Figure 3.

BRCA1 regulates transcription initiation through a histone intermediate. (A) 2.5 ng/μl pActin was incubated in mock- or BRCA1-depleted extract with increasing amounts of carrier plasmid. After 120 min, RNA was quantified by RT-qPCR (n = 2). For comparison, values were normalized to ΔMock reactions at each concentration. (B) pActin was incubated in mock- or BRCA1-depleted extract. Samples were withdrawn after 60 min and analyzed by ChIP with histone H3 antibodies (n = 3). (C) pActin was incubated in extract supplemented with buffer or 200 nM BRCA1-BARD1. Samples were withdrawn after 60 min and analyzed by ChIP with histone H3 antibodies (n = 3). (D, E) TBP- and RNAPII-ChIPs were performed as in (B) (n = 3). (F–H) Sperm chromatin was incubated in mock- or BRCA1-depleted extract. At the indicated time points, samples were visualized by phase contrast light microscopy (F). Chromatin volume (G) and density (H) were calculated at 150 min (n ≥ 12).