The Rb-Related p130 Protein Controls Telomere Lengthening through an Interaction with a Rad50-Interacting Protein, RINT-1  Ling-Jie Kong, Alison R. Meloni, Joseph R. Nevins  Molecular Cell  Volume 22, Issue 1, Pages 63-71 (April 2006) DOI: 10.1016/j.molcel.2006.02.016 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Effect of Loss of p130 Function on Telomere Length (A) si-p130 constructs effectively knock down p130. HFF cells were infected with control retrovirus (pSuper) or viruses harboring si-p130 hairpin structures (sh-p130: 1∼4) at passage eight. Cells were selected with puromycin. Total protein extracts were isolated, and the efficiency of siRNA viruses in eliminating p130 expression was measured by Western blot analysis using polyclonal anti-p130 antibody. Anti-actin antibody was used to probe the same blot to confirm the equal loading. (B) Loss of p130 results in telomere lengthening. HFF cells infected with different viruses as described in (A) were cultured under selection of puromycin for 3 months. Genomic DNA was isolated, and telomere length was measured by Southern blot analysis. The genomic DNA from HFF cell at passage eight (HFF p8) was used to show the initial telomere length. (C) Loss of p130 does not change the telomerase activity in HFF cells. HFF cells infected with viruses as described in (A) were harvested, and telomerase activity was measured by TRAP assay. The telomerase-positive cell line T98G and the PCR template control TSR8 were used as positive controls. Heat-inactivated (HI) protein extracts and CHAP buffer (buffer) were used as negative controls. (D) Loss of p130 does not change growth rate in HFF cells. HFF cells infected with viruses described in (A) were used to measure growth rates after 2 weeks of puromycin selection. Each cell line was cultured in triplicate. Population doublings were calculated by using the average of these three cultures. After 48 days in culturing, the population doubling for cells infected with pSuper and four different sh-p130 viruses were 33.14 ± 0.27, 32.91 ± 0.13, 33.03 ± 0.09, 32.42 ± 0.10, and 32.81 ± 0.21, respectively. Molecular Cell 2006 22, 63-71DOI: (10.1016/j.molcel.2006.02.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 p130 Interacts with RINT-1 (A) GST pull-down assay with in vitro-translated pocket proteins. E. coli-expressed GST-tagged RINT-1 protein was used for protein interaction assays with in vitro-translated p130 (left three lanes), p107 (middle three lanes), and Rb (right three lanes). Similar amounts of in vitro-translated proteins were used for each reaction as measured by radioactive activity (see also input lane). E. coli-expressed GST protein was used as control in all experiments. The starting amount of in vitro-translated protein for each GST pull-down assay is 30× of the input lane. (B) GST-RINT-1 interacts with the hypophosphorylated form of p130 in vitro. Total protein extract was isolated from T98G cells and used for GST pull-down assay similarly to (A). Low percentage (4%) SDS-PAGE was used to separate different phosphorylation forms of p130 protein. Anti-p130 polyclonal antibody was used for Western blot. The amount of protein in input lane is from 1 × 105 cells. The GST pull-down lanes start with protein extracts from 1.5 × 106 cells. (C) p130 and RINT-1 interact within the cell. Myc-tagged RINT-1 construct was transfected into 293 cells. Immunoprecipitations were performed with anti-p130 polyclonal antibody to recover the endogenous p130 protein. Anti-Gal4 antibody was used as control. Immunoprecipitates were assayed by Western blot with monoclonal anti-p130 (top row) or anti-Myc (bottom row) antibodies. The input lane contains total protein from 2.5 × 105 cells. The IP lanes represent immumoprecipitates starting with protein extracts from 1.5 × 106 cells. (D) p130 and RINT-1 form a complex in HFF cells. Nuclear extracts from HFF cell were used to perform immunoprecipitation with either control serum or anti-RINT-1 antiserum. The products were separated by SDS-PAGE and probed with anti-p130 or anti-Rad50 monoclonal antibodies. The input lane contains nuclear protein from 1 × 105 cells. The IP lanes represent immumoprecipitates starting with nuclear protein extracts from 2 × 106 cells. Molecular Cell 2006 22, 63-71DOI: (10.1016/j.molcel.2006.02.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 An N-Terminal Domain of p130 Is Required for RINT-1 Interaction (A) GST pull-down assay with in vitro-translated p130 mutants. In vitro-translated untagged (1, 2, and 3) or Gal4-fusion (4, 5, and 6) p130 proteins were used for protein interaction assays with GST-tagged RINT-1 protein as described in Figure 2A. Similar amounts of in vitro-translated proteins were used for each reaction as measured by radioactive activity (see also input lane). E. coli-expressed GST protein was used as a control in all experiments. (B) Diagram of p130 clones used for GST pull-down assay. The shaded areas indicate the pockets (“A” and “B”). The amino acid numbers are marked to show the positions of the p130 subclones. Molecular Cell 2006 22, 63-71DOI: (10.1016/j.molcel.2006.02.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 p130 Interacts with Rad50 in a RINT-1-Dependent Manner (A) Endogenous p130 interacts with Rad50 in the cell. Nuclear protein extracts from HFF cells were used for immunoprecipitation with control, anti-p130, anti-RINT-1, or anti-Rad50 polyclonal antibodies. Proteins were separated by SDS-PAGE and detected by Western blot using anti-Rad50 (top row) or anti-p130 (bottom row) monoclonal antibodies at dilution factors of 1:5000 and 1:1000, respectively. The input lanes contain nuclear proteins from 1 × 105 cells. The p130 IP lane in the bottom panel represents immumoprecipitates starting with nuclear protein extracts from 1.25 × 105 cells. All other IP lanes represent immumoprecipitates starting with nuclear protein extracts from 5 × 106 cells. (B) siRINT-1 construct effectively depletes RINT-1 protein. HFF cells were infected with control retrovirus (pSuper) or a virus harboring siRINT-1 hairpin structure and selected with puromycin. Total protein extracts were isolated, and the efficiency of siRNA viruses was measured by Western blot using anti-RINT-1 antibody. Anti-tubulin antibody was used to probe a parallel blot to show similar amounts of proteins were loaded in each lane. (C) Loss of RINT-1 abolishes the p130-Rad50 interaction. HFF cells were infected with control virus (pSuper) or siRINT-1 virus and selected with puromycin. Total protein extracts from either control cells (pSuper) or RINT-1 knockdown cells (siRINT-1) were used for immunoprecipitation with anti-p130 or anti-Rad50 polyclonal antibodies. Anti-Gal4 antibody was used as a control. Proteins were separated by SDS-PAGE and detected by Western blot using monoclonal anti-Rad50 or anti-p130 antibodies. Molecular Cell 2006 22, 63-71DOI: (10.1016/j.molcel.2006.02.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 RINT-1 Is Required for Telomere Length Control (A) Loss of RINT-1 leads to telomere elongation. HFF cells were infected with control retrovirus (pSuper) or siRINT-1 virus. DNA was isolated after puromycin selection for 2 months. Telomere length was measured by Southern blot. The DNA of HFF cell at passage eight (HFF p8) was used to show the initial telomere length. (B) Loss of RINT-1 does not change the telomerase activity in HFF cells. HFF cells infected with viruses as described in (A) were harvested, and telomerase activity was measured by TRAP assay. The telomerase-positive T98G cell line and a PCR template control TSR8 were used as positive controls. HI protein extracts and CHAP buffer (buffer) were used as negative controls. (C) Loss of RINT-1 does not change growth rate in HFF cells. HFF cells were infected with viruses at passage eight as described in (A) and cultured under puromycin selection. Passage 16 cells were used to initiate the measurement of growth rates. Every cell line was cultured in triplicate. Population doublings were calculated by using the average of the three cultures. After 56 days of culturing, the population doubling for cells infected with pSuper and siRINT-1 viruses were 31.23 ± 0.45 and 30.31 ± 0.08, respectively. (D) Telomere elongation is not caused by nonspecific targeting of siRNA. HFF cells were infected with either control MSCV virus (lanes 1 and 2) or RINT-1 rescue virus MSCV-RINT-r expressing an siRNA-resistant form of RINT-1 (lanes 3 and 4) and selected with hygromycin. Those cells were then infected with control retrovirus pSuper (lanes 1 and 3) or siRINT-1 virus (lanes 2 and 4). Genomic DNA was isolated after puromycin selection for 2 months, and telomere length was measured by Southern blot. The effectiveness of siRNA viruses and rescue construct was measured by Western blot using anti-RINT-1 antibody. Anti-tubulin antibody was used to show similar amounts of protein were loaded in each lane. Molecular Cell 2006 22, 63-71DOI: (10.1016/j.molcel.2006.02.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Telomere Length Control Requires Both p130 and RINT-1 (A) The p130 interaction domain in RINT-1 was mapped to within amino acids 358–440. The yeast two-hybrid system was used to measure the interaction between p130 and RINT-1. Full-length p130 was fused to Gal4 DNA binding domain. A series of RINT-1 truncations were fused to Gal4-activation domain. Yeast strain AH109 was used in the experiment. We define positive clones as those that grew well in SD minus histidine plates (LTH), gave white (W) not red (R) or pink (P) colonies to show adenine-positive in low adenine YPD plates, and gave blue (B) not white (W) colonies in plates with α-gal (LTα). (B) A D387G mutation in RINT-1 abolishes its interaction with p130 in yeast. We used the yeast reverse two-hybrid system to isolate a D387G mutant of RINT-1, which lost interaction with p130. This mutant maintains ability to interact with Rad50 in the yeast two-hybrid assay. (C) The D387G mutant of RINT-1 does not interact with p130 in human cells. HFF cells were infected with either RINT-1 rescue virus MSCV-RINT-r (left three lanes) or RINT-1 rescue virus with the D387G mutation MSCV-D387G-r (right three lanes) and selected with hygromycin. Those cells were then infected with siRINT-1 retrovirus and selected with puromycin. Both of the two rescue constructs effectively rescued RINT-1 at protein level (D). Total protein extracts from these cells were used for immunoprecipitation with anti-RINT-1 antibody. Rabbit serum was used as control. Proteins were separated by SDS-PAGE and detected by Western blot using anti-Rad50 (top row) or anti-p130 (bottom row) monoclonal antibodies. (D) D387G mutant of RINT-1 lost ability to control telomere length. HFF cells were infected with viruses as described in Figure 6C. Genomic DNA was isolated after puromycin selection for 2 months. Telomere length was measured by Southern blot. Western blots with anti-RINT-1 antibody showed the protein levels of RINT-1 in the rescued cell lines. Blots were subsequently blotted for tubulin to confirm equal loading of protein in each lane. Molecular Cell 2006 22, 63-71DOI: (10.1016/j.molcel.2006.02.016) Copyright © 2006 Elsevier Inc. Terms and Conditions