Fig 2: ISG induction in HR-deficient human cells and tumors and olaparib impact on this process. a Upregulation of innate immune response genes in BRCA2-deleted ovarian tumors (n = 4) versus tumors with median BRCA2 mRNA expression (n = 145). b Upregulation of innate immune response genes in RAD51-deleted ovarian tumors (n = 11) versus tumors with median RAD51 mRNA expression (n = 145). Dots in graphs represent individual tumors. Middle line (white), median; box limits 25 and 75 percentiles; whiskers, minimum and maximum values. c H1299 cells expressing a DOX-inducible BRCA2 shRNA were grown in the presence or absence of DOX for 4 days. Then, olaparib (1 or 10 µM) was added for 72 h, followed by quantitative RT-PCR analyses. Primers specific for the indicated genes were used. mRNA levels were expressed relative to the gene encoding ß-actin and to untreated (-DOX) control cells (2-??CT). Error bars represent SD of n = 3 independent experiments. *, p < 0.05 (unpaired two-tailed t test). d Whole-cell extracts from cells treated as in (c) were immunoblotted as shown. GAPDH and SMC1 were used as loading controls. Phosphorylation sites are indicated in red. e Cells treated as in (c) were pulse-labeled with EdU for 30 min. Frequency of cells in G1, S, and G2/M stages of the cell cycle were determined using FACS analyses of EdU incorporation and propidium iodide staining. Error bars represent SD of n = 3 independent experiments

Fig 3: Tissue-specific enhancer relies on cohesin to create self-interacting domains.4C-seq contact profile overlays comparing contacts of the integrated GFP reporter gene promoter (P), µLCR enhancer (E) and 3x-hCTCF (CBS), in the indicated (E100, EC100 and E407) cell lines treated with a control sgRNA (blue) versus a RAD21 or SMC1A sgRNA (causing partial depletion, in pink). Shared contacts are in light gray. Y-axis: 4C coverage normalized to 1 million cis-reads.

Fig 4: Cohesin distribution in human genome.(A) Cohesin binding at active genes in normal human lymphoblastoid cells represented as percentage of sites detected at promoter, downstream, 5'UTR, 3'UTR and coding exons. Cohesin peaks were aligned to RefSeq gene annotations by the use of CEAS tool. We compared binding of cohesin to a selected region to the average genome-wide binding (B) Genomic binding of SMC1A cohesin subunit at a selected region of chromosome 6 as determined by ChIP-sequencing. The Pol II Ser 2P binding profile and the RNA-seq data are also shown. This region was chosen to illustrate typical features of cohesin and Pol II binding patterns throughout the human genome.

Fig 5: Analysis of SMC1A occupancy at the promoter regions.(A) Cohesin binding increases in the CdLS cell lines compared to control cell lines at the promoter of downregulated genes and (B) upregulated genes. I: genes virtually active in all cell types, II: genes preferentially expressed in lymphoblastoid cells, and III: our best targets based on ChIP-Seq enrichment. Since no difference was found, control cell line data were pooled. IgG was used as negative control. Results represent three independent ChIP assays and the average values of the experiments and the relative standard errors are shown. *p < 0.05.