Model for the concurrent inhibition of DNA replication and histone gene expression by the G₁ checkpoint. See text for details.

IR results in p53/p21-dependent inhibition of NPAT phosphorylation. The phosphorylation state of NPAT in the indicated cells before or 24 h after irradiation (12 Gy) was analyzed as described in Materials and methods. λ-PPase: Lambda protein phosphatase. Treatment of NPAT from nonirradiated cells with Lambda protein phosphatase was used as a control to locate the migration of unphosphorylated NPAT on SDS–PAGE gels.

IR-induced dispersion of NPAT protein from histone gene loci depends on p53 and p21. HCT116 cells, p53^−/− or p21^−/− HCT116 cells were analyzed for the localization of NPAT protein by IF at 24 h after irradiation (12 Gy). The NPAT staining is shown in red and the nuclei are in blue. The percentage of cells that lost NPAT foci after irradiation is shown at the bottom. Without IR treatment, less than 4% of these cells lost NPAT foci (data not shown).

IR induces downregulation of histone gene transcription. (A) Parental HCT116 or p21^−/− HCT116 cells were treated with or without IR (12 Gy) as indicated. At 10 h after irradiation, the nuclei were harvested and nuclear run-on assays were carried out as described in Materials and methods. (B) The hybridization signals from the nuclear run-on assays, such as the one shown in panel A, were quantitated using a phosphorimager and expressed as the percentage of the nonirradiated controls.

Effects of p21 and p53 deficiency on the IR-induced downregulation of histone gene expression. p21^−/− or p53^−/− HCT cells were treated with IR (12 Gy). At the indicated times after irradiation, histone RNA levels were analyzed on Northern blots (A, C), and the cell cycle distribution was analyzed by FACS. The percentage of control is calculated as described in Figure 2D (B, D). (A, B) Data from p21^−/− cells , and (C, D) data from p53^−/− cells.

Inhibition of Cdk2 activity prevents NPAT foci formation. (A) Effect of ectopic expression of Cdk2 inhibitors on NPAT localization. U2OS cells were transfected with the indicated expression plasmids, together with a GFP-expressing plasmid to monitor the transfected cells. At 36 h after transfection, the cells were fixed and the localization of NPAT was analyzed by IF. The percentages of the transfected cells (green) that lost NPAT foci (red) are indicated. (B) Effect of Cdk2 kinase inhibitor roscovitine on NPAT localization. U2OS cells were treated with roscovitine (20 μM) or DMSO for 24 h, and then fixed and examined for the localization of NPAT (red) by IF. The percentage of cells that lost NPAT foci after treatment is indicated.

IR-induced downregulation of histone gene expression parallels inhibition of DNA synthesis. HCT116 were γ-irradiated (12 Gy) and harvested at the indicated times for the analysis of p53 and p21 protein levels and cyclin E–Cdk2 kinase activity (A), cell cycle distribution (B) and histone mRNA levels (C) as described in Figure 1. (D) Comparison of changes in S-phase population and histone mRNA levels after IR. The data from FACS analysis shown in panel B are expressed as percentage of control for S phase, and the Northern blot signals from panel C were quantitated using a phosphorimager and expressed as percentage of control for histone mRNAs. Data from nonirradiated samples (0 h) are set at 100%.

IR induces downregulation of histone gene expression. (A) Western blot analysis of p53 and p21 proteins. HCT 116 cells were untreated or irradiated with 6 Gy. At the indicated times after irradiation, the cells were harvested and the protein levels of p53 and p21 in the cell extract were analyzed. The analysis of β-tubulin was used as the loading control. (B) Analysis of cyclin E–Cdk2 kinase activity. HCT 116 cells were treated as described in panel A. The kinase activity associated with cyclin E or Cdk2 was assayed as described in Materials and methods. A Western blot of immunoprecipitated Cdk2 was used as the loading control. (C) Cell cycle profiles of cells treated with or without IR. HCT116 cells were treated as in panel A. BrdU was added to the culture medium 30 min before the cells were harvested for the FACS analysis as described in Materials and methods. (D) Analysis of histone mRNA levels. HCT116 cells were treated as described in panel A, and the mRNA levels of five subtypes of histones were analyzed on Northern blots. The signal of GAPDH mRNA was used as the loading control. The relative levels of histone mRNAs after IR, in comparison with histone mRNA levels in the nonirradiated cells, are shown on the right.

Effect of IR on NPAT localization. (A) WI38 cells were γ-irradiated (12 Gy) and the localization of NPAT protein was examined by immunofluorescence staining (IF) 24 h after the treatment. The NPAT staining is shown in red. The nuclei were stained with DAPI (blue). The cell cycle profiles of the irradiated cells and the control nonirradiated cells are depicted at the top. The percentage of cells that lost NPAT foci is shown at the bottom. (B) WI38 cells were first arrested at G₀ by serum starvation, and then stimulated to enter the cell cycle by serum addition. At 4 h after stimulation, the cells were fixed (4 h, control), left untreated (28 h, control), treated with IR (28 h, irradiated) or treated with aphidicolin (28 h, aphidicolin). After 24 h, the cells were fixed and analyzed for the localization of NPAT by IF. The cell cycle profiles of the cells are shown at the top. The percentage of cells that lost NPAT foci is shown at the bottom. (C) The NPAT and p21 protein levels in the cells treated as described in panel B were analyzed on Western blots. A Western blot of β-tubulin was used as a loading control. (D) MRC5 cells were first arrested at G₀ by serum starvation and then stimulated to enter the cell cycle with serum. At 10 and 28 h after stimulation, the association of NPAT protein with the histone H4/e promoter in the cells was determined by using ChIP assays as described previously (Zhao et al, 2000). PCR products from total DNA (0.5%) were used as the input controls.

Induction of p21 disperses NPAT protein from histone gene loci and inhibits histone gene expression. (A) Effect of p21 induction on NPAT localization. EGFP–p21 H1299 cells were treated with or without doxycycline (2 μg/ml) for 24 h. The expression of GFP–p21 was examined by the green fluorescence of the fusion protein (top panels) and the localization of NPAT was analyzed by IF (bottom panels). NPAT staining is shown in red and the nuclei are in blue. (B) Analysis of GFP–p21 and NPAT protein levels before and after induction. EGFP–p21 cells were treated with doxycycline for the indicated times, and the protein levels of GFP–p21 and NPAT were examined on Western blots using antibodies specific for GFP and NPAT, respectively. As a loading control, a Western blot of β-tubulin is also shown. (C) Cell cycle analysis following GFP–p21 induction. The cell cycle distribution of the EGFP–p21 cells at the indicated times following addition of doxycycline was analyzed by FACS. (D) Analysis of histone gene expression after GFP–p21 induction. The levels of histone mRNA in EGFP–p21 cells at the indicated times after GFP–p21 induction were examined on Northern blots. (E) Induction of p21 causes parallel loss of NPAT foci, downregulation of histone gene expression and the decrease of the S-phase population. EGFP–p21 cells were examined by IF at the indicated time following p21 induction to assess the presence or absence of the NPAT foci. The uninduced EGFP–p21 cells typically contain 6–9 NPAT foci, and the cells that contain more than three detectable NPAT foci were scored as NPAT-foci-positive cells. The percentage of cells that contain NPAT foci was compared with that of cells in S phase, as well as with that of histone mRNA levels, at the indicated time after p21 induction. The levels of uninduced cells were set at 100%. The FACS data from panel C and quantitation of hybridization signals from panel D by a phosphorimager were used for panel E.