Volume 36, Issue 1, Pages 110-120 (October 2009) BBAP Monoubiquitylates Histone H4 at Lysine 91 and Selectively Modulates the DNA Damage Response  Qingsheng Yan, Shilpee Dutt, Rong Xu, Katherine Graves, Przemyslaw Juszczynski, John P. Manis, Margaret A. Shipp  Molecular Cell  Volume 36, Issue 1, Pages 110-120 (October 2009) DOI: 10.1016/j.molcel.2009.08.019 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 BBAP Ubiquitylates Core Histones In Vitro Recombinant wild-type BBAP (BBAPWT) or BBAP with a deleted RING domain (BBAPRDdel) was incubated with individual core histone proteins (H3, H2A, H2B, or H4), E1, E2, and His ubiquitin in in vitro ubiquitylation assays. Samples were size-fractionated, immunoblotted, and analyzed with anti-multihistidine (ubiquitin) (A) or antibodies directed against the individual histones (H2A, H2B, H3, H4) (B). Monoubiquitylated histone proteins are indicated with arrows in (B). Molecular Cell 2009 36, 110-120DOI: (10.1016/j.molcel.2009.08.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 BBAP Monoubiquitylates Histone H4 In Vivo (A) Histone H4 monoubiquitylation in HEK293 cells cotransfected with FLAG-tagged histone H4 and HA-tagged BBAPWT, BBAPRDdel, or no BBAP construct. Samples were size-fractionated, immunoblotted, and analyzed with anti-FLAG (H4). (B) Coimmunoprecipitation of BBAP and histone H4. HEK293 cells cotransfected with HA-tagged BBAPWT and FLAG-tagged histone H4 were lysed and immunoprecipitated (IP) with anti-HA or control Ig and immunoblotted with anti-HA (BBAP) (top) or anti-FLAG (histone H4) (bottom). LC indicates light chain (bottom). Cotransfected HEK293 cell lysate (input, left lane) was also directly immunoblotted. (C) Coimmunoprecipitation of nuclear chromatin-associated BBAP and histone H4. DNA-bound nuclear protein complexes were isolated from HeLa cells, immunoprecipitated with BBAP antiserum or control IgG, and immunoblotted with monoclonal anti-BBAP antibody (top) or anti-H4 (bottom). The nuclear protein complexes (Nuc. Ext., left lane) were also directly immunoblotted. (D) BBAP depletion by siRNA. Untreated HeLa cells and cells treated with a scrambled control siRNA (ctl) or one of two independent BBAP siRNAs (#1, #2) were lysed, size-fractionated, and immunoblotted with anti-BBAP (top). Blots were subsequently reprobed with anti-actin to confirm equal loading (bottom). (E) Monoubiquitylated histone H4 is decreased in BBAP-depleted cells. After HeLa cells were transfected with either BBAP siRNA (#1 or #2) or the control siRNA (ctl), histones were purified, size-fractionated, and immunoblotted with anti-H4. Histone samples were also immunoprecipitated with anti-ubiquitin and immunoblotted with anti-histone H4 to confirm that the 17 kd H4 protein is monoubiquitylated (Figure S2). Molecular Cell 2009 36, 110-120DOI: (10.1016/j.molcel.2009.08.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 BBAP Monoubiquitylates Histone H4 Lysine 91 In Vivo (A) Covalent modifications of histone H4. Serine phosphorylation (P) and acetylation (Ac) and/or methylation (Me) of lysine and arginine residues and ubiquitylation of H4K91 are shown. (B) Histone H4K91 is targeted by the BBAP E3 ligase. Site-directed mutagenesis of each histone H4 lysine residue (5, 8, 12, 16, 20, 31, 44, 59, 77, 79, and 91) was performed, and the resulting histone H4 sequences were cloned into a FLAG-tagged expression vector. Thereafter, the individual FLAG-tagged wild-type or K→A histone H4 mutants were cotransfected with HA-tagged BBAP and His ubiquitin, and the resulting transfectants were lysed, size-fractionated, and immunoblotted with anti-FLAG (histone H4). (C) BBAP ubiquitylates H4 in histone octamers (core histones) in vitro. Recombinant wild-type BBAPWT or BBAPRDdel was incubated with either free H4 or intact histone octamers (core histones) in the presence with E1, E2, and His ubiquitin in in vitro ubiquitylation assays. Samples were size-fractionated, immunoblotted, and analyzed with anti-multihistidine (ubiquitin). Monoubiquitylated histone H4 and free His-ubiquitin are indicated with arrows. In Figure S3, the same samples were immunoblotted with anti-H4. Molecular Cell 2009 36, 110-120DOI: (10.1016/j.molcel.2009.08.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 BBAP Modulates the Cellular Response to DNA Damage (A) BBAP overexpression protects cells against Hu or Dox treatment. HEK293 cells transfected with BBAPWT, BBAPRDdel, or vector alone were subsequently treated with Hu or Dox for 24 hr and counted. Although BBAPWT did not alter the proliferation of untreated cells, BBAPWT protected cells against Hu and Dox toxicity (p < 0.01 for both, ANOVA). (B) BBAP depletion augments the cellular response to DNA-damaging agents. HeLa cells were transfected with control or BBAP siRNAs (siRNA#1, #2), treated with Dox at 50, 200, and 400 ng/ml or left untreated for 1–96 hr and subsequently evaluated by MTS assay. Although BBAP RNAi reduced the growth of untreated cells, the consequences of BBAP depletion were most striking in cells treated with low-dose Dox (50 ng) (p < 0.001, two-way ANOVA). After 48 hr of treatment with low-dose Dox (50 ng), cellular proliferation (as assessed by MTS assay) was ∼70%–80% lower in BBAP-depleted cells than in control RNAi or parental cells. (C) Cellular apoptosis following BBAP depletion and Dox treatment. Parental, control, and BBAP siRNA-transfected HeLa cells were untreated or treated with Dox at 50 and 200 ng/ml for 24 hr and analyzed for apoptosis with Annexin V/PI staining. Error bars in (A) and (B) represent SD of the mean for three replicates in a representative experiment. Molecular Cell 2009 36, 110-120DOI: (10.1016/j.molcel.2009.08.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 BBAP Selectively Modulates 53BP1 Foci Formation HeLa cells were transfected with BBAP or control siRNAs and either left untreated or treated with doxorubicin (Dox) for 1–24 hr. Thereafter, DDR foci formation—p1981-ATM, γ-H2AX, and MDC1 (A) and 53BP1 and BRCA1 (B)—was evaluated at serial time points. In both (A) and (B), representative photographs (left) and accompanying plots of the percentage of cells with greater than 10 DDR-foci/nuclear capture (right) are shown. A minimum of 100 BBAP siRNA#1, siRNA#2, and control siRNA-treated cells were analyzed at each time point. At 1, 2, and 4 hr following induction of DNA damage with Dox, there were significantly fewer 53BP1 foci in BBAP-depleted cells than in controls (with a maximal reduction at 1 hr; p < 0.01 for siRNA1 or siRNA2 versus control, B). At the earliest time point following DNA damage, there was also a minor reduction in BRCA1 repair foci in BBAP-depleted cells (at 1 hr, p < 0.01 for siRNA1 or siRNA2 versus control, B). Error bars on the right represent the SD of the mean for three independently stained slides for each time point and condition. Molecular Cell 2009 36, 110-120DOI: (10.1016/j.molcel.2009.08.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Histone H4 Modifications following DNA Damage and BBAP Depletion Parental, BBAP-depleted, and control siRNA HeLa cells were treated with Dox (50 ng/ml) for 1–24 hr. Thereafter, total cell lysates were prepared from one set of samples and immunoblotted to assess BBAP abundance and were reblotted for actin (to confirm equal loading) (immunoblots, A, and scanning densitometric tracings, B, top). Histones were purified from a second set of identically treated samples and immunoblotted with anti-histone H4, antibodies directed against mono- or dimethyl H4K20 or H4K91Ac or anti-H2A (to confirm equal loading) (immunoblots, A, and scanning densitometric tracings, B, additional panels). Molecular Cell 2009 36, 110-120DOI: (10.1016/j.molcel.2009.08.019) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 BBAP Depletion Decreases the Abundance of Nuclear Chromatin-Associated Set8 After confirming the specificity of the Pr-SET7/SET8 antibody (Figure S6), chromatin-associated nuclear proteins were prepared from parental control siRNA or BBAP-depleted HeLa cells that were untreated (−) or treated with Dox (50 ng/ml) for 1 hr (+). Thereafter, the samples were immunoblotted for BBAP, PR-SET7/SET8, and H2A (to confirm the enrichment of chromatin-associated proteins and equal loading). Molecular Cell 2009 36, 110-120DOI: (10.1016/j.molcel.2009.08.019) Copyright © 2009 Elsevier Inc. Terms and Conditions