BRCA1 is a histone-H2A-specific ubiquitin ligase

Abstract

The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3) ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.

Graphical abstract

Figure 1

BRCA1/BARD1 Specifically Ubiquitylates Histone H2A in Nucleosomes (A and B) Ubiquitylation of recombinant Xenopus laevis histones (H2A, H2B, H3, and H4), reconstituted nucleosomes, and chromatin isolated from HeLa cells by RING1B/MEL18 (A) or BRCA1/BARD1 (B) ubiquitin ligases. ¹²⁵I-labeled ubiquitin is covalently linked to its substrate and detected after SDS-PAGE. Specificity for a single histone occurs only within a nucleosomal context. Small amounts of diubiquitylated histone were observed as indicated (^∗). See also Figure S1.

Figure 2

Ubiquitylation of Histone H2A by BRCA1/BARD1 E3 Activity In Vivo (A and C) Site-specific recruitment of the BRCA1/BARD1 E3 to a single genomic location in HEK293 2-6-3 cells results in ubiquitylation of histone H2A. U2OS 2-6-3 cells containing 200 tandem copies of a 256 LacO sequence integrated at a specific site were transiently transfected with plasmids expressing mCherryLacI, BDfBC-mCherryLacI, or mutant BDfBC-I26A-mCherryLacI. (A and B) Cells were stained with antibodies against H2Aub (E6C5; A) and scored for colocalization with mCherry (B). (C and D) Cells were stained with antibodies against ubiquitin (FK2; C) and scored for colocalization with mCherry (D). Representative images are shown. Values represent the mean from two independent experiments (n = 100). Error bars represent 1 SD. Scale bar represents 10 μm. See also Figure S2.

Figure 3

BRCA1/BARD1 Ubiquitylates K127-129 of Histone H2A In Vitro and In Vivo (A) Ubiquitylation of nucleosomes by RING1B/MEL18 is impaired by mutation of lysines 118 and 119 of histone H2A. (B) Ubiquitylation of nucleosomes by BRCA1/BARD1 is reduced for histone H2A mutated at lysines K124, K127, and K129. Small amounts of diubiquitylated histone were observed as indicated (^∗). (C) Ubiquitylation of recombinant Xenopus nucleosomes by BRCA1/BARD1 and RING1B/MEL18, showing the difference in migration of the H2Aub product. (D) FLAG-H2A was stably expressed in DT40 cells and immunoprecipitated under denaturing conditions as described previously (Wang et al., 2004). Cells expressing wild-type or mutant H2A are indicated. Immunoprecipitated proteins were analyzed by western blot and probed with antibodies against FLAG, ubiquitin (FK2), and ubiquityl-H2A (E6C5). (E) DT40 cell lines expressing wild-type or mutant FLAG-H2A (K118/119R, K124/127/129, and K118/119/124/127/129) were transfected with the RING domains of BRCA1 and BARD1. Cells were harvested after 48 hr and histone proteins were isolated by acid extraction. FLAG-H2Aub was detected by western blot with anti-FLAG after separation by PAGE. H2Aub was detected after expression of the BRCA1 and BARD1 RING domains (band 1) and/or by endogenous E3 activity (band 2). (F) HEK293 cells expressing BDfBC-EGFP-NLS protein under the control of a Tet-responsive promoter was induced overnight with 1 μg/ml doxycycline. Nuclei were prepared from the cells and acid-extracted histones were separated on a 12% BisTris Novex gel and probed with the indicated antibodies. Increased H2Aub dependent on expression of BDfBC is indicated for two independent clones (#8 and #1). (G) Top, BRCA1/BARD1 ubiquitylates chicken histone H2A at lysine 126 (equivalent to K127 in humans) in nucleosomes. The modified peptide fragments identified by mass spectrometry are indicated. Data supporting this modification are provided in Figure S3E. Bottom, BDfBC expressed in HEK293 cells ubiquitylates human histone H2A at lysine K127-129. H2Aub was recovered from chromatin as described in the Experimental Procedures. The modified peptide fragments identified by mass spectrometry are indicated in the illustration. Data supporting this modification are provided in Figure S3F.