University of Dundee BRCA 1 is a histone-H 2 A-specific ubiquitin ligase

Summary 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.

mutants were expressed from the pDest12.2 expression vector under the control of the CMV promoter. C-terminal tail mutants were generated by amplification of the H2A coding sequence using a reverse primer containing the relevant mutation(s). Sequences of the mutagenic primers are available on request. The mutant cDNA was then re-cloned back into pDest12.2 using standard molecular biology techniques. All plasmids were verified by sequencing.
Microscopy 2-6-3 cells were seeded at a density of 2.5 x 10 4 cells/cm 2 in a 12 well dish containing a coverslip. 24 hours later the cells were transfected with the relevant mCherryLacI constructs using XtermeGENE HP (Roche) according to manufacturers instructions. After 24 h cells were washed with PBS and then incubated with CKS buffer (10 mM PIPES pH7.0, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl 2 ) supplemented with 0.5% Triton-X-100 and protease inhibitors for 3 minutes at room temperature. Washed cells were fixed with 4% Paraformaldehyde for 10 minutes at room temperature, permeabilised in 0.5% Triton-X-100 in PBS for 3 x 5 minutes and blocked for 30 minutes at 37˚C in blocking buffer (1% BSA in PBS). Fixed cells were incubated with primary antibody at the relevant dilution in blocking buffer for 1 hour at 37˚C, washed with PBS and incubated with the appropriate secondary antibody, 250 µg/ml RNase A (Qiagen) and 1µM TO-PRO-3 for 30 minutes. After another wash in the dark the coverslips were mounted onto slides using ProLong GOLD (Invitrogen). Antibodies used were anti-H2Aub (Clone E6C5) (Millipore) 1:100 and FK2 (Enzo Life Science) 1:500. Appropriate AlexaFluor 488 secondary antibodies (Invitrogen) were used at 1:1000. Images were captured on a Zeiss LSM510 confocal microscope using the 40x oil immersion objective (N/A 1.3). At least 100 mCherry foci were counted for each condition.

Proteins
C-terminal tagged FLAG/12xHis (FH) full-length human tagged BRCA1 and BARD1 cDNA was cloned into pDEST8 and expressed in Sf9 using the Bacto-Bac baculovirus expression system (Invitrogen) and purified essentially as described in Mallery et al. (2002). In short, viruses with high expression levels for BRCA1-FH and BARD1-FH were co-infected in Sf9 insect cells for 48 h.

Nucleosome Aassembly
Nucleosomes were assembled using purified octamers (recombinant X. laevis or chicken) and polyglutamic acid (PGA; Sigma P4886) according to Stein et al. (Stein et al., 1979). PGA and histones were mixed in a 2:1 ratio in 150 mM NaCl and incubated for 1-2 h at room temperature. Precipitates were removed by centrifugation and the supernatant (HP-mix) was stored at 4 °C. Different ratios of 32 P-body labeled DNA (Widom 601 sequence, 161 nt) and HP-mix were empirically tested to reveal the optimal conditions for nucleosome assembly. Nucleosomes were separated by native polyacrylamide gel electrophoresis (4.5% polyacrylamide, 0.25x TBE, 5% glycerol), assembled nucleosomes were cut out and eluted from the gel.

Binding assay
Approx. 10-40 fmol of gel eluted nucleosomes were incubated for 10 min on ice with BRCA1/BARD1 full-length proteins. The sample was then loaded on a native polyacrylamide gel (4.5% polyacrylamide, 0.4x TBE, 5% glycerol) and separated by gel electrophoresis. Migration of radiolabelled nucleosomes were detected by autoradiography of the dried gel (Phosphoimager, GE Healthcare).

Mass spectrometry
H2Aub was purified from cells using Actif motif histone purification kit followed by gel purification of H2Aub after PAGE in a 12% Bis-Tris acrylamide gel using MES buffer. H2Aub was digested in gel and analyzed as described in Figure S3.     The signals matched to b11, b12, b13, b15, y3, y4, y5 and y8 ions suggest that the modification is located at either K127 or K129. In addition, the presence of y1 ion at 147.4, y2 ion at 204.2 and the absence of four y2 ions observed in E suggest that the modification site is not located at K129. K127 is therefore the likely site of modification in G. The indicated amino acid positons in E and F refer to the translated cDNA sequence including the post-translationally removed initial methionine.