Fig. 6: BRCA2 also requires PRC2 for its binding to the bivalent chromatin.
From: ZFP281-BRCA2 prevents R-loop accumulation during DNA replication
a Heat maps of the BRCA2 binding profile in WT and EED KO ES cells. Shown are ±50 kb of the center of BRCA2 peaks. b ChIP-qPCR showing that the occupancies of BRCA2 are reduced at the tested bivalent regions (labeled with red color) in EED KO ES cells, while remains unchanged at the tested active Pura and Hspd1 loci (labeled with green color). c ChIP-qPCR showing that the occupancies of ZFP281 remains unchanged after EED KO. b, c The HEMO gene (Hba2) serves as a negative control for ChIP-qPCR. Mean ± SEM from three independent experiments. Multiple t tests were performed. (b, Hba2, p = 0.3989; Hoxb13, p = 0.0006; Gata6, p = 0.0019; Meis2, p < 0.0001; Pou3f2, p = 0.0012; Vrtn, p = 0.0023; Lhx4, p = 0.0055; Pura, p = 0.1818; Hspd1, p = 0.0564. c Hba2, p = 0.1929; Hoxb13, p = 0.3279; Gata6, p = 0.1113; Meis2, p = 0.1445; Pou3f2, p = 0.0102; Vrtn, p = 0.0657; Lhx4, p = 0.2136; Hspd1, p = 0.5652.) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant. d Endogenous immunoprecipitation analyses showing the interaction of SUZ12 with ZFP281 and BRCA2. Three independent biological repeats with similar results. e RT-qPCR showing that the induction of Hoxb5 and Hoxb1 by RA is impaired in ZFP281 KO ES cells. Results shown are technical replicates from representative biological replicates. Mean ± SEM from three independent experiments. Two-tailed, unpaired Student’s t tests were performed. (Hoxb1 and Hoxb5 in NonT shRNA, DMSO vs. RA24, p < 0.0001. Hoxb1 in RA24, NonT shRNA vs. ZFP281 shRNA, p < 0.0001; NonT shRNA vs. BRCA2 shRNA, p < 0.0001. Hoxb5 in RA24, NonT shRNA vs. ZFP281 shRNA, p < 0.0001; NonT shRNA vs. BRCA2 shRNA, p = 0.0001.) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. = not significant. f Cartoon model showing that ZFP281 functions together with BRCA2 to facilitate DNA replication through preventing R-loop accumulation in the bivalent chromatin (upper panel). ZFP281 depletion impairs the binding of BRCA2 to the bivalent chromatin, leads to aberrant R-loop accumulation and subsequent replication defects (middle panel). RNase H1 overexpression in ZFP281 knockout cells is able to rescue the DNA replication defects caused by ZFP281 knockout (lower panel).
