Identification of universal and cell-type specific p53 DNA binding

Abstract Background The tumor suppressor p53 is a major regulator of the DNA damage response and has been suggested to selectively bind and activate cell-type specific gene expression programs. However recent studies and meta-analyses of genomic data propose largely uniform, and condition independent p53 binding and thus question the selective and cell-type dependent function of p53. Results To systematically assess the cell-type specificity of p53, we measured its association with DNA in 12 p53 wild-type cancer cell lines, from a range of epithelial linages, in response to ionizing radiation. We found that the majority of bound sites were occupied across all cell lines, however we also identified a subset of binding sites that were specific to one or a few cell lines. Unlike the shared p53-bound genome, which was not dependent on chromatin accessibility, the association of p53 with these atypical binding sites was well explained by chromatin accessibility and could be modulated by forcing cell state changes such as the epithelial-to-mesenchymal transition. Conclusions Our study reconciles previous conflicting views in the p53 field, by demonstrating that although the majority of p53 DNA binding is conserved across cell types, there is a small set of cell line specific binding sites that depend on cell state. </jats:sec

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1(ATR)/Tel1(ATM)-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments

Additional file 4 of Identification of universal and cell-type specific p53 DNA binding

Additional file 4: Figure S1. Comparison of p53 DNA binding across cell lines and published p53 ChIP-seq datasets. Heatmap showing p53 binding intensity in 8742 locations in the genome in 12 IR treated cell lines (our data, same as Fig. 1c) as well as published datasets (listed in Methods and Table S1). Figure S2. Comparison of p53 levels between IR and Nutlin3A treatments. p53 levels were detected by western blot in MCF7 and UACC257 cells. Cells were either untreated, treated with 5 μM Nutlin3A or 4Gy IR for 2 h. Figure S3. Reproducibility of in vitro measurements of p53 DNA binding and comparison with in vivo binding. (A) Quantitative agreement in binding strength at p53 binding sites between two replicate p53 in vitro IP datasets using different p53 protein preps. (B) UCSC browser shots of three key binding sites for p53 showing agreement between in vitro binding datasets and divergence with in vivo data. Figure S4. Basal gene expression, but not DNA damage induced gene expression, correlates with cell-type specific p53 DNA binding. (A) Boxplots showing the distribution of Pearson’s correlation coefficients of either basal or DNA damage induced fold change of gene expression with p53 binding at the p53 target or variable p53 binding gene sets. (B) Box plots of the fold change of three canonical p53 target genes 3 h after IR. Each dot represents a cell line. CDKN1A/p21 is induced in all cell lines, while MDM2 and BBC3/Puma are cell line dependent. Figure S5. p53 binds to IL1A and IL1B in mesenchymal cell lines. UCSC browser screen shot of the p53 ChIP signal. In A549 cells, p53 binds in the proximity of IL1A/IL1B only after TGFβ treatment. Binding of p53 in this region can also be observed in another mesenchymal, CAL51, but not epithelial, HCT116, cell line. Figure S6. Knockdown of p53 in LOXIMVI cells reduces expression of inflammatory genes. Expression of p53, IL1A, IL1B, and CXCL1 by qPCR in cells treated with p53 siRNA compared to control siRNA (N = 4)

Additional file 2 of Identification of universal and cell-type specific p53 DNA binding

Additional file 2: Table S2. Table of 8742 p53 ChIP peaks reported in this study. Table includes chromosome number, peak start and end coordinates, whether the peak was variably bound across 12 cell lines in this study (494 peaks marked by ‘1’ in the ‘variable peak’ column), the nearest associated gene to each peak as well as the normalized peak intensities for each cell line in this study and in published datasets (Additional file 4: Figure S1, Additional file 1: Table S1)

Additional file 1 of Identification of universal and cell-type specific p53 DNA binding

Additional file 1: Table S1. p53 ChIP data generated in this study and the public datasets used (in Additional file 4: Figure S1). For each cell line and condition, we also individually called peaks and report the number of peaks identified in each condition

Additional file 4 of Identification of universal and cell-type specific p53 DNA binding

Additional file 4: Figure S1. Comparison of p53 DNA binding across cell lines and published p53 ChIP-seq datasets. Heatmap showing p53 binding intensity in 8742 locations in the genome in 12 IR treated cell lines (our data, same as Fig. 1c) as well as published datasets (listed in Methods and Table S1). Figure S2. Comparison of p53 levels between IR and Nutlin3A treatments. p53 levels were detected by western blot in MCF7 and UACC257 cells. Cells were either untreated, treated with 5 μM Nutlin3A or 4Gy IR for 2 h. Figure S3. Reproducibility of in vitro measurements of p53 DNA binding and comparison with in vivo binding. (A) Quantitative agreement in binding strength at p53 binding sites between two replicate p53 in vitro IP datasets using different p53 protein preps. (B) UCSC browser shots of three key binding sites for p53 showing agreement between in vitro binding datasets and divergence with in vivo data. Figure S4. Basal gene expression, but not DNA damage induced gene expression, correlates with cell-type specific p53 DNA binding. (A) Boxplots showing the distribution of Pearson’s correlation coefficients of either basal or DNA damage induced fold change of gene expression with p53 binding at the p53 target or variable p53 binding gene sets. (B) Box plots of the fold change of three canonical p53 target genes 3 h after IR. Each dot represents a cell line. CDKN1A/p21 is induced in all cell lines, while MDM2 and BBC3/Puma are cell line dependent. Figure S5. p53 binds to IL1A and IL1B in mesenchymal cell lines. UCSC browser screen shot of the p53 ChIP signal. In A549 cells, p53 binds in the proximity of IL1A/IL1B only after TGFβ treatment. Binding of p53 in this region can also be observed in another mesenchymal, CAL51, but not epithelial, HCT116, cell line. Figure S6. Knockdown of p53 in LOXIMVI cells reduces expression of inflammatory genes. Expression of p53, IL1A, IL1B, and CXCL1 by qPCR in cells treated with p53 siRNA compared to control siRNA (N = 4)