CBP and p300 Histone Acetyltransferases Contribute to Homologous Recombination by Transcriptionally Activating the <em>BRCA1</em> and <em>RAD51</em> Genes

<div><p>Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the <em>BRCA1</em> and <em>RAD51</em> genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated <em>BRCA1</em> and <em>RAD51</em> at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the <em>BRCA1</em> and <em>RAD51</em> genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.</p> </div

Involvement of CBP and p300 in the transcription of the <i>BRCA1</i> and <i>RAD51</i> genes.

<p>(<b>A</b>) 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>B, C</b>) Reduction of <i>BRCA1</i> and <i>RAD51</i> 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 <i>BRCA1</i> (B) and <i>RAD51</i> (C) mRNAs. Expression levels were normalized against the levels of <i>GAPDH</i> mRNA. Data represent the mean ± SD. (<b>D</b>) 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.</p

Impairment of RPA foci formation and the DNA damage checkpoint in CBP and p300 depleted cells.

<p>(<b>A</b>) 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>B, C</b>) 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. (<b>D, E</b>) 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. (<b>F, G</b>) 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. (<b>H, I</b>) 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. (<b>J</b>) 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. (<b>K</b>) 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.</p

Histone acetylation and E2F1 binding at the <i>BRCA1</i> and <i>RAD51</i> promoter regions.

<p>(<b>A, B</b>) CBP and p300 recruitment to the <i>BRCA1</i> and <i>RAD51</i> 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 <i>BRCA1</i> (A) or <i>RAD51</i> (B) and for the detection of the non-promoter region of <i>GAPDH</i>. 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 (<i>BRCA1</i> or <i>RAD51</i> promoter and the <i>GAPDH</i> 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. (<b>C–J</b>) Impaired histone acetylation and changes in E2F1 and E2F4 binding at <i>BRCA1</i> and <i>RAD51</i> 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 <i>BRCA1</i> (C, E, G, I) and <i>RAD51</i> (D, F, H, J), and the gene body region of <i>GAPDH</i>. 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 <i>BRCA1</i> or <i>RAD51</i> promoter and the <i>GAPDH</i> gene body region in the siRNA-treated sample by the ChIP enrichment at the <i>BRCA1</i> or <i>RAD51</i> promoter in the siNT-treated sample. Data represent the mean ± SD.</p

Involvement of CBP and p300 in HR.

<p>(<b>A</b>) HR Assay design. I-<i>Sce</i>I sites are indicated by white triangle heads. The locations of the PCR primers used for quantitative PCR to monitor DSB introduction by I-<i>Sce</i>I (uncut DNA) and the subsequent repair (repaired DNA) are indicated by blue and red arrows, respectively. (B) Suppression of <i>I-Sce</i>I-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>I-Sce</i>I 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. (<b>C, D</b>) Assessment of DSB generation and DNA repair. (C) Proportion of repaired product 48 hr after transfection of the I-<i>Sce</i>I 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-<i>Sce</i>I site 24 hr after transfection of the I-<i>Sce</i>I expression plasmid. The proportion of uncut product is expressed as a ratio to the amount of uncut product present before I-<i>Sce</i>I expression plasmid transfection. Data represent the mean ± SD. (<b>E, F, G, H</b>) 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.</p

The centromeric binding of kinetochore components is partially impaired in mutants

<p><b>Copyright information:</b></p><p>Taken from "Actin-related protein Arp4 functions in kinetochore assembly"</p><p></p><p>Nucleic Acids Research 2007;35(9):3109-3117.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888834.</p><p>© 2007 The Author(s)</p> 3HA-tagged or untagged wild-type and cells were grown in YPAD at 23°C for 3 h with 15 μg/ml nocodazole to ensure that both populations had an equivalent cell cycle distribution since a higher proportion of cells are in G2/M phase. The culture was shifted to 37°C and incubated in the presence of nocodazole for 1 h. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA coimmunoprecipitated with the anti-HA antibody (IP) were amplified with primer sets corresponding to sequences around centromeres (). Quantitative data were obtained by real-time PCR. To ensure the linearity of the PCR signal, appropriate dilutions of IP samples were used in PCR amplifications. In each case, ChIP enrichment is expressed relative to that for a subtelomeric region of chromosome V (9716–9823). Results are expressed as the mean and SD of two independent ChIP experiments. Dashed lines indicate the background level of ChIP signal intensity in an untagged strain. () Schematic of kinetochore components. () The centromere-specific histone H3 variant Cse4p (wild-type cells: YHO805; cells: YHO825), a representative protein of the inner kinetochore Mif2p (wild-type cells: YHO806; cells: YHO826) and Ndc10p (wild-type cells: YHO807; cells: YHO827) were analyzed by ChIP at . () A representative protein of the outer kinetochore Mtw1p (wild-type cells: YHO808; cells: YHO828), Nuf2p (wild-type cells: YHO809; cells: YHO829) and Ctf3p (wild-type cells: YHO810; cells: YHO830) were analysed by ChIP at . () The cohesin component Scc1p (wild-type cells: YHO811; cells: YHO831) was analysed by ChIP at

Arp4p and Arp4 containing complexes associate with the centromere and telomere

<p><b>Copyright information:</b></p><p>Taken from "Actin-related protein Arp4 functions in kinetochore assembly"</p><p></p><p>Nucleic Acids Research 2007;35(9):3109-3117.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888834.</p><p>© 2007 The Author(s)</p> Cells with 13Myc-tagged Arp4 (YHO312) were grown in YPAD at 20°C for 3 h with 100 ng/ml α-factor. Cells were released by washing in YPAD and incubated in fresh YPAD medium at 20°C. Samples were taken at the time points indicated and analyzed by flow cytometry () and ChIP (). () Flow cytometry analysis of cell cycle profiles. () Input DNA and DNA coimmunoprecipitated with the anti-Myc antibody (IP) were amplified with primer sets corresponding to sequences around centromeres ( and ), the inner region of a large ORF (), a telomere (), and a sub-telomeric region (). To ensure the linearity of the PCR signal, appropriate dilutions of IP samples were used in PCR amplifications. ChIP PCR products were separated by agarose gel electrophoresis. Representative data are shown. () Arp4p, Ino80p, Esa1p and Swr1p interact with and localize to , , , but not to and a subtelomeric region. Flag-tagged Arp4 (YHO311), Ino80 (YHO313), Esa1 (YHO314), Swr1 (YHO315) or untagged (YK402) cells were arrested in G2/M by treatment with nocodazole at 30°C. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA coimmunoprecipitated with the anti-FLAG antibody (IP) were amplified with primer sets corresponding to sequences around , , , , and a subtelomeric region. The templates used were total chromatin (Input) or immunoprecipitate (IP)

Sensitivity of p53^+/+ and p53^-/- HCT116 cells to X-ray and carbon-ion beam irradiation as assessed by clonogenic survival assays.

<p>Cells were seeded in 6-well plates, incubated overnight, and then exposed to X-ray or carbon-ion beam irradiation. After incubation for a further 10 days, the cells were fixed, stained, and counted. The surviving fraction was normalized to the value of the corresponding controls. Data are expressed as the mean ± SD. C-ion, carbon-ion.</p

Mode of cell death induced by X-ray or carbon-ion beam irradiation in p53^+/+ and p53^-/- HCT116 cells.

<p>Cells seeded on glass coverslips were incubated overnight, exposed (or not; 0 h) to X-ray (4 Gy) or carbon-ion beam (1.5 Gy) irradiation, and then stained with DAPI. Apoptosis, mitotic catastrophe, and senescence were determined according to the characteristic nuclear morphologies (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115121#s2" target="_blank">Materials and methods</a>” for the definitions). (<b>a–c</b>) Representative images showing the nuclear morphology of cells undergoing apoptosis (a), mitotic catastrophe (b), or senescence (c). The images of p53^-/- cells were taken 72 h after carbon-ion beam irradiation. (<b>d, e</b>) Mode of cell death in p53^+/+ (d) and p53^-/- (e) cells at 0, 12, 24, 48, 72, 96 and 120 h after X-ray irradiation. (<b>f, g</b>) Mode of cell death in p53^+/+ (f) and p53^-/- (g) cells at 0, 12, 24, 48, 72, 96 and 120 h after carbon-ion beam irradiation. IR, irradiation; C-ion, carbon-ion.</p

Mode of cell death induced by X-ray or carbon-ion beam irradiation in isogenic H1299 cells expressing different p53 missense mutations.

<p>Cells were seeded on glass coverslips, incubated overnight, irradiated with X-rays (10.9 Gy, D₁₀ for X-rays; or 3.8 Gy, D₁₀ for carbon-ion beams) or carbon-ion beams (3.8 Gy, D₁₀ for carbon-ion beams), and then stained with DAPI 72 h later. Apoptosis, mitotic catastrophe, and senescence were determined according to the characteristic nuclear morphologies (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115121#s2" target="_blank">Materials and methods</a>” for the definitions). Data are expressed as the mean ± SD. MC, mitotic catastrophe; C-ion, carbon-ion; IR, irradiation. Note that a part of p53-null H1299 panel is the same as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115121#pone-0115121-g004" target="_blank">Fig. 4</a> (but the context is now different).</p