Figure 1.
Involvement of CBP and p300 in HR.
(A) HR Assay design. I-SceI sites are indicated by white triangle heads. The locations of the PCR primers used for quantitative PCR to monitor DSB introduction by I-SceI (uncut DNA) and the subsequent repair (repaired DNA) are indicated by blue and red arrows, respectively. (B) Suppression of I-SceI-induced HR upon CBP and p300 depletion. HeLa DR-GFP cells, after transfection for 48 hr with non-targeting (siNT), CBP (siCBP), p300 (sip300), CBP+p300 (siC+p), BRCA1 (siBRCA1), or BRCA2 (siBRCA2) siRNAs, were transfected with an I-SceI expression plasmid or mock-transfected. Forty-eight hours after transfection, cells were harvested and assayed for GFP expression by flow cytometry. Data represent the mean ± SD. (C, D) Assessment of DSB generation and DNA repair. (C) Proportion of repaired product 48 hr after transfection of the I-SceI expression plasmid. The proportion of repaired product detected after targeting siRNA treatment is expressed as a ratio to that detected after non-targeting siRNA (siNT) treatment. (D) Proportion of uncut product at the I-SceI site 24 hr after transfection of the I-SceI expression plasmid. The proportion of uncut product is expressed as a ratio to the amount of uncut product present before I-SceI expression plasmid transfection. Data represent the mean ± SD. (E, F, G, H) Suppression of CPT-induced foci formation upon depletion of CBP and/or p300. HeLa or H1299 cells were transfected with non-targeting (siNT), CBP (siCBP), p300 (sip300), or CBP+p300 (siC+p) siRNAs for 48 hr. Cells were then treated with DMSO (NT) or 1 μM camptothecin (CPT), fixed and processed for immunofluorescence detection of RAD51 (E, H1299; F, HeLa), γH2AX (G, HeLa), or 53BP1 (H, HeLa). Data represent the mean ± SD.
Figure 2.
Involvement of CBP and p300 in the transcription of the BRCA1 and RAD51 genes.
(A) Down-regulation of BRCA1 and RAD51 proteins upon depletion of CBP and/or p300. H1299 cells were transfected for 48 hr with non-targeting (siNT), CBP (siCBP), p300 (sip300), or CBP+p300 (siC+p) siRNAs. The cells were harvested and whole cell extracts were subjected to immunoblotting. (B, C) Reduction of BRCA1 and RAD51 transcripts in CBP- and p300-depleted cells. H1299 cells were transfected for 48 hr with non-targeting (siNT), CBP (siCBP), p300 (sip300), or CBP+p300 (siC+p) siRNAs. Cells were harvested and subjected to quantitative real-time PCR for the detection of BRCA1 (B) and RAD51 (C) mRNAs. Expression levels were normalized against the levels of GAPDH mRNA. Data represent the mean ± SD. (D) H1299 cells were transfected for 48 hr with non-targeting (siNT), CBP (siCBP), p300 (sip300), or CBP+p300 (siC+p) siRNAs, and stained with propidium iodide (PI). The percentage of cells in each cell cycle phase was determined by FACS. Percentages of cells in G1, S, and G2/M are shown.
Figure 3.
Histone acetylation and E2F1 binding at the BRCA1 and RAD51 promoter regions.
(A, B) CBP and p300 recruitment to the BRCA1 and RAD51 promoter regions. H1299 cells were subjected to ChIP using either control IgG or antibodies against CBP and p300. Immunoprecipitated DNAs were subjected to quantitative PCR for the detection of the promoter region of BRCA1 (A) or RAD51 (B) and for the detection of the non-promoter region of GAPDH. Normalized ChIP enrichment was calculated to show the degree of enrichment following pulldown with anti-p300 and anti-CBP antibodies. First, ChIP enrichment at the test sites (BRCA1 or RAD51 promoter and the GAPDH gene body) was calculated as the fraction of total input DNA that was pulled down by the specific antibody or the non-specific control IgG. Then, normalized ChIP enrichment at the test sites was calculated by dividing the ChIP enrichment of the specific antibody by that of the non-specific control IgG. Data represent mean values ± SD. (C–J) Impaired histone acetylation and changes in E2F1 and E2F4 binding at BRCA1 and RAD51 promoter regions caused by knockdown of CBP and p300. H1299 cells pre-transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNAs were subjected to ChIP assays. DNA immunoprecipitated with anti-acetylated H3 K18 (H3 AceK18) (C, D), anti-acetylated H4 (H4 AceK5/8/12/16) (E, F), anti-E2F1 (G, H), or anti-E2F4 (I, J) antibodies was subjected to quantitative PCR to detect the promoter regions of BRCA1 (C, E, G, I) and RAD51 (D, F, H, J), and the gene body region of GAPDH. Relative ChIP enrichment was calculated to show the decrease/increase in enrichment at these sites following gene knockdown. The relative ChIP enrichment was calculated by dividing the ChIP enrichment at the BRCA1 or RAD51 promoter and the GAPDH gene body region in the siRNA-treated sample by the ChIP enrichment at the BRCA1 or RAD51 promoter in the siNT-treated sample. Data represent the mean ± SD.
Figure 4.
Impairment of RPA foci formation and the DNA damage checkpoint in CBP and p300 depleted cells.
(A) Effects of CBP and p300 depletion on DSB-induced RPA and CHK1 activation. H1299 cells were pre-transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, and treated with DMSO (NT), 1 μM camptothecin (CPT), or 500 ng/ml neocarzinostatin (NCS) for 2 hr. Whole cell extracts were subjected to immunoblot analysis. (B, C) Decreased DSB-induced chromatin binding of RPA proteins. Chromatin enriched fractions from H1299 cells pre-transfected for 48 hr with non-targeting (siNT), BRCA1 (B), or CBP and p300 (siC+p) (C) siRNAs and treated with DMSO (NT) or 1 μM camptothecin (CPT) for 2 hr were subjected to immunoblot analysis. (D, E) Decreased DNA damage-induced chromatin binding of RPA proteins in H1299 cells. Chromatin enriched fractions from cells pre-transfected for 48 hr with non-targeting (siNT) or CBP+p300 (siC+p) siRNA were treated or not treated (NT) with (D) 500 ng/ml neocarzinostatin (NCS) for 1 or 2 hr, or (E) incubated for 3 hr after 50 Gy ionizing irradiation (IR) and subjected to immunoblot analysis. (F, G) Decreased DSB-induced RPA32 foci formation. H1299 cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with DMSO (NT) or 1 μM camptothecin (CPT) for 3 hr, and subjected to immunofluorescence analysis. The percentages of cells with RPA32 foci (F) or phosphorylated RPA32 (pS4/S8) foci (G) are shown. Data represent the mean ± SD. (H, I) CPT- and IR-induced formation of BRCA1 foci. H1299 cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with 1 μM camptothecin (CPT) for 3 hr (H) or 10 Gy ionizing irradiation (IR) for 4 hr (I), and subjected to immunofluorescence analysis. The percentages of cells showing >10 BRCA1 foci are shown. Data represent the mean ± SD. (J) Suppression of the G2/M DNA damage checkpoint. H1299 cells were pre-transfected for with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. The cells were then irradiated (IR, 3 Gy) for 1 hr or not irradiated (No IR), and stained with anti-phospho-histone H3 antibody and propidium iodide (PI). The percentage of mitotic cells (mitotic index) was determined by flow cytometry. Data represent the mean ± SD. (K) Colony formation of H1299 cells treated with CPT. H1299 cells were pre-transfected for with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. siRNA-treated cells were replated and incubated for 10 days. Survival is expressed as a percentage representing the number of colonies in treated samples relative to the number in DMSO-treated samples.