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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

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).
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pone.0141014.g001: 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).

Mentions: As exposure is reported to induce global alteration of histone modification in human cells [12,13,24,25]. To clarify these effects on histone H4 specific lysine sites, we used immunofluorescent staining with acetylation-specific antibodies in As2O3-exposed HeLa cells, and we observed that H4K16ac was reduced (Fig 1A, upper panel), but other histone modifications of H4 did not change much (Figure A in S1 Text). Data were consistent with previous reports that showed histone H4K16ac is decreased after As treatment [24]. To understand whether histone H4K16ac reduction by As2O3 is due to reduced hMOF, we measured hMOF protein expression and observed that hMOF protein did not decline (Fig 1A, lower panel).


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)

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).
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pone.0141014.g001: 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).
Mentions: As exposure is reported to induce global alteration of histone modification in human cells [12,13,24,25]. To clarify these effects on histone H4 specific lysine sites, we used immunofluorescent staining with acetylation-specific antibodies in As2O3-exposed HeLa cells, and we observed that H4K16ac was reduced (Fig 1A, upper panel), but other histone modifications of H4 did not change much (Figure A in S1 Text). Data were consistent with previous reports that showed histone H4K16ac is decreased after As treatment [24]. To understand whether histone H4K16ac reduction by As2O3 is due to reduced hMOF, we measured hMOF protein expression and observed that hMOF protein did not decline (Fig 1A, lower panel).

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