Barrows JK et al. (2021), BRCA1-BARD1 regulates transcription through BRD...

XB-ART-57897

Nucleic Acids Res 2021 Apr 06;496:3263-3273. doi: 10.1093/nar/gkab111.

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BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract.

Barrows JK , Fullbright G , Long DT .

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The tumor suppressor BRCA1 is considered a master regulator of genome integrity. Although widely recognized for its DNA repair functions, BRCA1 has also been implicated in various mechanisms of chromatin remodeling and transcription regulation. However, the precise role that BRCA1 plays in these processes has been difficult to establish due to the widespread consequences of its cellular dysfunction. Here, we use nucleoplasmic extract derived from the eggs of Xenopus laevis to investigate the role of BRCA1 in a cell-free transcription system. We report that BRCA1-BARD1 suppresses transcription initiation independent of DNA damage signaling and its established role in histone H2A ubiquitination. BRCA1-BARD1 acts through a histone intermediate, altering acetylation of histone H4K8 and recruitment of the chromatin reader and oncogene regulator BRD4. Together, these results establish a functional relationship between an established (BRCA1) and emerging (BRD4) regulator of genome integrity.

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Species referenced: Xenopus laevis
Genes referenced: bard1 brca1 brd4 h2ac21 tbp

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	Graphical Abstract
	Figure 1. BRCA1-BARD1 is necessary and sufficient to suppress transcription in NPE. (A) Different amounts of mock-depleted (δMock) or BRCA1-depleted (δBRCA1) NPE was analyzed by western blot with the indicated antibodies. (B) pActin was incubated in mock- or BRCA1-depleted extract and RNA was quantified by RT-qPCR over time (n = 3). (C) Purified BRCA1-BARD1 (WT) and BRCA1I26A-BARD1 (I26A; used in Figure 4E) were resolved by SDS-PAGE and visualized by silver stain. (D) pActin was incubated in mock-depleted extract, BRCA1-depleted extract, or BRCA1-depleted extract supplemented with recombinant 200 nM BRCA1-BARD1. After 120 min, RNA was quantified by RT-qPCR (n = 2) (E) pActin was incubated in NPE supplemented with increasing amounts of BRCA1-BARD1. After 120 min, RNA was quantified by RT-qPCR (n = 3).
	Figure 2. BRCA1-BARD1 is necessary and sufficient to suppress transcription in NPE. (A) Different amounts of mock-depleted (ΔMock) or BRCA1-depleted (ΔBRCA1) NPE was analyzed by western blot with the indicated antibodies. (B) pActin was incubated in mock- or BRCA1-depleted extract and RNA was quantified by RT-qPCR over time (n = 3). (C) Purified BRCA1-BARD1 (WT) and BRCA1I26A-BARD1 (I26A; used in Figure 4E) were resolved by SDS-PAGE and visualized by silver stain. (D) pActin was incubated in mock-depleted extract, BRCA1-depleted extract, or BRCA1-depleted extract supplemented with recombinant 200 nM BRCA1-BARD1. After 120 min, RNA was quantified by RT-qPCR (n = 2) (E) pActin was incubated in NPE supplemented with increasing amounts of BRCA1-BARD1. After 120 min, RNA was quantified by RT-qPCR (n = 3).
	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).
	Figure 4. Ubiquitination is dispensable for BRCA1-mediated transcription suppression. (A) pActin was incubated in mock- or BRCA1-depleted extract. Samples were withdrawn after 60 min and analyzed by ChIP with histone H2A-Ub or H3 antibodies. Recovery of H2A-Ub over H3 is graphed (n = 2). (B) pActin was incubated in extract supplemented with buffer or 200 nM BRCA1-BARD1. Samples were analyzed by ChIP as in (A) (n = 2). (C, D) pActin was incubated in extract pre-treated with buffer or UbVS and then supplemented with buffer or 200 nM BRCA1-BARD1. Samples were withdrawn after 60 min and analyzed by western blot (C), or 120 min and RNA was quantified by RT-qPCR (D) (n = 2). (E) pActin was incubated in NPE supplemented with increasing amounts of BRCA1I26A-BARD1. After 120 min, RNA was quantified by RT-qPCR (n = 3). Results from Figure 1E are also shown for comparison.
	Figure 5. BRCA1-BARD1 regulates DNA-binding of multiple transcription regulators. (A) pActin was incubated in extract that was mock-depleted, BRCA1-depleted, or supplemented with 200 nM BRCA1-BARD1. DNA-bound proteins were isolated by plasmid pull-down and analyzed by mass spectrometry. Results were normalized to ΔMock samples and ranked by enrichment of ΔBRCA1 over +BRCA1-BARD1 samples. Representative complex members are labeled. An extended graph showing distribution of ranked proteins is shown in Supplementary Figure S5. (B) Schematic showing the relationship between BRCA1, BRD4, and complexes identified in (A).
	Figure 6. BRCA1-BARD1 suppresses H4K8 acetylation and BRD4 binding. (A) pActin was incubated in mock- or BRCA1-depleted extract. At the indicated time points, DNA-bound proteins were isolated by plasmid pull-down and analyzed by western blot. Total recovery of plasmid DNA from mock- and BRCA1-depleted extracts is shown in Supplementary Figure S6. (B) pActin was incubated in extract supplemented with buffer or 200 nM BRCA1-BARD1 and DNA-bound proteins were analyzed as in (A). (C) pActin was incubated in extract supplemented with buffer or JQ1 (BETi). At the indicated time points, DNA-bound proteins were analyzed as in (A). (D) pActin was incubated in extract with increasing amounts of JQ1. After 120 min, RNA was quantified by RT-qPCR (n = 3). (E) pActin was incubated in NPE supplemented with buffer or JQ1. At the indicated time points, RNA was quantified by RT-qPCR. (F) pActin was incubated in mock- or BRCA1-depleted extract. Samples were withdrawn after 20 min and analyzed by ChIP with the indicated antibodies (n = 2). (G) pActin was incubated in extract supplemented with buffer or JQ1. Samples were withdrawn after 20 min and analyzed by ChIP with the indicated antibodies (n = 2).

References [+] :

Amodeo, Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition. 2015, Pubmed, Xenbase