Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

Abstract

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.

Figure 1.

NME2-ChIP-seq peaks with telomere repeats. (A) Representative peaks that are made of almost entirely Tel_rep units—TTAGGG/CCCTAA. Lower panel shows reads (with or without Tel_rep) mapped to peaks; full sequence for one peak is shown below (non-Tel_rep reads in blue). Percentage identity is with respect to a stretch of TTAGGG/CCCTAA of similar length; fold-coverage is the ratio of total bp count of reads with Tel_rep that map within a peak over length of the peak. Supplementary Table S2A shows all peaks with Tel_rep units. Fraction of reads with either four Tel_rep (B) or three Tel_rep units (C) that mapped within NME2-ChIP peaks are shown in comparison to reads generated computationally from human promoter or ENCODE regions.

Figure 2.

NME2 associates with telomere ends. (A and B) Immunoprecipitated DNA using anti-NME2, -TRF2, -MYC or specific isotype was hybridized with telomere-specific double strand or Alu sequence using dot blot (A). Quantification of TTAGGG repeat DNA recovered in each ChIP is shown in (B). Results are average of experiments performed in triplicate; **P < 0.01, *P < 0.05. (C) ChIP-PCR of immunoprecipitated DNA with probes specific for telomeric region. PCR amplified telomere fragments migrated as a smear (50 to ∼500 bp); primer dimers migrated as a single band [as observed in blank (water)]. TRF2 ChIP was used as a positive control in both the cases.

Figure 3.

NME2 interacts with telomere binding factor TRF2. (A) Co-immunoprecipitation of NME2 with TRF2. HT-1080 nuclear lysate immunoprecipitated with anti-TRF2 antibody, followed by immunoblotting with anti-NME2 or anti-TRF2 antibody. (B) Reverse co-immunoprecipitation TRF2 by NME2. HT-1080 nuclear lysate immunoprecipitated with anti-NME2 or specific isotype and immunoblotted with anti-TRF2 or anti-NME2 antibody. (C) Interaction of NME2 with TRF2 in vitro. Ni-NTA only or Ni-NTA NME2 (purified his-tagged) beads were incubated with cell extracts from HT-1080 following by detection of bound TRF2 by immunoblot using TRF2-specific antibody. (D) Association of TRF2 and NME2 in HT-1080 cells was not affected after treatment with DNase I, ethidiumbromide (EtBr) or RNase A. Quantification is shown for IP with anti-TRF2 and anti-NME2 antibodies with respect to respective input fractions; average of three independent pull-down experiments is shown.

Figure 4.

Inhibition of telomerase catalytic activity by NME2. Real time quantification of change in telomerase activity with respect to that at 0 ng NME2 is shown for either His-tag NME2 (A) or His-tag NME2 (K12A) (B) after incubating with telomerase extracts from HT-1080 cells for 10 min at 30°C. All experiments were performed in triplicate. **P < 0.01, *P < 0.05.

Figure 5.

NME2 directly interacts with hTERT (A–C). Co-immunoprecipitation of NME2 and hTERT. HT-1080 nuclear lysate was subjected to immunoprecipitation with anti-NME2 (A), -hTERT (B), -HA-hTERT (C) antibody, followed by immunoblotting with either anti-hTERT, anti-NME2 or anti-HA-tag antibody (A, B, C, respectively). (D) Interaction of NME2 with hTERT in vitro. Ni-NTA only or Ni-NTA NME2 (His-tagged) beads were incubated with cell extracts from HT-1080 following by detection of bound hTERT by immunoblot using anti-hTERT antibody. (E) Association of endogenous hTERT with NME2 in HT-1080 cells was not changed after treatment with DNase I, ethidiumbromide (EtBr) or RNase A. Quantification is shown for IP with anti-NME2 and anti-hTERT antibodies with respect to respective input fractions; average of three independent pull-down experiments is shown.

Figure 6.

Sustained expression of NME2 decreases telomere length. (A and B) RT-PCR (A) and western blot (B) for stable expression of HA-tagged NME2 in HT-1080 cells. (C) Flow-FISH analysis of telomere length in HA-NME2 cells compared to HA-vector cells with increasing population doubling time [each point represent 10 000 cells from population doubling time points: closed squares, 45, closed diamonds, 90, closed triangles, 130, closed circles, 170 and asterisk 200)]; three independent experiments were performed, a representative analysis is shown. (D) Telomerase activity in HA-NME2 cells using real time quantification of telomerase activity; *P < 0.05. (E and F) RT–PCR (E) and western blot analysis (F) shows no change in hTERT level in HA-NME2 expressing cells.