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. Author manuscript; available in PMC: 2019 May 9.
Published in final edited form as: Cell Rep. 2017 May 30;19(9):1758–1766. doi: 10.1016/j.celrep.2017.05.027

The Histone Variant MacroH2A1 Is a BRCA1 Ubiquitin Ligase Substrate

Beom-Jun Kim 1, Doug W Chan 2, Sung Yun Jung 1,2, Yue Chen 1, Jun Qin 1,2,3,*, Yi Wang 1,2,4,*
PMCID: PMC6507409  NIHMSID: NIHMS1019499  PMID: 28564596

SUMMARY

The breast- and ovarian-cancer-specific tumor suppressor BRCA1 and its heterodimeric partner BARD1 contain RING domains that implicate them as E3 ubiquitin ligases. Despite extensive efforts, the bona fide substrates of BRCA1/BARD1 remain elusive. Here, we used recombinant GST fused to four UBA domains to enrich ubiquitinated proteins followed by a Lys-ε-Gly-Gly (diGly) antibody to enrich ubiquitinated tryptic peptides. This tandem affinity purification method coupled with mass spectrometry identified 101 putative BRCA1/BARD1 E3 substrates. We identified the histone variant macroH2A1 from the screen and showed that BRCA1/BARD1 ubiquitinates macroH2A1 at lysine 123 in vitro and in vivo. Primary human fibroblasts stably expressing a ubiquitination-deficient macroH2A1 mutant were defective in cellular senescence compared to their wild-type counterpart. Our study demonstrates that BRCA1/BARD1 is a macroH2A1 E3 ligase and implicates a role for macroH2A1 K123 ubiquitination in cellular senescence.

In Brief

Using a tandem affinity purification method coupled with mass spectrometry, Kim et al. identified 101 putative substrates of the BRCA1/BARD1 E3 ubiquitin ligase. They report that, among these substrates, ubiquitination at Lys123 of macroH2A1 plays an important role in replicative senescence.

Graphical Abstract

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INTRODUCTION

The breast and ovarian tumor suppressor BRCA1 plays an important role in maintaining the genome stability. BRCA1 is a key mediator in DNA-damage response. It is recruited to sites of DNA damage, where it functions with DNA-repair factors to activate the cell-cycle checkpoint, promote DNA repair, and regulate transcription (Huen et al., 2010). BRCA1 has also been implicated in regulating cellular senescence (Cao et al., 2003, 2009; Tu et al., 2013; Sedic et al., 2015), as constitutive DNA damage due to BRCA1 loss leads to premature senescence in cells and embryonic lethality in animals.

BRCA1 contains a highly conserved amino-terminal RING domain, which mediates protein ubiquitination. The RING domain also mediates its interaction with the BRCA1-associated RING domain 1 (BARD1), whose own RING domain plays an integral part in the BRCA1/BARD1 E3 function. Although the in vitro activity of BRCA1/BARD1 E3 ligase has been demonstrated, few in vivo substrates have been convincingly characterized (Huen et al., 2010; Ohta et al., 2011). Recently, Zhu and colleagues reported that histone H2A ubiquitination at K118 by BRCA1 mediates transcription silencing (Zhu et al., 2011). Defects in BRCA1 E3 function are linked to transcription derepression of satellite DNA and loss of histone H2A ubiquitination at satellite repeats. Importantly, exogenous expression of a H2A-ubiquitin fusion protein could suppress the elevated satellite DNA transcripts due to BRCA1 loss. Additionally, BRCA1 can specifically ubiquitinate histone H2A in its C-terminal tail on K127 and 129 when H2A is within a nucleosomal context (Kalb et al., 2014). Together, these reports indicate that BRCA1 is a histone H2A-specific ubiquitin (Ub) ligase and that the E3 activity is essential for its function.

Identification of E3 ligase substrates is a challenging biochemical problem, in part due to the difficulties in detecting ubiquitinated proteins (Harper and Tan, 2012). Two recent technical advancements have accelerated ubiquitin proteomic research (Shi et al., 2011; Udeshi et al., 2012; Kim et al., 2011). One is the development of the antibody that can specifically recognize ubiquitinated peptides (Xu et al., 2010); the other is the use of the recombinant tandem Ub-binding domains (UBDs) as affinity reagents to enrich poly-ubiquitinated proteins (Shi et al., 2011; Hjerpe et al., 2009). Here, we applied an improved affinity purification method by combining these two techniques. We used glutathione S-transferase (GST) fused with four Ub-associated domains (UBAs) of ubiquilin (qUBA) and Lys-ε-Gly-Gly (diGly) antibody (qUBA-diGly) immunoprecipitation (IP) to enrich ubiquitinated peptides in BRCA1/BARD1-overexpressing cells, and we identified BRCA1/BARD1-dependent ubiquitinated proteins with quantitative mass spectrometry (MS). We further investigated in vitro and in vivo ubiquitination of a histone variant macroH2A1 (mH2A1) by BRCA1 and characterized the functional consequence of mutation at the mH2A1.1 ubiquitination sites.

RESULTS

A Tandem Affinity Enrichment Workflow to Identify Putative BRCA1/BARD1 E3 Substrates

To identify potential BRCA1 substrates, we overexpressed BRCA1/BARD1 in 293T cells and searched for ubiquitinated peptides whose abundances were increased by overexpression. We performed tandem affinity purifications to enrich ubiquitinated proteins and peptides respectively (Figure 1A). First, GST fused to four tandem repeats of the UBA domain of ubiquilin-1 (Shi et al., 2011) was used to pull down ubiquitinated proteins, which were then subject to on-beads trypsin digestion to generate ubiquitinated peptides; next, anti-diGly antibody-conjugated agarose beads were used to enrich the GG remnant peptides (Xu et al., 2010). The resulting GG remnant peptides were analyzed and quantified by label-free MS. In all, MS analysis identified 5,563 ubiquitinated peptides belonging to 2,482 gene products from the control pcDNA3 and BRCA1/BARD1-transfected cells. Among them, 5 and 38 GG peptides were derived from BARD1 and BRCA1, respectively, indicating an extensive auto-ubiquitination of the E3 ligases. We searched for high-confidence peptides (peptide spectrum match [PSM] > 2, area under the curve [AUC] > 106, posterior error probability [PEP] < 0.01) whose abundances were increased to more than 3-fold upon E3 overexpression, resulting in 108 peptides from 101 proteins excluding BRCA1 and BARD1 (Table S1).

Figure 1. Sequential qUBA/diGly Dual-Affinity Purification Coupled with MS Identifies BRCA1/BARD1 E3 Ubiquitination Substrates.

Figure 1.

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(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 chromatographytandem 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.

Previous studies reported several proteins–including NPM1, estrogen receptor α, CtIP, and histone H2A–as BRCA1/BARD1 substrates (Sato et al., 2004; Eakin et al., 2007; Yu et al., 2006). Our dataset contains ubiquitinated peptides belonging to NPM1, estrogen receptor α, and various isoforms of histone H2A (Table S2); however, their levels were not increased upon BRCA1/BARD1 expression. BRCA1 catalyzes K6-, K48-, and K63-linked poly-Ub chains in an E2-dependent manner in vitro (Christensen et al., 2007). We found that, while K6-, K11-, K48- and K63-linked Ub chains were readily detectable, their abundances upon BRCA1/BARD1 overexpression were not significantly changed (Table S3), suggesting that BRCA1/BARD1 does not significantly impact global polyubiquitination linkages but may assemble specific Ub chains on its substrates.

We validated three proteins (HLTF, GADD45GIP1, and macroH2A1) that are known to be involved in DNA-damage response and transcription, two processes that BRCA1 regulates (Figure 1B) (Achar et al., 2015; Lin et al., 2011; Chung et al., 2003; Gamble and Kraus, 2010). We also included KDM4B as a negative control. We co-transfected hemagglutinin (HA)-Ub, BRCA1/BARD1, and putative BRCA1 substrates and determined the ubiquitination levels of immunoprecipitated substrates by immunoblotting with an anti-HA antibody (Figures S1A and S1B). HLTF, which was ubiquitinated at multiple lysine residues without BRCA1/BARD1 overexpression (Figure 1B), displayed poly-ubiquitination ladders with increased intensity upon BRCA1/BARD1 overexpression (Figure S1A). GADD45GIP1, which was not ubiquitinated at detectable levels without BRCA1/BARD1, appeared to be mainly mono-ubiquitinated (Figure S1B); moreover, mutating Lys59 to Arg largely abolished the ubiquitination signal, suggesting that Lys 59 is the predominant ubiquitination site (Figure S1C). BRCA1/BARD1 overexpression also efficiently increased the ubiquitination signals of mH2A1.1 (Figure 2B) but did not noticeably change the ubiquitination of KDM4B, consistent with the qUBA-diGly IP results (Figures 1B and S1A). These results suggest that qUBA-diGly IP is an effective method to screen for putative substrates of E3 Ub ligases whose substrates are not well defined.

Figure 2. BRCA1/BARD1 Ubiquitinates Histone mH2A In Vivo and In Vitro.

Figure 2.

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(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 H2Aor 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.

BRCA1 Ubiquitinates K123 of mH2A1 in a Ligase-Activity-Dependent Manner

The histone variant mH2A1 (encoded by the H2AFY gene) caught our attention because histone H2A has been shown as a BRCA1 substrate. Moreover, mH2A1.2, together with PRDM2, regulates double-strand-break (DSB)-associated chromatin reorganization and recruitment of BRCA1 for DSB repair (Khurana et al., 2014). We next carried out an in-depth characterization of BRCA1-dependent mH2A ubiquitination. Three mH2A gene products are encoded in humans: mH2A1.1 and mH2A1.2, which are produced by alternative splicing of H2AFY gene; and mH2A2, which is produced by H2AFY2 (Figure 2A). Upon BRCA1/BARD1 overexpression, all three isoforms showed increased ubiquitination in mainly mono-ubiquitinated forms (Figure 2B). Recombinant RING domains of BRCA1 and BARD1 could ubiquitinate mH2A1.1-GFP in vitro (Figure 2C), but not mH2A1.1–4KR mutant (K116/117/121/123R) (Figure S2), suggesting that mH2A1 can be ubiquitinated directly by RING domains of BRCA1/BARD1 heterodimer.

Of the two ubiquitination sites identified by MS, Lys 116 ubiquitination was detected at relatively high levels in the absence of BRCA1/BARD1 overexpression, but its level was moderately increased (by 0.8-fold) upon BRCA1/BARD1 overexpression (Figures 1B and 2A). Sequence alignment shows that K116 of mH2A1.1 corresponds to K118 of canonical H2A, which is the reported ubiquitination site by BRCA1 (Zhu et al., 2011). The other site, Lys 123, which was detected only in BRCA1/BARD1-overexpressing cells, is located at the boundary between the H2A-like and the linker regions and corresponds to H2A-K129, the ubiquitination site reported by Kalb and colleagues (Kalb et al., 2014).

To obtain more accurate quantification on their dependency on BRCA1, we quantified these ubiquitination events using a SILAC (stable isotope labeling with amino acid in cell culture)- based method (Figure 3A). We extracted histones from BRCA1/BARD1-overexpressing or BRCA1-knockdown cells and mixed them with equal amount of heavy lysine and arginine (13C6 Lys and 13C6 Arg) labeled histones. Trypsin-digested histone peptides were directly subject to diGly IP followed by MS (Figure 3A). BRCA1/BARD1 overexpression resulted in a 2-fold increase in mH2A1 K123-Ub, whereas it affected neither mH2A1 K116-Ub nor H2A K118-Ub (Figures 3B and S3A); moreover, small interfering RNA (siRNA)-mediated silencing of BRCA1 reduced mH2A1 K123 ubiquitination to ~30% of that in a control knockdown but had no effect on mH2A1 K116-Ub or H2A K118-Ub (Figure 3B). Consistent with results obtained from tandem affinity enrichment, the global distribution of Ub-K6, K11, K48, and K63 linkages in the histone extracts was not changed by BRCA1/BARD1 overexpression or BRCA1 knockdown (Table S3).

Figure 3. BRCA1/BARD1 Ubiquitinates K123 of mH2A1.

Figure 3.

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(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.

We generated mH2A1.1 ubiquitination-deficient mutants and validated the MS results by western blotting. Since K117 is adjacent to K116, and K123 is very close to K121, we mutated K117 and K123 to prevent them from serving as alternative ubiquitination sites and generated two 2KR mutants (K116R/K117R and K121R/K123R) and a 4KR mutant. We co-transfected the mH2A1.1 mutants with HA-Ub in the presence or absence of BRCA1/BARD1 and examined the ubiquitination of GFP-tagged mH2A1.1 by their mobility shifts (Figure 3C). The ubiquitination signal of the K121/123R mutant was similar to that of the wildtype mH2A1.1 (comparing lane 5 to lane 1), suggesting that K121/123 ubiquitination makes a minor contribution to the overall ubiquitination and that K116 and K117 are the dominant ubiquitination sites. Importantly, overexpression of BRCA1/BARD1 did not appear to increase ubiquitination of the mH2A1-K121/123R mutant, consistent with the MS results that K116/117 modification is not dependent on BRCA1/BARD1. Although the Ub signal of K116R/K117R mutant was weak, longer exposure revealed that the ubiquitination of the remaining sites (K121/123) was increased by BRCA1/BARD1 overexpression. Overall, these results suggest that K116 is the dominant but BRCA1/BARD1-independent ubiquitination site, whereas K123 is the secondary but BRCA1/BARD1-dependent ubiquitination site.

We tested whether mH2A1 ubiquitination depends on BRCA1 E3 ligase activity using breast-cancer-associated C61G and I26A mutants (Figures 3D, 3E, S3B, and S3C). Overexpression of wild-type (WT) BRCA1 or BARD1 alone had no effect on the ubiquitination of any histone residues (Figure 3D), confirming that formation of BRCA1/BARD1 heterodimer is required for its ubiquitin ligase activity. While BRCA1 heterodimer increased the K123-Ub of mH2A1 by more than 2-fold, overexpression of BRCA1-C61G or I26A with BARD1 had no effect on mH2A1 K123-Ub (Figures 3D and 3E), confirming that BRCA1 E3 activity is required for mH2A ubiquitination in vivo.

Ubiquitination of mH2A on K123 Plays a Role in Cellular Senescence

While mH2A is mainly characterized as a transcription repressor, Chen and colleagues recently reported that mH2A1 is localized in SASP (senescence-associated secretory phenotype)-promoting genes and is a critical component of the positive-feedback loop that maintains SASP gene expression (Chen et al., 2015). We tested whether mH2A ubiquitination plays a role in replicative cellular senescence in primary human fibroblast IMR90 cells. While all GFP-mH2A1 variant proteins were expressed at comparable levels, the endogenous mH2A1 protein levels were greatly reduced (Figure S4A), allowing for examination of knockin-like phenotypes. Compared to GFP control cells, WT mH2A1.1 overexpression clearly induced cellular senescence at passage 18 when this phenotype was not obvious in control GFP cells (Figure 4A); the K116R/117R mutant cell line exhibited a similar phenotype as that of the WT-mH2A1.1-expressing cells, suggesting that ubiquitination at K116/117 does not affect senescence. However, mH2A1 K121R/123R-expressing cells exhibited phenotypes that resembled GFP control cells (Figure 4A). Consistent with this phenotype, the levels of mH2A1 K123-Ub at passage 35 were increased by 10-fold than those at passage 13 and 2-fold when normalized by the increased total mH2A1 levels (Figure 4C). Growth rates measured by MTT assays confirmed that WT-mH2A1- and K116R/117R-expressing cells exhibited a slower growth rate than control GFP and mH2A1 K121R/123R cells (Figures 4B and S4B). p21CIP1, a key senescence marker, was expressed at elevated levels in WT and K116R/117R mH2A1 cells than in control cells, but its level in K121R/K123R cells was comparable to that in control cells (Figure S4C). We further examined the SASP gene expression by proteomic profiling of GFP, WT, and K121/123R-mH2A1.1 cell lines at passages 13 and 19 with MS. Among the 51 reported SASP genes (Chen et al., 2015), we detected 11 and 12 at protein levels at passages 13 and 19, respectively (Table S4). As shown in Figure 4E, with few exceptions (C3, IGFBP5, and MMP1), the SASP protein levels in WT and K121R/123R mH2A1.1 cells were largely comparable to those in GFP cells at passage 13; at passage 19,9 of the 11 SASP protein levels were higher by more than 2-fold in WT mH2A1.1 cells than in GFP cells; and interleukin-6 (IL-6), which was undetectable at passage 13, was detected at passage 19. The increased level of SASP proteins was consistent with the senescent phenotype observed by senescence-associated β-galactosidase (SA-β-gal) staining. In K121/123R cells at passage 19, seven of the nine SASP proteins induced in mH2A1.1 WT cells were induced at markedly lower levels (Figure 4E); IL-6, which was also undetectable at passage 13, was detectable at passage 19 but was at 44% of the level in mH2A1.1 WT cells. Although the levels of several SASP transcripts in K121R/K123R cells were lower than those in WT mH2A1.1 cells (Figure S4D), data from chromatin IP (ChIP) followed by qPCR showed that K121R/123R mutant cells bound to SASP gene promoters at comparable levels to that by WT mH2A1.1 (Figure 4D). Together, our data indicate that the reduction of SASP gene expression in mH2A1.1 K121/123R cells is likely caused by impaired recruitment of RNA polymerase, transcription factors, or coregulators.

Figure 4. MacroH2A1 Ubiquitination at K123 Is Important for Cellular Senescence.

Figure 4.

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(A) Replicative senescence in mH2A1 WT-, K116R/K117R-, and K121R/123R-expressing IMR90 cells. An SA-β-gal assaywas 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 ofGFP, WT-, and K121/123R-mH2A1.1-expressing IMR90 at passages 13and 19was performed with MS. 11 and 12SASP 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.

DISCUSSION

In this study, we combined two powerful affinity enrichment methods to sequentially enrich ubiquitinated proteins and peptides to increase specificity and efficiency. Our approach is similar to a dual-affinity pull-down method termed TR-TUBE (trypsin-resistant tandem Ub-binding entity), in which a tandem Ub-binding entity is exogenously expressed in cell lines as the first affinity reagent (Yoshida et al., 2015). Our approach differs in that the GST-qUBA domains used in the first affinity enrichment step is expressed in E. coli with very high yield and at low cost, making it a feasible method for tissues and cells that cannot be easily transfected.

Our study also demonstrates the necessity of MS for quantitative characterization of protein ubiquitination. Of the two mH2A1.1 ubiquitination sites, K116 is the dominant but BRCA1-independent site; the Ub signal of GFP-mH2A1-K121/123R by western blotting is almost indistinguishable from that of WT mH2A1. Clearly, the attenuated K123-Ub signal by BRCA1 depletion would be difficult to detect without MS or a Ub-site-specific antibody that is hard to generate.

Loss of BRCA1 is associated with premature senescence (Cao et al., 2003). Our finding that BRCA1-dependent ubiquitination promotes senescence appears to contradict to this function. This difference may reflect the multiple functions BRCA1 plays in the cell. Supporting this notion, BRCA1 contains several distinct structural domains; serves as a scaffold protein to assemble protein complexes; and, as an E3 ligase, may catalyze the ubiquitination of multiple substrates that lead to various functional outcomes. Complete loss of BRCA1 results in loss of all its functions, and the accumulation of DNA damage drives the cell into senescence. This, in fact, may reflect the dominant function of BRCA1 in DNA-damage repair. On the other hand, mH2A1 K123-Ub-dependent replicative senescence represents the function of a BRCA1 substrate. As a tumor suppressor, BRCA1 may promote or maintain senescence by regulating transcription through mH2A ubiquitination in cell types that do not proliferate, such as breast cells. This would be consistent with the role of BRCA1 as a breast cancer tumor suppressor.

Histone H2A has been shown previously as a BRCA1 substrate. In our study, H2A-Ub levels remained relatively unchanged when BRCA1 was overexpressed or depleted. The reason for the lack of BRCA1 dependency is not clear. Since H2A can also be ubiquitinated by other E3s, such as polycomb (Buchwald et al., 2006; Wang et al., 2004), it is possible that BRCA1’s contribution to total H2A-Ub levels is too small for our assays to detect. Our present data do not necessarily suggest that mH2A1 is a better substrate than H2A for BRCA1. As both proteins function in the context of chromatin in vivo, the specificity may be evaluated by direct comparison of BRCA1 E3 activity on bona fide nucleosome substrates with either H2A or mH2A1 incorporated.

The K123-containing tryptic peptide is identical in both mH2A1.1 and −1.2. Although transfection experiments demonstrate that both isoforms can be ubiquitinated by BRCA1, it is likely that only the mH2A1.1-Ub has the senescence-promoting function, as binding of poly(ADP-ribose) by its macro domain is necessary for this function (Chen et al., 2015). It was reported recently that mH2A1 is recruited to DSBs and that the mH2A1.2 isoform promotes chromatin reorganization and subsequent recruitment of BRCA1 (Khurana et al., 2014). This raises the possibility that recruited BRCA1 may also ubiquitinate mH2A1.2 to modulate chromatin reorganization or mediate DSB repair. Further investigation to determine whether mH2A1 ubiquitination is involved in DSB repair, and determining which splicing isoform is responsible, will provide more insights into BRCA1’s role in tumor suppression.

EXPERIMENTAL PROCEDURES

qUBA-diGly IP and MS Analysis

GST-qUBA protein was prepared as described previously (Shi et al., 2011). Mammalian cells were lysed in 2× packed cell volume (PCV) of NETN buffer containing protease and phosphatase inhibitors and iodoacetamide. The pellet after centrifugation was lysed with 1 × PCV of RIPA and sonicated. The combined lysates were incubated with GST-qUBA beads at 4°C for 1 hr and washed with NETN. Bound proteins were digested with trypsin in 50 mM NH4HCO3 at 37°C. Peptides were extracted with 50% acetonitrile, and dried peptides were resuspended with IAP Buffer Plus Detergent (Cell Signaling Technology). For histone extraction, cells were lysed with PBS containing 0.5% Triton X-100 and protease inhibitors. Pelleted nuclei were incubated with 0.2 N HCl at 4°C overnight and neutralized with 1 M Tris. Same amounts of SILAC-labeled histones were mixed with the light-labeled histones and digested with trypsin.

diGly-containing peptides were immunoprecipitated with PTMScan Ubiquitin Remnant Motif(K-ε-GG) antibody bead conjugate (Cell Signaling Technology). Peptides were eluted with 0.15% trifluoroacetic acid after washing with IAP Buffer Plus Detergent and distilled water. Dried peptides were resuspended with 5% methanol, 0.1% formic acid for MS analysis.

Samples were run on a LTQ Orbitrap Elite mass spectrometer (ThermoFisher Scientific). The mass spectrometer was operated in the data-dependent acquisition mode acquiring the fragmentation spectra of the top 50 strongest ions. Tandem MS (MS/MS) spectra were searched against the target-decoy human RefSeq database in Proteome Discoverer 1.4 interface with the Mascot algorithm. Peptides werevalidated with a 5% false discovery rate (FDR) and subject to manual verifications. The iBAQ algorithm was used to calculate protein abundance.

In Vitro Ubiquitination

Recombinant 6xHis-BRCA1 (amino acids [aa] 1–304) and 6xHis-BARD1 (aa 26–327) were co-expressed and purified. In vitro ubiquitination reaction was performed with UBE1, UbcH5a, HA-Ub, and immunoprecipitated mH2A1.1-GFP.

mRNA Quantitation by qPCR

Total RNAwas prepared using the RNAeasy Mini Kit (QIAGEN). 5 μg total RNA was used for reverse transcription reaction using random hexamers with Superscript II Reverse Transcriptase (Invitrogen). Each real-time PCR reaction was performed in technical duplicates using the SYBR Green PCR Master Mix (Thermo Fisher Scientific). Primer information was described in the previous study (Chen et al., 2015).

MTT Assay and BrdU Incorporation Assay

Cells were seeded at 5 × 103 perwell in a 96-well plate, and cell viability was determined using an MTT assay kit (Roche Life Science). Bromodeoxyuridine (BrdU) incorporation was measured using the BrdU Cell Proliferation ELISA Kit (Abcam). 5 × 104 cells were plated in a 96-well plate and treated with BrdU for 24 hr.

SA-β-Gal Assay

IMR90 cells at passage 18 were plated in a six-well plate (104 cells per well), and an SA-β-gal assaywas performed asdescribed previously(Debacq-Chainiaux et al., 2009).

ChIP Assay

GFP-mH2A1.1-or GFP-expressing IMR90 cells at passage 18 were used for ChIP assays, as previously described (Kim et al., 2010). GFP-Trap beads (Chromotek) were used for IP. Primer information for qPCR was described in the previous study (Chen et al., 2015).

Statistical Analyses

All experiments except for the proteome profiling in Figure 4E were performed independently at least two times. Differences between groups were compared using an unpaired, two-tailed Student’s t test. Data are presented as means ± SD. Values of p < 0.05 were considered statistically significant.

Additional experimental procedures are included in the Supplemental Experimental Procedures.

Supplementary Material

TableS1
TableS2
TableS3
TableS4
1

Highlights.

  • Tandem affinity purification was used to enrich ubiquitinated proteins/peptides

  • Quantitative mass spectrometry identified ubiquitination of macroH2A1 K123 by BRCA1

  • MacroH2A1.1 ubiquitination positively regulates SASP genes

  • MacroH2A1.1 K123R mutant is defective in replicative senescence

ACKNOWLEDGMENTS

We thank Drs. E. Bernstein, J. Chen, B. Grimes, K. Cimprich, I. Bae, and T. Westbrook for sharing reagents; and X. Ni, Y. Yu, and J. Yang for technical assistance. This work was supported in part by CPRIT grant RP110784 and National Basic Research Program of China (973 Program) 2012CB910300. Additional support was provided by the Cytometry and Cell Sorting Core at Baylor College of Medicine, with funding from the NIH (P30 AI036211, P30 CA125123, and S10 RR024574), and the expert assistance of Joel M. Sederstrom.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, four figures, and four tables and can be found with this article online at http://dx.doi.org/10.10167j.celrep.2017.05.027.

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Associated Data

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Supplementary Materials

TableS1
NIHMS1019499-supplement-TableS1.xlsx (17.1KB, xlsx)
TableS2
NIHMS1019499-supplement-TableS2.xlsx (11.1KB, xlsx)
TableS3
NIHMS1019499-supplement-TableS3.xlsx (11.9KB, xlsx)
TableS4
NIHMS1019499-supplement-TableS4.xlsx (461.9KB, xlsx)
1
NIHMS1019499-supplement-1.pdf (403.5KB, pdf)

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