1. Volume 44, Issue 1, Pages 39-50 (October 2011)
HAT4, a Golgi Apparatus-Anchored B-Type Histone Acetyltransferase, Acetylates Free Histone H4 and Facilitates Chromatin Assembly 
Xiaohan Yang, Wenhua Yu, Lei Shi, Luyang Sun, Jing Liang, Xia Yi, Qian Li, Yu Zhang, Fen Yang, Xiao Han, Di Zhang, Jie Yang, Zhi Yao, Yongfeng Shang 
Molecular Cell 
Volume 44, Issue 1, Pages (October 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 Cloning and Characterization of HAT4
(A) Schematic representation of the structure of HAT4 protein.
(B) HAT4 transcribes in multiple normal and cancer tissues. Electronic Northern: For the set of nonfetal normal and cancer human tissues shown, NCBI's UniGene dataset is mined for information about the number of unique clones per gene per tissue. Clones are assigned to particular tissues by applying data-mining heuristics to UniGene's library information file. Electronic expression results were calculated by dividing the number of clones per gene by the number of clones per tissue. They were then normalized by multiplying by 1M, and the obtained normalized counts are presented on the same root scale as the experimental tissue vectors.
(C) Real-time RT-PCR and western blotting analysis of HAT4 expression in different cancer cell lines. GAPDH or β-actin was analyzed as an internal control. Each bar represents the mean ± SD for three independent experiments.
(D) Amino-acid sequence alignment of HAT4 from different species. The same amino acids are starred and shown in red, and the conserved amino acids are shown in green and marked with two points.
(E) Subcellular localization of HAT4 protein. HeLa cells on coverslips were stained with anti-HAT4 or double stained with anti-HAT4 and anti-58K Golgi protein with or without brefeldin A (BFA) treatment.
(F) Subcellular localization of HAT4 and HAT4 mutants. HeLa cells were transfected with FLAG-HAT4 or FLAG-HAT4 mutants (WT: wild-type; ΔA: deletion of transmembrane region A [52–69aa]; ΔB: deletion of transmembrane region B (84–103aa); Δ(A+B) plus: deletion of transmembrane regions A and B and the amino acids between [52–103aa]) and double stained with anti-FLAG and anti-58K Golgi protein. The expression of FLAG-HAT4 deletion mutants was analyzed by western blotting.
(G) HeLa cells were transfected with control or HAT4 siRNA or HAT4 shRNA plasmids together with green fluorescent protein as a transfection efficiency monitor. Western blotting and immunofluorescent imaging were performed to verify the specificity of anti-HAT4 antibodies.
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3. Figure 2
HAT4 Possesses HAT Activity toward Free, but Not Nucleosomal, Histones
(A and B) Bacterially expressed recombinant HAT4 (reHAT4) (A) or mammalian cell-purified HAT4 (B) was incubated with calf thymus histones or nucleosomes in the presence of [3H]acetyl-CoA. The incorporation of [3H]acetate was detected by liquid scintillation counting (left). The reactions were also resolved on SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining to detect total proteins and by autoradiography to detect [3H]acetate-labeled proteins (right). Each bar represents the mean ± SD for three independent experiments. 0, 100 ng, 200 ng, 500 ng, or 1 μg reHAT4 were used.
(C) Mutation of the conserved amino acid (glycine) in acetyl-CoA binding site of HAT4 resulted in the loss of its HAT activity. Bacterially expressed or mammalian cell-purified wild-type HAT4 and HAT4G111A mutant were incubated with calf thymus histones in the presence of [3H]acetyl-CoA. The incorporation of [3H]acetate was detected by liquid scintillation counting (left). The reaction mixtures were also resolved on SDS-PAGE followed by CBB staining to detect total proteins and by autoradiography to detect 3H-acetylated proteins (right). Each bar represents the mean ± SD for three independent experiments.
(D) Experiments were performed as described in (A) and (B), except that recombinant wild-type Xenopus H2A, H2B, H3, and H4 were used as substrates. Each bar represents the mean ± SD for three independent experiments.
(E) Drosophila Hat4b but not Hat4a possess HAT activity. Experiments were performed as described in (C), except that bacterially expressed recombinant Hat4a and Hat4b was used. Each bar represents the mean ± SD for three independent experiments. 0, 100 ng, 200 ng, 500 ng, or 1 μg of GST-Hat4b were used.
(F) reHAT4 was incubated with calf thymus histones and [3H]acetyl-CoA in the presence of FLAG-HAT4G111A-tranfected and anti-FLAG-purified cellular extracts. The reaction mixtures were resolved on SDS-PAGE followed by CBB staining and autoradiography.
(G) Golgi extracts display HAT activity. Subcellular organelles of HeLa cells–Golgi, mitochondria (MT), endoplasmic reticulum (ER), and nuclei–were extracted using the OptiPrep protocol. Calf thymus histones or nucleosomal histones were used as substrates for HAT assays. Each bar represents the mean ± SD for three independent experiments. Western blotting was performed using antibodies against the indicated proteins and the organelle-specific markers; 58K: Golgi; Calreticulin: ER; Tim23: MT; Lamin A: Nuclei.
(H) Acetylation of reHAT4 and FLAG-HAT4 identified by mass spectrometry. Different acetylation profiles were shown. In vitro HAT assays were performed as described in (A) and (B), except for using anti-FLAG-purified immunoprecipitates from FLAG-HAT4-, HAT4K79R-, HAT4K105R-, HAT4K109R-, HAT4K121R-, or HAT4K156R-transfected HeLa cells.
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4. Figure 3 HAT4 Acetylates Free Histone H4 in a Site-Specific Manner
(A) Mass spectrometry analysis of lysine residues of histones acetylated by bacterially expressed HAT4 or mammalian cell-purified HAT4 with bacterially expressed Xenopus histones as substrates.
(B) The effect of overexpression or knockdown of HAT4 on histone acetylation in U2OS cells. U2OS cells were transfected with HAT4 or treated with HAT4 siRNA and/or HAT1 siRNA. The soluble histones were prepared and their acetylation levels were analyzed by western blotting.
(C) Mass spectrometry analysis of H4 acetylation by HAT4 in vivo. U2OS cells were treated with control or HAT4 siRNA. Cytoplasmic histones were extracted and resolved on SDS-PAGE followed by CBB staining. Histone bands were retrieved and analyzed by LC-MS-MS and MOLDI-MS-TOFF for acetylation modifications. Each bar represents the mean ± SD for three independent experiments.
(D) Verification of HAT4 knockdown in U2OS and MCF-7 cells by real-time RT PCR, western blotting, or immunofluorescent imaging. Each bar represents the mean ± SD for three independent experiments.
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5. Figure 4 HAT4 Facilitates Proper Nucleosome Assembly
(A) HAT4 facilitates CAF-1-mediated nucleosome formation onto replicating DNA. Human 293 cells were treated with HAT1 and/or HAT4 siRNA. Plasmid pSV011 was replicated in siRNA-treated human 293 S100 extracts in the presence of T antigen, [α-32P]-dATP, and CAF-1. Total DNA was visualized by ethidium bromide staining and by autoradiography. Migration of form I negatively supercoiled and form II/I0 relaxed monomer circular DNA is indicated. Middle panel: Radioactively labeled supercoiled products were quantified by densitometry scan of the autoradiogram. Right panel: Micrococcal nuclease digestion analysis of chromatin assembled onto newly synthesized DNA. Mononucleosome (Mono-) and dinucleosome (Di-) are indicated.
(B) U2OS cells were treated with HAT1 siRNA and/or HAT4 siRNA, arrested by double thymidine block, and then released to S phase. Nuclei were then prepared and treated with 2.5 or 5 units/ml of MNase as indicated. Genomic DNA was purified and resolved by agarose gel electrophoresis. Mononucleosome (Mono-), dinucleosome (Di-), and trinucleosome (Tri-) are indicated.
(C) The expression profile of HAT4. HeLa cells were synchronized in S phase by double thymidine block, and the expression of HAT4 mRNA and protein were measured at the indicated times after release by real-time RT PCR and western blotting, respectively. Each bar represents the mean ± SD for three independent experiments.
(D) HAT4 knockdown led to the accumulation of soluble histones in cells. HeLa cells were synchronized in S phase by double thymidine block and released. Histone H3 expression levels were analyzed by western blotting with β-actin as internal control.
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6. Figure 5 Depletion of HAT4 Inhibits Cell Growth
(A) Depletion of HAT4 is associated with inhibition of cell proliferation. Control or HAT1 and/or HAT4 siRNA-treated MCF-7 cells were split into 96-well plates and then harvested at day 1, 2, 3, 4, or 5. The growth curves of the cells were measured by Alamar blue cell viability assays. Each point represents the mean ± SD for three independent experiments.
(B) Hat4b but not Hat4a partially rescued the effect of HAT4 knockdown. MCF-7 cells were transfected with pGCsi-Nonsilencer, pGCsi-HAT4 shRNA, pGCSi-HAT4 shRNA plus pcDNA3.1-FLAG-Hat4a, or pGCSi-HAT4 shRNA plus pcDNA3.1-FLAG-Hat4b. Cells were maintained in G418-containing medium for 14 days before staining with crystal violet and counting for colony numbers. Each bar represents the mean ± SD for three independent experiments.
(C) MCF-7 cells treated with control siRNA or HAT4 siRNA were synchronized by serum starvation, double thymidine block, or thymidine nocodazole block at G0/G1, G1/S, or the G2/M boundary, respectively. Cell-cycle progression after releasing was analyzed by flow cytometry.
(D) Hat4b but not Hat4a could partially rescue the delay of the cell cycle associated with HAT4 knockdown. MCF-7 cells were transfected with control, HAT4 shRNA, HAT4 shRNA plus Hat4a, or HAT4 shRNA plus Hat4b. Transfected cells were subjected to cell-cycle analysis by flow cytometry.
Molecular Cell  , 39-50DOI: ( /j.molcel )
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7. Figure 6
Depletion of HAT4 Sensitizes Cells to DNA Damage and Induces Apoptosis
(A) U2OS cells were treated with control siRNA or HAT1 siRNA and/or HAT4 siRNA. Thirty-two hours later, cells were treated with UV irradiation, 2 mM hydroxyurea, or 100 nM CPT for 12 or 24 hr followed by Alamar blue staining to detect their viability. Each bar represents the mean ± SD for three independent experiments. Post hoc tests were performed in conjunction with one-way ANOVA using SPSS 17.0 software for statistical analysis. Stars indicate that the differences of those groups are significant compared with the control (p < 0.01).
(B) HAT4 knockdown promotes cell apoptosis. siRNA-treated U2OS cells were double stained with annexin V and propidium iodide. Cell apoptosis was determined by flow cytometry. Each bar represents the mean ± SD for three independent experiments. The protein levels of p53 and H2AXP-S139 in siRNA-treated and double thymidine-synchronized U2OS cells were measured by western blotting.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions