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. 2021 Jun 10;12:3542. doi: 10.1038/s41467-021-23716-6

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2021

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Fig. 1 — a Interaction of BRCA1 with the shelterin complex in HeLa cells. The chromatin fraction (CF) was IPd with BRCA1 antibody, and the immune complex was analyzed by immunoblotting. n = 5 independent experiments. b BRCA1 ChIP analyses of telomeric regions performed in synchronized T98G cells. Each dot represents a biological replicate (n = 3), and the median values are connected by a line, ss - serum-starved. c A Venn diagram revealing an overlap between proteins (n = 90) found in the endogenous pulldowns (CF) of BRCA1 and TRF2. d A pulldown revealing interaction of endogenous XRN2 with BRCA1 and the shelterin complex, using XRN2 antibody from CFs of HME cells. n = 4 independent experiments. e R-loop-dependent interactions between BRCA1, XRN2, and the shelterin proteins. CFs from ±RNAse H1-treated HeLa cells were used for IPs of endogenous BRCA1, TRF2, TRF1, and POT1. n = 5 independent experiments. f Schematic of CpG-island promoters and telomeric regions. Arrows indicate positions of primers used for ChIP, DRIP, and qRT-PCR. g BRCA1 ChIP analyses of promoter and telomeric regions performed in ±RNAse H1-treated U2OS cells. Bars represent the average value from n = 3 biological replicates for each sample, ±SD. P values were obtained using a two-tailed Student’s t test. h DRIP analyses of promoter-telomeric regions performed in siCtrl and BRCA1-depleted U2OS cells. Bars represent the average value from n = 3 biological replicates for BRCA1-depleted sample over siCtrl, set at 1, ±SD. P values were calculated using a two-tailed Student’s t test. i Immunoblot of CRISPR-modified HME cells confirming efficient transfection with V5-tagged RNAse H1-encoding vector. n = 3 independent experiments. j Representative images of cytospun HME CRISPR metaphase spreads in i, which were stained with an antibody for γH2AX and for telomeric DNA. Cells were mock- or RNAse H1-treated prior to the assay. Chromosome ends displaying γH2AX signals are identified by arrows. n = 3 independent experiments. k Mean percentage of CRISPR HME BRCA1^+/+, BRCA1^+/−, and BRCA1^−/− cells with ≥5 positive γH2AX-telomere DNA colocalizations and the effects of RNAse H1 treatment from three independent experiments. At least n = 30 chromosome spreads scored/experiment, ±SD. P values were obtained using a two-tailed Student’s t test. Source data are provided as a Source data file.