFP-tagged protein.

FIG. 2.

hTRF2-hTERT fusion proteins retain in vitro telomerase activity. (A) Stable expression of hTRF2 and hTRF2-hTERT in HA5 cells. Lysates isolated from HA5 cells expressing the described constructs were immunoblotted with anti-flag (α-flag) and anti-myc antibodies to detect ectopic hTERT and hTRF2, respectively. Actin served as a loading control. (B) Fusion of hTRF2 to hTERT does not affect telomerase activity. Extracts isolated from HA5 cells stably expressing the described proteins were assayed for in vitro telomerase activity. As a negative control, duplicate lysates were heat treated (+HT) to inactivate telomerase prior to the assay. The internal standard (IS) served as a positive control for PCR amplification.

FIG. 3.

In vivo function of hTERT₊₁₂₈ is rescued by fusion with hTRF2. (A) hTRF2-hTERT₊₁₂₈ arrests telomere shortening. Restriction enzyme-digested genomic DNA isolated from late-passage HA5 cells expressing hTERT, hTRF2-hTERT, or hTRF2-hTERT₊₁₂₈ (left) or from HA5 cells at the time of infection or once a vector control population was selected (right) were hybridized with a telomeric probe to visualize telomere-containing fragments. Molecular size markers are shown on the left. (B) hTRF2-hTERT₊₁₂₈ can immortalize human cells. The life spans of HA5 cell lines infected with vectors expressing hTRF2-hTERT (□) or hTRF2-hTERT₊₁₂₈ (○) or controls infected with vector alone (▴) or expressing hTERT (▪) or hTERT₊₁₂₈ (•) are plotted against time.

FIG. 4.

In vivo functions of other hTERT DAT mutants are rescued by fusion with hTRF2. (A) Telomerase activities of other DAT mutants of hTERT alone or fused to hTRF2. Extracts isolated from HA5 cells stably expressing the described proteins were assayed for in vitro telomerase activity. The internal standard (IS) served as a positive control for PCR amplification. (B) Fusion of hTRF2 to other DAT mutants can immortalize human cells. The life spans of HA5 cell lines expressing hTERT_+128-3A (▪), hTRF2-hTERT_+128-3A (□), hTERT_+125NAA (•), hTRF2-hTERT_+125NAA (○), hTERT_+134NAA (▴), hTRF2-hTERT_+134NAA (▵), vector (♦), or hTRF2-hTERT (⋄) are plotted against time.

FIG. 5.

DNA binding activity of hTRF2 is required to rescue the in vivo function of an hTERT DAT mutant. (A) Telomeric localization of hTRF2 is lost upon mutation of the DNA binding and dimerization domains. An example of an LM217 cell(s) transiently expressing hTRF2_DPV-YFP or hTRF2-YFP is shown as a differential-interference contrast image (right) to visualize the cells and as a fluorescent image (left) to visualize the YFP-tagged protein. (B) Stable expression of hTRF2_DPV-hTERT in HA5 cells. Lysates isolated from HA5 cells expressing the described constructs were immunoblotted with an anti-flag (α-flag) antibody to detect ectopic hTERT. Actin served as a loading control. (C) Fusion of hTRF2_DPV to hTERT does not affect telomerase activity. Extracts isolated from HA5 cells stably expressing the described proteins were assayed for in vitro telomerase activity. The internal standard (IS) served as a positive control for PCR amplification. (D) hTRF2_DPV-hTERT₊₁₂₈ cannot immortalize human cells. The life spans of HA5 cell lines expressing hTRF2-hTERT₊₁₂₈ (•), hTRF2_DPV-hTERT₊₁₂₈ (○), hTRF2-hTERT (▪), hTRF2_DPV-hTERT (□), or an empty vector (▴) are plotted against time.

FIG. 6.

Fusion with hTRF2 underlies the rescue of DAT mutants of hTERT. (A) Endogenous hTERT expression is absent in HA5 cells expressing hTRF2-hTERT₊₁₂₈. Total RNAs isolated from early (pd 1) infected HA5 cells and in some cases late-passage (pd 44 [asterisk]) HA5 cells expressing hTRF2-hTERT₊₁₂₈ were RT-PCR amplified with primers specific for ectopic or endogenous hTERT or, as a control, GAPDH mRNA content. Telomerase-positive CWR22-RV1 cells served as a positive control, whereas vector-infected HA5 cells or water alone served as a negative control for endogenous hTERT expression. (B) Equivalent expression of hTERT₊₁₂₈ and hTRF2-hTERT₊₁₂₈. Lysates isolated from HA5 cells expressing the described constructs were immunoblotted with an anti-flag (α-flag) antibody to detect ectopic hTERT or hTRF2-hTERT protein. Hygro indicates cells infected with pBabehygro constructs, which express proteins at lower levels. Actin served as a loading control. (C) Reduced expression of hTRF2-hTERT₊₁₂₈ still immortalizes HA5 cells. The life spans of HA5 cell lines infected with the low-expression pBabehygro vectors encoding hTRF2-hTERT (•), hTRF2-hTERT₊₁₂₈ (○), or vector alone (▴) are plotted against time.

FIG. 7.

In vivo activity of hTERT₊₁₂₈ is restored only when directly fused to hTRF2. (A) Expression of YFP, hTRF2, or hTRF2-YFP in either hTERT- (wt) or hTERT₊₁₂₈ (+128)-infected HA5 cells. Lysates isolated from HA5 cells expressing the described constructs were immunoblotted with anti-GFP (α-GFP) or anti-myc antibodies to detect ectopic hTRF2. Actin served as a loading control. (B) Ectopic expression of hTRF2 does not immortalize HA5 cells expressing hTERT₊₁₂₈. The life spans of HA5 cells coexpressing hTERT and vector (▪), YFP (▴), hTRF2 (•), or hTRF2-YFP (⧫) or those coexpressing hTERT₊₁₂₈ and vector (□), YFP (▵), hTRF2 (○), or hTRF2-YFP (◊) are plotted against time.