Acetylation Regulates Transcription Factor Activity at Multiple Levels Evi Soutoglou, Nitsa Katrakili, Iannis Talianidis  Molecular Cell  Volume 5, Issue 4, Pages 745-751 (April 2000) DOI: 10.1016/S1097-2765(00)80253-1

Figure 1 HNF-4 Is Acetylated by CBP (A and B) One microgram of purified recombinant 6× His-tagged HNF-4 (HisHNF-4) or crude core histones (Histones) or the indicated GST-HNF-4 fusion proteins were subjected to in vitro acetylation reaction with 50 ng of baculovirus-expressed wild type (wtCBP) or its L1690K;C1691L mutated form (CBP HAT−) and analyzed by SDS-PAGE. (C) The domain structure of HNF-4 and sequence of the 80–121 aa region encompassing HNF-4 NLS (Sladek et al. 1990). Mutated residues are underlined. Wild-type and mutant GST-HNF-4 (91–142) were acetylated and analyzed as above. (D) In vivo acetylation of HNF-4. HepG2 cells and Cos1 cells transfected with pCDNA expression vectors containing the indicated cDNAs were metabolically labeled with [3H]acetate, and whole-cell extracts were prepared, immunoprecipitated with HNF-4 antibody, and analyzed by SDS-PAGE (Autoradiograph). Parts of the extracts were used for Western blot analysis with the same antibody (Western blot). Molecular Cell 2000 5, 745-751DOI: (10.1016/S1097-2765(00)80253-1)

Figure 2 CBP-Mediated Acetylation Is Important for HNF-4-Dependent Gene Activation In Vivo (A) NIH3T3 cells were transfected with the indicated linearized expression vectors and selected with G418 (0.6 mg/ml) for 16 days. Stably transfected colonies were pooled, whole-cell extracts were prepared as described, and the expression of HNF-1 and HNF-4 was detected in Western blots with HNF-1 (αHNF-1) and HNF-4 (αHNF-4) antibodies, respectively. (B) NIH3T3 cells were transiently transfected with 2 μg of HNF-4-responsive reporter 1xA-TK-CAT, 0.2 μg of wild-type or the indicated mutant HNF-4 plasmids, and 2 μg of wtCBP and CBP HAT− expression vectors as indicated. (C) NIH3T3 cells were transfected with 2 μg G4-CAT reporter, 0.2 μg of the indicated Gal-4 HNF-4 fusion constructs, and 2 μg of wtCBP and mutant vectors. Bars represent means ± SE of normalized CAT activities from at least five independent experiments and expressed as fold induction above the activity obtained by wtHNF-4 alone (B), or by Gal4 HNF-4 (1–455) (C). Molecular Cell 2000 5, 745-751DOI: (10.1016/S1097-2765(00)80253-1)

Figure 3 Acetylation Increases HNF-4 DNA Binding Activity and the Affinity of Its Interaction with CBP In Vitro (A) The indicated amounts of recombinant HNF-4 were incubated with 50 ng of baculovirus-expressed CBP in the absence or presence of acetyl CoA (1 mM) for 1 hr at 30°C, and subjected to electrophoretic mobility shift assays (EMSA) using the siteA probe. Protein-bound and unbound fractions were quantified by phosphorimage analysis, and the ratios of bound/total radioactivities were compared. (B) Whole-cell extracts from Cos1 cells transfected with the indicated expression vectors were analyzed by EMSA. Two different amounts of the extracts containing equal quantities of HNF-4 were tested. (C) Glutathione-Sepharose beads containing 2 μg of GST HNF-4FL fusion protein were incubated with CBP containing crude Sf9 extracts in interaction buffer for 5 hr at 4°C. After extensive washing to remove noninteracted proteins, the beads were incubated in acetylation buffer with or without cold acetyl-CoA (1 mM) for 1 hr at 30°C. After the acetylation reaction, the beads were extensively washed with interaction buffer containing the indicated KCl concentrations. HNF-4-bound CBP was detected in Western blots using αCBP-22 antibody (Santa Cruz). (D) Whole-cell extracts from (B) were incubated with the indicated amounts of alkaline protease (Promega) for 10 min at 25°C, and the cleavage products were analyzed in Western blots using HNF-4 antibody. Arrows indicate a protease-induced double band. Molecular Cell 2000 5, 745-751DOI: (10.1016/S1097-2765(00)80253-1)

Figure 4 CBP-Mediated Acetylation Is Important for the Retention of HNF-4 in the Nucleus (A) Cos1 cells were transfected with the indicated plasmids, transferred to glass coverslips, and stained with αHNF-4 antibody. Parallel samples were treated with 10 ng/ml leptomycin B (LMB) for 4 hr before fixing. Typical examples of the immunofluorescent images are shown (magnification 300×). (B) Nuclear and cytoplasmic extracts from Cos1 cells transfected with the indicated plasmids were prepared and analyzed in Western blots with αHNF-4 antibody. (C) Cos1 cells were cotransfected with the indicated plasmids together with CRM1 expression vector. Samples were treated and processed as above. (D) Direct interaction of CRM-1 with HNF-4. Whole-cell extracts from Cos1 cells transfected with the indicated plasmids were incubated with TALON resin-associated His-tagged CRM1 (HisCRM1). Aliquots corresponding to 3% of the input (Input) and the entire bound fractions were analyzed in Western blots with αHNF-4 antibody. Molecular Cell 2000 5, 745-751DOI: (10.1016/S1097-2765(00)80253-1)