ING2 controls the progression of DNA replication forks to maintain genome stability

Abstract

Inhibitor of growth 2 (ING2) is a candidate tumour suppressor gene the expression of which is frequently lost in tumours. Here, we identified a new function for ING2 in the control of DNA replication and in the maintenance of genome stability. Global replication rate was markedly reduced during normal S-phase in small interfering RNA (siRNA) ING2 cells, as seen in a DNA fibre spreading experiment. Accordingly, we found that ING2 interacts with proliferating cell nuclear antigen and regulates its amount to the chromatin fraction, allowing normal replication progression and normal cell proliferation. Deregulation of DNA replication has been previously associated with genome instability. Hence, a high proportion of siRNA ING2 cells presented endoreduplication of their genome as well as an increased frequency of sister chromatid exchange. Thus, we propose for the first time that ING2 might function as a tumour suppressor gene by directly maintaining DNA integrity.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

ING2 ensures normal DNA synthesis. U2OS cells were transfected with siCT or siING2. (A) FACS analysis showing BrdU incorporation. (B) Representative immunofluorescence images of BrdU incorporation. Pictures were acquired with the same exposure time. (C) Graphic ImageJ (NIH) representation of BrdU intensity measured for immunostained cells shown in (B). (D) Number of replication foci per nucleus counted on 10 nuclei for each condition. BrdU, bromodeoxyuridine; FACS, fluorescence-activated cell sorting; ING2, inhibitor of growth 2; siCT, small interfering RNA control.

Figure 2

ING2 controls the progression of the replication fork. (A) U2OS cells transfected with siCT or siING2 were pulsed sequentially with BrdU and IdU, as shown in the experimental protocol. DNA fibres were spread onto microscopic slides (Jackson & Pombo, 1998) and immunostained with antibodies recognizing BrdU or IdU to distinguish between (i) ongoing forks, (ii) newly initiated forks, and (iii) terminated forks. (B) Representative immunofluorescence images showing IdU-positive tracks (pink) on DNA fibres (blue). White arrows indicate short IdU-positive tracks. (C) Fork length re-partition measured in micrometres (μm) using ImageJ software (NIH) for siCT (n=520) and siING2#1 (n=601). (D) Boxplots represent interquartile ranges and extreme values of fork lengths for siCT and siING2. Horizontal bar denotes the median. The P-value was measured using analysis of a variance test followed by a Mann–Whitney test. AS, asynchronous; BrdU, bromodeoxyuridine; IdU, iododeoxyuridine; ING2, inhibitor of growth 2; siCT, small interfering RNA control.

Figure 3

ING2 interacts with PCNA. (A) Western blotting analysis of the WCE or the CEF proteins in U2OS cells. Actin and histone H3 were used as loading controls. (B) Immunoprecipitation (IP) of endogenous PCNA. Actin and IgG were used as the loading control. CEF, chromatin-enriched fraction; ING2, inhibitor of growth 2; PCNA, proliferating cell nuclear antigen; WCE, whole-cell extract.

Figure 4

ING2 regulates the amount of PCNA in the chromatin fraction. (A) PCNA was immunoprecipitated in U2OS cells transfected with the truncated forms of ING2 (top panel). The proteins were detected using the indicated antibodies. MCM6 was used as a negative control of the IP. (B) GST pull-down experiment of recombinant GST–PCNA with ING2 in vitro translated constructs. Actin and IgG were used as loading controls for the Input and the IP, respectively. (C) Cells were collected from early (T0) to the end (T8 h) of S-phase. (D) The total expression of ING2 and PCNA was assessed in WCE. In parallel, the chromatin-bound proteins were detected in CEF. CEF, chromatin-enriched fraction; GST, glutathione S-transferase; ING2, inhibitor of growth 2; IP, immunoprecipitation; MCM6, mini chromosome maintenance 6; PCNA, proliferating cell nuclear antigen, WCE, whole-cell extract.

Figure 5

ING2 protects DNA integrity. Stable U2OS clones were established using miRNA. (A) Propidium iodide (left panels) and BrdU incorporation (right panels) were analysed by FACS to detect genome instability (>4C cells), endoreduplication (8C cells) and apoptosis (sub-G1 population). (B) Western blot analysis of caspase 3 and PARP in miRNA cells shown in (A). (C) Representative images of SCEs assessed in BrdU-labelled, giemsa-stained chromosome spreads from miRNA CT and miRNA ING2 cells. (D) At least 45 metaphases were counted for each condition and the number of SCE was normalized to the number of chromosomes per metaphase. The boxplots represent interquartile ranges and extreme values of SCEs per cell. The horizontal bars denote the median. The P-value was measured using an analysis of variance test followed by a Mann–Whitney U-test. (E) Western blot analysis of γH2AX and phospho-Chk1 (P-Chk1) in the CEF in siCT and siING2 cells. Histone H3 was used as a loading control. BrdU, bromodeoxyuridine; FACS, fluorescence-activated cell sorting; PARP, poly(ADP-ribose) polymerase; miRNA, microRNA; SCE, sister chromatid exchange; siCT, small interfering RNA control.