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Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells.

Liu D, Wu D, Zhao L, Yang Y, Ding J, Dong L, Hu L, Wang F, Zhao X, Cai Y, Jin J - PLoS ONE (2015)

Bottom Line: Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells.However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation.In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jilin University, Changchun, Jilin 130012, China; School of Pharmacy, Changchun University of Traditional Chinese Medicine, Changchun 130117, China.

ABSTRACT
Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0141014.g007: 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.

Mentions: Histone acetylation, one of the best characterized epigenetic modifications, is controlled by histone HATs and histone deacetylases (HDACs). Recent studies suggest that the global modification status of H4K16ac is also affected by some HDAC complexes, such as SIRT1 and HDCA2 [26,36]. To know whether As2O3-induced decreases in histone H4K16ac was related with HDACs, we measured HDACs expression using Western blot and observed that As2O3-dependent increases of HDAC4 occurred. As shown in Fig 7A and 7B, elevation co-incident HDAC4 expression was confirmed and increased with increasing As concentration. Also, elevated HDAC4 was reversed by over-expressing hMOF (1 μg) (Fig 7C), providing evidence that hMOF may regulate cellular HDAC4 protein. To confirm this, ChIP assays were performed for hMOF in 293T cells with/without As2O3-treatment. Fig 7D shows that hMOF enriched at -0.47 kb upstream of the HDAC4 transcriptional start site on HDAC4 was blocked in As2O3-exposed cells. To learn whether increased HDAC4 is involved in hypo-acetylation of histone H4K16, HDAC4 knockdown experiments were performed with specific siRNAs, but global histone H4K16ac did not increase after HDAC4 knock down (Fig 7E). Moreover, depletion of HDAC4 could not block reduction of histone H4K16ac in As2O3-exposed cells, suggesting that HDAC4 may not be directly involved in deacetylation of H4K16 (Fig 7F).


Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells.

Liu D, Wu D, Zhao L, Yang Y, Ding J, Dong L, Hu L, Wang F, Zhao X, Cai Y, Jin J - PLoS ONE (2015)

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.
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pone.0141014.g007: 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.
Mentions: Histone acetylation, one of the best characterized epigenetic modifications, is controlled by histone HATs and histone deacetylases (HDACs). Recent studies suggest that the global modification status of H4K16ac is also affected by some HDAC complexes, such as SIRT1 and HDCA2 [26,36]. To know whether As2O3-induced decreases in histone H4K16ac was related with HDACs, we measured HDACs expression using Western blot and observed that As2O3-dependent increases of HDAC4 occurred. As shown in Fig 7A and 7B, elevation co-incident HDAC4 expression was confirmed and increased with increasing As concentration. Also, elevated HDAC4 was reversed by over-expressing hMOF (1 μg) (Fig 7C), providing evidence that hMOF may regulate cellular HDAC4 protein. To confirm this, ChIP assays were performed for hMOF in 293T cells with/without As2O3-treatment. Fig 7D shows that hMOF enriched at -0.47 kb upstream of the HDAC4 transcriptional start site on HDAC4 was blocked in As2O3-exposed cells. To learn whether increased HDAC4 is involved in hypo-acetylation of histone H4K16, HDAC4 knockdown experiments were performed with specific siRNAs, but global histone H4K16ac did not increase after HDAC4 knock down (Fig 7E). Moreover, depletion of HDAC4 could not block reduction of histone H4K16ac in As2O3-exposed cells, suggesting that HDAC4 may not be directly involved in deacetylation of H4K16 (Fig 7F).

Bottom Line: Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells.However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation.In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jilin University, Changchun, Jilin 130012, China; School of Pharmacy, Changchun University of Traditional Chinese Medicine, Changchun 130117, China.

ABSTRACT
Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity.

No MeSH data available.


Related in: MedlinePlus