Role of progerin-induced telomere dysfunction in HGPS premature cellular senescence

Abstract

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature-aging syndrome caused by a dominant mutation in the gene encoding lamin A, which leads to an aberrantly spliced and processed protein termed progerin. Previous studies have shown that progerin induces early senescence associated with increased DNA-damage signaling and that telomerase extends HGPS cellular lifespan. We demonstrate that telomerase extends HGPS cellular lifespan by decreasing progerin-induced DNA-damage signaling and activation of p53 and Rb pathways that otherwise mediate the onset of premature senescence. We show further that progerin-induced DNA-damage signaling is localized to telomeres and is associated with telomere aggregates and chromosomal aberrations. Telomerase amelioration of DNA-damage signaling is relatively rapid, requires both its catalytic and DNA-binding functions, and correlates in time with the acquisition by HGPS cells of the ability to proliferate. All of these findings establish that HGPS premature cellular senescence results from progerin-induced telomere dysfunction.

Fig. 1.

TERT rescues HGPS premature senescence through inhibition of tumor-suppressor pathway activation. (A) Growth curves of HGPS fibroblasts expressing ectopic TERT or vector control. HGPS fibroblasts near the end of their proliferative lifespan (two remaining PDs) were transduced and marker selected for 2 weeks with retroviral TERT or control vectors, and the number of cumulative PDs from the time of selection was calculated. (B) Cell-cycle analysis of HGPS fibroblasts expressing ectopic TERT or vector control. Propidium iodide was measured by flow cytometry at 2 weeks after retroviral transduction and selection with vector control or TERT. Error bars indicate s.d. for a representative experiment performed in triplicate. (C) SA-β-gal staining of HGPS cultures expressing ectopic TERT or vector control at 2 weeks after retroviral transduction and selection. (D) Western blot showing levels of endogenous progerin in HGPS fibroblasts before, 6 PDs after, and 70 PDs after TERT transduction and selection. Protein levels of lamin A, lamin C and β-actin are shown as controls. (E) Western blot showing levels of p53, p21, p16 and total Rb (top band indicates Rb-P) in HGPS fibroblasts expressing TERT compared with vector controls at 2 weeks after infection and selection. β-actin levels are shown as a control. (F) Growth curves of HGPS fibroblasts expressing CDK4, DNp53, both CDK4 and DNp53, or vector alone. HGPS fibroblasts near the end of their proliferative lifespan (two remaining PDs) were transduced with retroviral vectors encoding the indicated proteins, and cumulative PDs from the time of infection and selection were calculated. In the case of CDK4+DNp53, DNp53 was added to cells expressing CDK4 at 64 days.

Fig. 2.

TERT blocks progerin-induced DNA-damage signaling. (A) Confocal immunofluorescence microscopy of γH2AX (green) and ATM-P (red) DNA-damage foci in HGPS fibroblasts expressing TERT or vector control at 2 weeks after retroviral transduction and marker selection. The merged images are shown superimposed on DAPI (blue) staining of DNA with co-localization of γH2AX and ATM-P in yellow. (B) Quantification of staining shown in A. The percentage of cells with either 0, 1, 2-5 or >5 γH2AX and ATM-P foci is shown. For each condition, at least 300 cells were counted. (C) Western blot showing levels of ATM-P in HGPS fibroblasts expressing TERT or vector control at 2 weeks after infection and selection. β-actin levels are shown as a control. (D) Western blots comparing levels of endogenous progerin in HGPS fibroblasts with ectopic progerin in NDFs. Lamin A, lamin C, and β-actin levels are shown as controls. (E) Confocal immunofluorescence microscopy of γH2AX (green) and ATM-P (red) DNA-damage foci in NDFs with or without ectopic TERT and expressing progerin or vector control immediately following lentiviral transduction and selection. The merged images are shown superimposed on DAPI (blue) staining of DNA with co-localization of γH2AX and ATM-P in yellow. (F) Quantification of staining shown in E. The percentage of cells with either 0, 1, 2-5 or >5 γH2AX and ATM-P foci is shown. For each condition, at least 300 cells were counted. (G) Time course of lifespan extension and DNA-damage reduction in HGPS fibroblasts with ectopic TERT expression. Late-passage HGPS fibroblasts were infected and selected for 2 weeks with a lentivirus expressing TERT or vector control. Immediately after selection, lifespan was measured by tracking PDs versus time. DNA-damage signaling was measured by flow-cytometry analysis for γH2AX using G1-gated cells. γH2AX positivity was measured on days 7, 17, 25 and 39. Error bars indicate s.d. for a representative experiment performed in triplicate.

Fig. 3.

Rescue of progerin-induced growth defects and DNA-damage phenotypes by TERT is specific to its function at telomeres. (A,B) TERTs catalytic and DNA-binding functions are required to rescue the HGPS premature senescence and DNA-damage phenotypes. (A) Comparison of the proliferative abilities of late-passage HGPS fibroblasts expressing wild-type TERT, DNA-binding-deficient TERT (N125A+T126A TERT), catalytically inactive TERT (D868A TERT), or vector control. Cumulative PDs of each culture were determined 28 days after infection and selection. Error bars indicate s.e.m. from three separate experiments. (B) Flow-cytometry analysis of γH2AX on G1-gated HGPS fibroblasts expressing the indicated TERT constructs at 2 weeks after infection and selection. Error bars indicate the range for a representative experiment performed in duplicate. (C) Western blot showing similar protein levels of ectopic wild-type and mutant TERT constructs in HGPS fibroblasts. β-actin was used as a loading control. (D) Effect of TERT on doxorubicin (DOX) treatment. NDFs (IMR90), either with or without ectopic TERT expression, were transduced and marker selected with progerin or vector control. Vector-control fibroblasts were either untreated or treated with 500 nM DOX for 1 hour. γH2AX positivity was measured by flow cytometry. Error bars indicate the range for a representative experiment performed in duplicate.

Fig. 4.

Progerin-induced DNA-damage signaling localizes to telomeres. (A) Telomere-dysfunction-induced foci (TIFs) were detected by confocal immunofluorescence microscopy of γH2AX (green) and TRF1 (telomere marker) (red) foci at 5 days after infection of NDFs with progerin, TRF2^ΔBΔM positive control or a vector control that was either untreated or treated with 500 nM DOX for 1 hour. The merged images are superimposed on DAPI (blue) staining showing DNA with co-localization of γH2AX and TRF1 in yellow. White boxes indicate areas that are enlarged 10× and shown to the right without DAPI. Scale bar: 10 μm. (B) Quantification of TIFs in cells from A. TIFs were determined by the co-localization of γH2AX and TRF1. The percentage of cells with DNA damage containing 0-1, 2-5, 6-10 or >10 TIFs are shown. For each condition, 100 cells were counted. (C) Additional merged images of TIFs in progerin-expressing cells, each of which show telomere aggregates. TIFs were detected as in A. White boxes indicate areas that are enlarged 10×. Scale bar: 10 μm.

Fig. 5.

Telomeric chromatin immunoprecipitation showing that progerin-induced DNA-damage signaling is enriched specifically at telomeres. (A) NDFs (IMR90) were infected and marker selected for 7 days with progerin, TRF2^ΔBΔM or vector-control viruses. Antibodies specific for γH2AX or IgG negative control were used for immunoprecipitation. DNA was loaded onto a nylon membrane using a slot blot and probed with a DIG-labeled telomere probe, stripped and re-probed with a DIG-labeled Alu probe. Input DNA represents 0.1% of total DNA. A light and a dark exposure are shown. (B) Quantification of the dark exposure shown in A. Signal density was measured by ImageJ, and histogram values represent the γH2AX telomeric or Alu ChIP signal normalized to input signal, and subtracted for background signal in the IgG control. The amount of DNA in each ChIP is expressed in arbitrary units (a.u.) after vector control sequences were normalized to 1.

Fig. 6.

Effect of progerin on the association of telomere-binding proteins with telomeric DNA. (A) NDFs (IMR90) were infected with viruses expressing progerin, TRF2^ΔBΔM, or vector control and marker selected for 7 days. For ChiP analysis, antibodies specific for TRF1, TRF2 or an IgG negative control were used for immunoprecipitation following crosslinking and DNA shearing. DNA was loaded onto a nylon membrane using a slot blot and hybridized with a DIG-labeled telomere probe. Input DNA represents 1% of total DNA. (B) Quantification of blot shown in A. Signal density was measured by ImageJ, and histogram values represent the TRF1 and TRF2 telomeric ChIP signal normalized to input signal. The amount of telomeric DNA in each ChIP is expressed in arbitrary units (a.u.) after vector control sequences were normalized to 1. (C) NDFs (IMR90) were infected with lentiviruses expressing progerin or vector control and marker selected for 5 days. For ChiP analysis, antibodies specific for TRF1, TRF2 or an IgG negative control were used for immunoprecipitation following crosslinking and DNA shearing. DNA was loaded onto a nylon membrane using a slot blot and hybridized with a DIG-labeled telomere probe, stripped and re-hybridized with a DIG-labeled Alu probe. Input DNA represents 1% of total DNA. (D) Quantification of blot in C performed as described in B.

Fig. 7.

Progerin-induced chromosomal aberrations. Telomeric FISH on metaphases derived from early passage NDFs (IMR90) exogenously expressing progerin for 5, 8 or 13 days. Telomeric PNA hybridization signals are shown in green and DAPI-counterstained chromosomes in blue. (A) Representative complete metaphase spread from NDFs (IMR90) expressing progerin. Arrow indicates the fusion of two chromosomes at the telomere, enlarged and shown at the bottom right. (B-H) Additional examples of progerin-induced chromosomal aberrations. Arrows indicate aberrations. (B) Sister-chromatid fusion. (C) Sister-telomere loss. (D) Telomere doublet. (E) Chromosomal break. (F) Extra-chromosomal telomeric signals. (G) Diplochromosome. (H) Chromatin bridge containing telomeric signals between two interphase nuclei. Scale bar: 2 μm.