Fig 1.
Decreased acetylation of histone H4K16 was observed in the presence of As2O3 HeLa or HEK293T cells.
(A) Declined global H4K16ac in As2O3-exposed HeLa cells. Global acetylation of histone H4K16 and hMOF protein expression in As2O3-treated HeLa cells were measured with immunofluorescence and indicated antibodies. (B) A significant reduction of H4K16ac in As2O3-exposed 293T cells occurred. Representative results from three independent experiments are shown. hMOF and H4K16ac were measured with anti-hMOF and anti-H4K16ac antibodies and final protein signals were visualized with ChemiScope5000 (CLINX, China). (C-D) Quantified protein. Error bars represent standard error of means of 3 independent experiments. Blot images were scanned and signals were densitometrically quantified using Quantity One Basic software (Bio-Rad). Signals of hMOF and H4K16ac were normalized to GAPDH. (E) No changes of hMOF mRNA levels and hMOF transactivation in As2O3-exposed HEK293T cells. Cells were cultured in DMEM medium containing 0.2, 0.4 or 0.8 μM As2O3 for 48 hours. Relative mRNA levels of hMOF and GAPDH (as control) were measured with qRT-PCR (left panel). In addition, luciferase activity of pGL4-hMOF in As2O3-exposed 293T cells was measured and firefly values were normalized by renilla values (right panel).
Fig 2.
As2O3 inhibits hMOF HAT activity in vitro.
(A) Recombinant hMOF. hMOF protein was measured with anti-hMOF antibody. (B) Insect cell expressed/purified HA-hMOF possessing HAT activity. Histones were visualized with Coomassie brilliant R250 blue (CBB) stain (middle panel) and H4K16ac was confirmed with acetylation-specific antibody (top panel). (C) Inhibitory effect of As2O3 on hMOF enzymatic activity.
Fig 3.
As2O3 can bind directly to hMOF.
(A) Schematic of As-immobilized agarose preparation. (B) As-immobilized agarose pulled down hMOF. (C&D) Competitive inhibition between As and hMOF. H2B protein is as negative control. Western blot images were scanned and signals were densitometrically quantified with Quantity One Basic software (Bio-Rad). The percentage of bound hMOF in the flow through appears in D.
Fig 4.
As2O3 binds directly to zinc finger (C2HC) peptide of hMOF.
(A) Schematic of domain structure of hMOF. Chromo, chromatin organization modifier domain; C2HC, zinc finger domain; MYST-HAT, MYST-family histone acetyltransferase domain. The sequences from 208–231 and 247–270 residues are synthetic peptides of hMOF. (B) Analysis of MALDI-TOF mass spectra on interaction between As2O3 and synthetic peptides of hMOF. The molar ratio between the peptides and arsenic is 1:2. (C) UV absorption spectra of the zinc finger peptide. (D) Simulation of binding between zinc finger of hMOF and arsenic atoms. Three-dimensional structure based on the X-ray structure of hMOF (PDB code: 2GIV) [35]. All Cys residues on hMOF are represented in a ball-and-stick depiction. The zinc finger region-C2HC-type is dark cyan. Color coding: green, C; red, O; blue, N; yellow, S. The arsenic atom is pink.
Fig 5.
Over-expression of hMOF inhibited cell sensitivity to As2O3 in 293T cells.
(A) Representative flow cytogram of Annexin V binding (X-axis) versus PI uptake (Y-axis) in 293T cells. Numbers in the upper left and right, lower left and right quadrants represent percentage of damaged, necrotic, live and apoptotic cells, respectively. (B) Quantified percentage of damaged and necrotic cells for (A). Statistical significant difference expressed as **p<0.01 (Student t-test). (C) Reversion of declined H4K16ac in As2O3-exposed 293T cells. 48 h after As2O3 treatment (0.2 and 0.4 μM), cells were harvested and lysed. hMOF and global modification of H4K16ac were measured.
Fig 6.
Knockdown hMOF promoted cell sensitivity to As2O3 in HeLa cells.
(A) Representative flow cytogram of Annexin V binding (X-axis) versus PI uptake (Y-axis) in HeLa cells. (B-C) Quantified percentage of damaged and necrotic cells for (A). Statistical significant difference is expressed as *p<0.05 and **p<0.01 (Student t-test).
Fig 7.
Reduction of global H4K16ac by As2O3 is due to the loss of enzymatic activity of hMOF, but not HDAC4.
(A) As2O3-induced high expression of HDAC4 confirmed by immunofluorescence. (B) Increased HDAC4 protein by As2O3. (C) Overturn of the HDAC4 protein levels by over-expression of hMOF in As2O3-treatedt cells. WCE analyzed by Western blot with anti-HDAC4 antibody. (D) As2O3 blocked recruitment of hMOF on HDAC4 promoter region. ChIP assays were performed using hMOF specific antibody in As2O3 exposed 293T cells. ChIP DNA was measured by qRT-PCR with designed primer sets. (E) Effect of knockdown HDAC4 on global histone H4K16ac. (F) Knockdown of HDAC4 did not stop As2O3-induced reduction of H4K16ac. 3 h after HDAC4 siRNA transfection, cells were exposed to cell culture medium containing 0.5 and 1.0 μM As2O3, and 48 h later, cells were harvested. Proteins were measured with Western blot.