1. The Histone Variant MacroH2A1 Is a BRCA1 Ubiquitin Ligase Substrate
Beom-Jun Kim, Doug W. Chan, Sung Yun Jung, Yue Chen, Jun Qin, Yi Wang 
Cell Reports 
Volume 19, Issue 9, Pages (May 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 1758-1766DOI: (10.1016/j.celrep.2017.05.027)
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3. Figure 1
Sequential qUBA/diGly Dual-Affinity Purification Coupled with MS Identifies BRCA1/BARD1 E3 Ubiquitination Substrates
(A) Ubiquitinated proteins from pcDNA3- or BRCA1/BARD1-transfected 293T cells were enriched by GST-qUBA-bound beads and digested with trypsin; GG peptides from Ub-conjugated proteins were immunoprecipitated with an anti-diGly antibody and analyzed by MS. LC-MS/MS, liquid chromatography-tandem MS.
(B) BRCA1/BARD1-dependent ubiquitinated peptides of DNA-damage response proteins and transcription factors.
See also Figure S1 and Tables S1, S2, and S3.
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4. Figure 2 BRCA1/BARD1 Ubiquitinates Histone mH2A In Vivo and In Vitro
(A) Sequence alignment of mH2A1 isoforms and of histone H2A. K115/116 and K121/122, which aligned with canonical H2A ubiquitination sites K118/119 and K127/129 sites, respectively, are highlighted in red.
(B) 293T cells were transfected with HA-Ub and GFP-H2A or with GFP-mH2A isoforms with or without BRCA1/BARD1. Immunoprecipitated H2A or mH2As and whole-cell lysates (input) were immunoblotted with indicated antibodies. Ubiquitinated H2A and mH2A are indicated with arrows. WB, western blot.
(C) In vitro ubiquitination was performed using immunoprecipitated mH2A1.1 with E1, UbcH5c, HA-Ub, and recombinant RING domains of BRCA1/BARD1. Ubiquitinated mH2A1.1 (red arrow) was detected with an anti-HA antibody.
BR/BD, BRCA1/BARD1. See also Figure S2.
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5. Figure 3 BRCA1/BARD1 Ubiquitinates K123 of mH2A1
(A) Histones extracted from DNA- or siRNA-transfected 293T cells were mixed with heavy-isotope-labeled histones extracted from 293T cells. Mixed histones were digested with trypsin, and peptides were subject to diGly IP-MS analysis. Peak areas of light peptides were normalized with areas of spiked heavy peptides. RT, retention time.
(B) Relative ubiquitination levels of mH2A1-K123 in BRCA1/BARD1 overexpression or knockdown cells. Error bars indicate SD of two measurements of knockdown and three measurements of overexpression.
(C) mH2A1.1 WT, K116R/K117R, K121R/K123R, or 4KR mutants were co-transfected with HA-Ub and BRCA1/BARD1. Ubiquitinated mH2A1.1 (indicated by the arrows) was detected by immunoblotting with GFP. SMC1 serves as a loading control.
(D) 293T cells were transfected with BRCA1 WT or C61G mutant, and histones were extracted for diGly IPs. Heavy-isotope-labeled histones were spiked for normalization.
(E) Similar experiments as in (D) were conducted with BRCA1 I26A mutant.
Data indicate mean ± SD. See also Figure S3.
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6. Figure 4
MacroH2A1 Ubiquitination at K123 Is Important for Cellular Senescence
(A) Replicative senescence in mH2A1 WT-, K116R/K117R-, and K121R/123R-expressing IMR90 cells. An SA-β-gal assay was performed at passage 18.
(B) Growth rates of the cell lines in (A) were measured by MTT assays.
(C) Relative levels of total Ub-K123 (left) and Ub-K123 normalized by total mH2A1 (right) at passages 13 and 35, measured by MS.
(D) ChIP-qPCR of GFP and GFP-mH2A1.1 (WT or K121R/123R) of IMR90 passage 18 at SASP gene promoters; satellite III (SATIII) was used as a control.
(E) Proteomic profiling of GFP, WT-, and K121/123R-mH2A1.1-expressing IMR90 at passages 13 and 19 was performed with MS. 11 and 12 SASP proteins were detected at passage 13 and passage 19, respectively. 2KR, mH2A1.1 K121R/K123R.
Data indicate mean ± SD. See also Figure S4 and Table S4.
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