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. 2009 Mar 15;8(6):931-9.
doi: 10.4161/cc.8.6.7941. Epub 2009 Mar 26.

A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix

Patrick G Kaminker  1 , Sahn-Ho Kim, Pierre-Yves Desprez, Judith Campisi
Affiliations

A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix

Patrick G Kaminker et al. Cell Cycle. .

Abstract

Telomeres are specialized heterochromatin at the ends of linear chromosomes. Telomeres are crucial for maintaining genome stability and play important roles in cellular senescence and tumor biology. Six core proteins-TRF1, TRF2, TIN2, POT1, TPP1 and Rap1 (termed the telosome or shelterin complex)-regulate telomere structure and function. One of these proteins, TIN2, regulates telomere length and structure indirectly by interacting with TRF1, TRF2 and TPP1, but no direct function has been attributed to TIN2. Here we present evidence for a TIN2 isoform (TIN2L) that differs from the originally described TIN2 isoform (TIN2S) in two ways: TIN2L contains an additional 97 amino acids, and TIN2L associates strongly with the nuclear matrix. Stringent salt and detergent conditions failed to extract TIN2L from the nuclear matrix, despite removing other telomere components, including TIN2S. In human mammary epithelial cells, each isoform showed a distinct nuclear distribution both as a function of cell cycle position and telomere length. Our results suggest a dual role for TIN2 in mediating the function of the shelterin complex and tethering telomeres to the nuclear matrix.

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Figures

Figure 1
Figure 1
Two isoforms of hTIN2 result from alternative splicing. (A) Alignment of exons encoding hTIN2 and mTIN2. Intron retention generates a translational stop sequence one nucleotide into the intron thereby encoding the smaller hTIN2S isoform. Hatched fill represents the 3' UTRs. Also shown are the coding regions known to be important for TIN2 interactions with TRF1, TRF2 and TPP1/PTOP, and the region deduced to be important for association with the nuclear matrix (NM). (B) RT-PCR products using primers spanning exons 6–9 (a), 5–9 (b) or 1–7 (c). We analyzed total RNA from human fibroblasts (lanes 1, 3 and 5) and epithelial cells (lanes 2, 4 and 6), described in the text. Sizes of the major PCR products were determined from the mobilities of markers (unlabeled right lane) and are consistent with transcripts that retain (upper bands) or splice (lower bands) introns separating exons 6–9. Lane 7 is a positive control using primers designed to identify β-actin mRNA and lane 8 is a negative control in which reverse transcription was omitted and PCR only was performed using primers spanning exons 5–9. (C) Western blot analysis confirming expression of two hTIN2 proteins in human cells. Total cell protein lysates were prepared from normal human fibroblasts (WI38, BJ, HCA2), human fibrosarcoma cells (HT1080), and immortal human mammary epithelial cells (184A1) using 2X Laemmli buffer (4% SDS). Blots were probed with an antibody raised against the N-terminal domain of hTIN2, then stripped and re-probed with an antibody against β-actin to control for protein loading. Identities of hTIN2L (~50 kDa) and hTIN2S (~40 kDa) were confirmed by expressing in 184A1 cells hTIN2L or hTIN2S cDNAs containing C-terminal epitope-tags (HA or V5, respectively) using the MSCV retroviral vector, as described in Materials and Methods, and analyzing lysates by western blotting using the indicated epitope-specific antibodies.
Figure 2
Figure 2
RNAi reduces both TIN2S and TIN2L. (A) Western analyses of hTIN2 proteins in cells expressing different shRNA constructs. HT1080 cells were transiently transfected with vectors expressing different shRNAs designed to target hTIN2 (shTIN2-1 to 5). The cells were lysed in 2X Laemmli buffer 36 h later and analyzed for hTIN2 proteins and β-actin (protein loading control). Controls for non-specific shRNA effects include a scrambled shRNA sequence and a pre-validated lamin A/C shRNA. (B) Quantification of the western blot shown in (A). The signals were quantified by densitometry and the hTIN2 signals were normalized to the β-actin signals. The normalized signals are displayed as a percentage of signals generated by the scrambled shRNA control. (C) Time course of hTIN2 knockdown. HT1080 cells were transiently transfected with the shRNA 5 vector. At the indicated intervals, the cells were lysed and analyzed for hTIN2 proteins and β-actin by western blotting. (D) Immunofluorescence analysis of hTIN2 depletion by shRNA. HT1080 cells were transfected with the vector expressing shTIN2-5. Transfected cells were identified by EGFP expression (green; a and d); the nuclei of all cells were identified by DAPI staining (blue; c and f). The cells were immunostained for hTIN2 (red; b) or lamin A/C (red; e). Cells were visualized by fluorescence microscopy.
Figure 3
Figure 3
Nuclear compartmentalization of hTIN2 isoforms. (A) Association of endogenous hTIN2 isoforms with nuclear fractions. Normal human fibroblasts (strain MJ90) were subjected to increasingly stringent extractions consisting of: 0.5% triton (lane 2), DNAse I digestion (lane 3), 0.25 M ammonium sulfate (lane 4) and 2 M NaCl (lane 5) extraction. The remaining pellet was solubilized in 2X Laemmli buffer (lane 6). Half the cell equivalents were solubilized directly in 2X Laemmli buffer (whole cell lysate) (lane 1) and used to determine the input. Equal cell equivalents were analyzed for each of the other fractions. Proteins were analyzed by western blotting using antibodies to detect the N-terminus of hTIN2, TRF1, TRF2, tankyrases (TNKS1/2), heat shock protein 60 (HSP60, a mitochondrial chaperone and cytoplasmic marker) and lamin A/C (insoluble nuclear matrix marker). (B) Solubilities of epitope-tagged hTIN2 isoforms. C-terminal epitope (HA)-tagged hTIN2 isoforms (hTIN2L-HA, hTIN2S-HA) were expressed in MJ90 cells, as described in the text and Materials and Methods. The cells were subjected to fractionation and analysis as described in A except that anti-HA antibodies were used for detection.
Figure 4
Figure 4
hTIN2 isoforms interact similarly with known telomere-associated partners. HT1080 cells were infected with retroviruses expressing either FLAG-TIN2S, FLAG-TIN2L, no insert (pLXSN) or FLAG-TREM1 (negative control), as indicated in the bottom panel. After selection, the cells were lysed and immunoprecipitated using either anti-FLAG or non-specific IgG resin. Proteins were eluted from resin as described in Materials and Methods, and analyzed by western blotting using antibodies (WB) against tankyrase, TRF1, TRF2 or FLAG. 2.5% of the lysate (input) were also analyzed by western blotting.
Figure 5
Figure 5
hTIN2L localizes to telomeres. (A) Immunolocalization of TIN2S-V5 expressed in 184A1 HMECs using antibodies against the hTIN2 N-terminal domain (TIN2; green) and the V5 epitope (V5; red). Arrows identify the telomere-independent nuclear domains, which co-stain (Merge; yellow). DAPI staining (blue) identifies nuclei. (B) Immunolocalization of TIN2L-HA expressed in 184A1 HMECs using antibodies against the hTIN2 N-terminal domain (green) and the HA epitope (red). Arrows identify telomere-independent nuclear domains, and DAPI staining identifies nuclei (blue). (C) Co-localization (Merge; yellow) of TIN2L-HA (HA; red) with TRF2 (TRF2; green) in nuclei (DAPI; blue).
Figure 6
Figure 6
hTIN2S, but not hTIN2L, localization depends on telomere length. (A) 184A1 HMECs were infected with a control retrovirus (pLXSN) or retrovirus expressing the catalytic subunit of telomerase (hTERT). The cells were immunostained for TIN2 (red) 2 and 11 population doublings (PD) after infection. Arrows indicate non-telomeric TIN2 domains. Parallel cultures were analyzed for telomere length using qFISH (green), as described in Materials and Methods. Nuclei were counterstained with DAPI (blue). (B) Quantification of average fluorescence from qFISH analyses. Fluorescence intensities from >300 cells were measured. The results are expressed in arbitrary units (a.u.) relative the fluorescence intensities of uninfected cells (PD 0). Arrow indicates the telomere length at which non-telomeric TIN2 domains are no longer visible.
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