1. Volume 41, Issue 4, Pages 384-397 (February 2011)
WAC, a Functional Partner of RNF20/40, Regulates Histone H2B Ubiquitination and Gene Transcription 
Feng Zhang, Xiaochun Yu 
Molecular Cell 
Volume 41, Issue 4, Pages (February 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 WAC Associates with RNF20/40
(A) Silver staining of affinity-purified RNF20/40 complex. Cell lysates of 293T cell stably expressing SFB-RNF20, SFB-WAC, or SFB-RNF40 were subjected to affinity purification. Eluted proteins were visualized by silver staining. Arrows indicate proteins corresponding to RNF20, RNF40, and WAC. Peptide coverage is shown in the upper-right table.
(B) WAC interacts with RNF20/ T cell lysates were analyzed by IP and western blotting with the antibodies indicated.
(C and D) 293T cell extracts (C) and purified RNF20/40/WAC complex from 293T cells stably expressing SFB-RNF20 (D) were analyzed by size-exclusion chromatography on a Superose 6 gel filtration column. Proteins eluted from the indicated fractions were blotted with the indicated antibodies.
(E) WAC directly binds RNF20/40. SF9 cells were coinfected with baculoviruses encoding SFB-RNF40 and SBP-RNF20 together with GST-WAC or GST-hPAF1. Protein complex was purified by glutathione agarose beads. The purified proteins were visualized by Coomassie blue staining and blotted with antibodies against RNF20 and RNF40.
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3. Figure 2 Mapping the Interaction Regions of WAC and RNF20/40
(A) SFB-tagged wild-type WAC and its deletion mutants were expressed in 293T cells. Cell lysates were IPed and blotted with the indicated antibody. The expression level of exogenous WAC was blotted with anti-FLAG antibody from whole cell lysates (WCL).
(B and C) A coiled-coil region of RNF20 (B) or RNF40 (C) interacts with WAC. HA-tagged wild-type RNF20 or RNF40 and their internal deletion mutants were expressed in 293T cells. The interaction between RNF20/40 and WAC were IPed and blotted with the indicated antibodies.
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4. Figure 3 WAC Regulates H2B Ubiquitination
(A) Upper panel: 293T cell lysates were analyzed by IP and western blotting with the indicated antibodies. Lower panel: 293T cell expressing wild-type or mutant WAC were analyzed by IP and western blotting with the antibodies indicated. The expression levels of WAC and its mutants were examined by anti-FLAG antibody.
(B–D) HCT116 cells were treated with indicated siRNA. Cell lysates were IPed and blotted with the indicated antibody. The level of WAC, RNF20 and hRAD6 was blotted from whole cell lysates. (B, right panel) The input level of RNF20 was normalized; the interaction between RNF20 and hRAD6 was examined.
(E) HCT116 cells were treated with the indicated siRNA. Histone marks were examined with the indicated antibodies. Blots with anti-actin and anti-H3 were used as protein loading controls.
(F) Coomassie blue staining of purified protein.
(G) WAC facilitates RNF20/40 and hRAD6-mediated H2B ubiquitination in vitro. In vitro chromatin ubiquitination assays contained nucleosomal histones, hE1, hRAD6A, RNF20/40, HA-tagged ubiquitin, and GST-WAC (0, 50, 0, 10, 25, 50 ng from lane 1 to 6). Nucleosomal histones were examined by western blotting with anti-ubH2B antibody (upper panel) and then anti-H2B antibody (lower panel).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
WAC Is Recruited to the p21 Gene Locus upon Transcription Activation
(A) DNA damage induces WAC to the p21 gene locus. HCT116 cells were treated with or without 0.5 μM doxorubicin for 12 hr. ChIP analyses on the p21 locus were performed by using the indicated antibodies. An irrelevant IgG was used for negative control shown as the dotted lines. Primer pairs of p21 used for quantitative PCR are indicated in schematic diagram.
(B) WAC regulates RNF20 and ubH2B enrichment at the p21 locus during p53-dependent transcription. HCT116 cells were transfected with the indicated siRNA followed by doxorubicin treatment. ChIP analyses on the p21 locus were performed by using the indicated antibodies. Error bars: standard deviation (SD) of triplicate experiments.
(C) Total RNA was extracted from HCT116 cells transfected with the indicated siRNA followed by doxorubicin treatment. Quantitative PCR analyses of p53-dependent transcription are shown. Error bars: SD of triplicate experiments.
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6. Figure 5 WAC Associates with RNA Polymerase II through the WW Domain
(A) GST-WAC or GST- ΔWW were used to pull down endogenous Pol II from WCL of 293T cells.
(B) GST-CTD was incubated with or without CDK9/Cyclin T1 (left panel). The nonphosphorylated GST-CTD or phosphorylated GST-CTD was immobilized onto glutathione beads and used to pull down cell lysates from 293T cell expressing SFB-WAC and ΔWW, respectively (right panel).
(C) Left panel: biotinylated nonphospho-CTD peptide, pSer5 or pSer2 CTD peptide was immobilized onto streptavidin beads and used to pull down endogenous WAC from WCL of 293T cells. Empty beads were used as a negative control. Middle panel: pSer2 CTD peptide was used to pull down cell lysates from 293T cells expressing FLAG-WAC or ΔWW mutant. Right panel: nonphospho-CTD peptide, pSer5 or pSer2 CTD peptide was immobilized onto streptavidin beads and incubated with recombinant GST-WAC, and then peptide pull-down assay was performed.
(D) Localization of phospho-Pol II at the p21 locus. HCT116 cells were treated with or without 0.5 μM doxorubicin for 12 hr. ChIP analyses on the p21 locus were performed by using the indicated antibodies. An irrelevant IgG was used for negative control presented as the dotted lines. Error bars: SD of triplicate experiments.
(E) WAC associates with phosphorylated Pol II in vivo. 293T cell lysates were analyzed by IP and western blotting with indicated antibodies.
(F) HCT116 cells were treated with the indicated siRNAs. Cell lysates were examined by IP and western blotting.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
RNA Polymerase II Is Important for WAC Enrichment at the p21 Locus
(A) WAC is dispensable for pSer2-Pol II occupancy at the p21 locus. HCT116 cells were transfected with control or WAC siRNA. ChIP analyses on the p21 locus were performed by using anti-Ser2P-Pol II antibody.
(B) Pol II regulates H2B ubiquitination. HCT116 cells were treated with or without 10 μg/ml α-amanitin for 12 hr. Total cells were subjected to western blotting with indicated antibodies.
(C) Pol II is important for WAC occupancy at the p21 locus. Cells were treated the same as in (B). ChIP analyses on the p21 locus were performed by using the indicated antibodies.
(D) HCT116 cells and HCT116 cells stably expressing SFB-ΔWW mutant were treated with control or WAC siRNA, respectively. Cell lysates were analyzed by western blotting with indicated antibodies to examine the protein levels of SFB-ΔWW mutant, endogenous WAC, and RNF20. IP and western blotting were performed to investigate the interactions between RNF20 and WAC or between RNF20 and SFB-ΔWW mutant.
(E) Cells were treated the same as in (D). ChIP analyses were performed by using the indicated antibodies at the p21 locus. An irrelevant IgG was used as the negative control shown as the dotted lines (A, C, and E). Error bars: SD of triplicate experiments.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
WAC Is Important for G1/S Checkpoint Activation in Response to DNA Damage
(A) HCT116 cells were transfected with the indicated siRNA followed by 15 Gy IR treatments. mRNA level of p21 was examined by qPCR 12 hr after IR. Error bars: SD of triplicate experiments.
(B) As in (A) except that 12 hr after IR, cells were pulse labeled with 20 μM BrdU for 30 min and stained with FITC-conjutated anti-BrdU and PI. Incorporation of BrdU and total DNA content were determined by FACS. Cells at G1/S transition and early S phase with positive BrdU staining were calculated.
(C) A model of WAC mediates transcription-coupled H2B ubiquitination. WAC recognizes RNA polymerase II C-terminal domain and recruits RNF20/40 and RAD6 to gene transcription sites for H2B ubiquitination.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions