Volume 43, Issue 1, Pages 85-96 (July 2011) Recruitment of TIF1γ to Chromatin via Its PHD Finger-Bromodomain Activates Its Ubiquitin Ligase and Transcriptional Repressor Activities  Eleonora Agricola, Rebecca A. Randall, Tessa Gaarenstroom, Sirio Dupont, Caroline S. Hill  Molecular Cell  Volume 43, Issue 1, Pages 85-96 (July 2011) DOI: 10.1016/j.molcel.2011.05.020 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 The Repressive Activity of TIF1γ on TGF-β Superfamily Transcriptional Responses Requires Its PHD Finger-Bromodomain (A and B) MDA-MB-231 cells containing integrated CAGA12-Luc (A) or BRE-Luc (B) and TK-Renilla reporters were transfected with individual siRNA oligos or pools and treated with the indicated ligands for 8 hr. (C) HaCaT-SRE-Luc cells were transfected with siRNAs and induced ± cytochalasin D for 7 hr. NT, nontargeting. (D) 293Ts were transfected with CAGA12-Luc and TK-Renilla along with the indicated pCS2-TIF1γ constructs. In all cases, luciferase levels were normalized to Renilla levels. In all cases the data are means and standard deviations for a representative experiment performed in triplicate. Protein overexpression and knockdown were verified by western blot. In (D), wild-type TIF1γ protein has a slower mobility due to its FLAG tag. Molecular Cell 2011 43, 85-96DOI: (10.1016/j.molcel.2011.05.020) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 TIF1γ Binding to the TGF-β-Induced PAI-1 Gene Depends on Both Ligand Activation and Smad4 (A) This figure shows qPCR of the PAI-1 Smad binding region (SBR), transcription start site (TSS), and coding region (CR) from ChIP assays using RNA Pol II, TIF1γ, and Smad4 antibodies. ChIPs were performed on extracts from MDA-MB-231 cells treated ± TGF-β for 1 hr. (B) As in (A) except extracts were additionally obtained from cells treated for 2 hr with SB-431542 after the TGF-β induction. (C) Extracts were prepared from HaCaT-TR and HaCaT-TRS4 cells treated with tetracycline (Tet) overnight and treated ± TGF-β for 1 hr. Smad4 knockdown was verified by western blot. In all cases data correspond to the average of duplicate qPCRs from a representative experiment normalized to input. Molecular Cell 2011 43, 85-96DOI: (10.1016/j.molcel.2011.05.020) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 TIF1γ-Smad Complex Formation Does Not Occur on Naked DNA (A and B) Extracts were prepared from treated HaCaT-TR and HaCaT-TRS4 cells (A) or 293Ts (B). Extracts were analyzed by western blot using the indicated antibodies, either directly (Input) or after IP with protein A beads (Beads) or anti-TIF1γ antibody-coupled beads (α-TIF1γ). Note that in (B), the antibody against phosphorylated Smad3 (PSmad3) also recognizes phosphorylated Smad1 (PSmad1). Only PSmad3 interacts with TIF1γ. (C) Nuclear extracts were prepared from HaCaT cells treated ± TGF-β for 1 hr and either analyzed by western blot directly (Input) or after DNA pull-down assay using wild-type (WT) c-Jun SBR oligos or those mutated in the Smad3/Smad4 binding site (Mut). Molecular Cell 2011 43, 85-96DOI: (10.1016/j.molcel.2011.05.020) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 TIF1γ Specifically Binds an Acetylated H3 Tail in which R2 and K4 Are Unmethylated (A) Sequences of histone H3 and H4 tails. Colored residues indicate modifications analyzed. (B) Peptide pull-down using differently modified H3 and H4 peptides and extracts made from untransfected 293Ts (upper panel) or 293Ts transfected with FLAG-tagged BPTF PHD finger (middle panel) or with FLAG-tagged BRDT bromodomain (BD1; lower panel). FLAG-tagged proteins were detected by western blot and binding of endogenous TIF1γ using a TIF1γ antibody. (C) As in (B) except that extract was prepared from 293Ts stably expressing FLAG-tagged TIF1γ ΔPHD/Bromo. Endogenous TIF1γ was detected with the anti-TIF1γ antibody. (D) Peptide pull-down using extract prepared from untransfected 293Ts and H3 peptides with the indicated mutations. (E) As in (D) except that H3 peptides with single acetylated lysines or a combination of two acetylated lysines were used. (F) As in (D) except that single H3 peptides or combinations of H3 peptides were used. In all cases the lanes marked Input are extract prior to pull-down, and those marked Beads were pulled down with beads alone. Beads conjugated with peptides were analyzed by SDS-PAGE, and peptides were detected by Coomassie (peptide loading). (G) A model indicating how the TIF1γ PHD finger-bromodomain binds to the H3 tail. Molecular Cell 2011 43, 85-96DOI: (10.1016/j.molcel.2011.05.020) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Histone Binding Induces TIF1γ Autoubiquitination Activity (A) In vitro ubiquitination assay. Recombinant E1, E2, and HA-Ubiquitin (HA-Ub) were incubated with purified core histones and TIF1γ purified from FLAG-TIF1γ 293Ts. Autoubiquitinated TIF1γ was detected with the HA antibody and unmodified TIF1γ with TIF1γ antibody. A Coomassie-stained gel shows purified TIF1γ (left panel). Molecular weight (MW) markers are shown in kDa. (B) Assays were performed as in (A) using 5 μl purified TIF1γ, but additionally the samples were IPed using FLAG and TIF1γ antibodies prior to western blot. (C) As in (B) but also including incubation with recombinant octamers. The histones used in the assay were western blotted using antibodies recognizing histone H3 acetylated at different lysines as indicated and are also shown stained with Coomassie. Note that the migration of H2B in the purified chicken erythrocyte histones differs slightly from that of the recombinant Xenopus histones, as previously observed (Luger et al., 1997). (D) As in (B) but including incubation with TIF1γ ΔPHD/Bromo purified from FLAG-TIF1γ ΔPHD/Bromo 293Ts. (E) 293Ts were transfected with HA-Ubiquitin and FLAG-TIF1γ and incubated overnight ± SB-431542. Extracts were IPed (IP) with FLAG-antibody and blotted as indicated. In (A) and (C), the migration of a 116 kDa marker is shown. Molecular Cell 2011 43, 85-96DOI: (10.1016/j.molcel.2011.05.020) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Smad4 Ubiquitination by TIF1γ Depends on Activation of TIF1γ by Binding to Modified Histones and on the TIF1γ PHD Finger-Bromodomain and the RING Finger (A) In vitro ubiquitination assay performed as in Figure 5A, but with the inclusion of Gal4-Smad4 as the TIF1γ substrate. Autoubiquitinated TIF1γ and ubiquitinated Gal4-Smad4 are detected in the HA blot. (B) In vivo ubiquitination assay. Extracts were prepared from 293Ts transfected with Myc-Smad4, FLAG-TIF1γ, or mutants thereof ± a plasmid expressing HA-Ubiquitin (HA-Ub). Extracts were IPed (IP) using anti-Myc antibody, and the IPs were western blotted with the indicated antibodies. Inputs and positions of molecular weight markers are shown. A lighter exposure of the Smad4 blots indicated equal loading. (C) The N-terminal TRIM domain and the C-terminal PHD-bromodomain interact in solution. 293T cells were transfected with the constructs shown. Extracts were IPed using anti-Myc antibody, and the IPs were western blotted with FLAG antibody. Inputs are shown. Molecular Cell 2011 43, 85-96DOI: (10.1016/j.molcel.2011.05.020) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Model of TIF1γ Action (A–C) Model for chromatin-dependent TIF1γ ubiquitination of Smad4. The TRIM domain is indicated in yellow when TIF1γ is inactive; in active TIF1γ, it is orange. The key histone H3 modifications are labeled on both the blue box and the pink circle. The arrow indicates H3 acetylation by a HAT. SBE, Smad binding element; Ub, ubiquitin. For discussion, see text. Molecular Cell 2011 43, 85-96DOI: (10.1016/j.molcel.2011.05.020) Copyright © 2011 Elsevier Inc. Terms and Conditions