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. 2022 Jun 17;13:3493. doi: 10.1038/s41467-022-31211-9

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Pie chart showing the percentage of BRCA2 peaks overlapping with a transcription start site (TSS), residing within a gene (inside), or upstream or downstream of the nearest gene. b G/C rich sequence is overrepresented in BRCA2 peaks. Statistical significance of the over-representation and the percentage of the G/C rich sequence in BRCA2 peaks are shown. p value from hypergeometric test. c Average occupancy plots of ZFP281, BRCA2, H3K4me1 and H3K4me3 at the BRCA2 and ZFP281 co-bound regions (left panel) and the BRCA2 only regions (right panel) in mouse ES cells. Shown are ±5 kb of the center of BRCA2 peaks. d Heat maps of the binding profiles in mouse ES cells for BRCA2, ZFP281, H3K4me3, and H3K27me3 are shown at the BRCA2 and ZFP281 co-bound regions, which were partitioned into two groups: the H3K4me3 and H3K27me3 co-bound (bivalent) group, and the H3K4me3 only (active) group. Shown are ±50 kb of the center of BRCA2 peaks. e BRCA2 occupancy log2 fold change after ZFP281 KO was measured at the BRCA2 and ZFP281 co-bound regions. Shown are ±50 kb around the center of BRCA2 peaks. f, g DRIP-qPCR showing that the levels of R-loop are reduced at the tested bivalent regions (labeled with red color) after ZFP281 knockout (f) or AID mediated BRCA2 knockdown (g), while remains unchanged at the tested active Pura and Hspd1 loci (labeled with green color). DRIP-qPCR analyses in samples pre-treated with RNase H were used as negative controls. Mean ± SEM from three independent experiments. Multiple t tests were performed. (f, Gata6, p = 0.0009; Kctd1, p = 0.0006; Pou3f2, p = 0.0092; Lhx4, p = 0.0069; Meis2, p = 0.0170; Hoxa1, p = 0.0051; Pura, p = 0.1255; Hspd1, p = 0.5828. g Gata6, p < 0.001; Kctd1, p = 0.0002; Pou3f2, p = 0.0003; Lhx4, p = 0.0012; Meis2, p = 0.0002; Hoxa1, p = 0.0005; Hspd1, p = 0.0611.) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant. h Reciprocal immunoprecipitation analyses showing the interaction between ZFP281 and DDX5. i Immunoprecipitation analyses showing that the interaction between BRCA2 and DDX5 was compromised after ZFP281 knockout. The experiments in h and i were performed three times show similar results.