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Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response.

Kao GD, McKenna WG, Guenther MG, Muschel RJ, Lazar MA, Yen TJ - J. Cell Biol. (2003)

Bottom Line: Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear.Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells.Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. kao@xrt.upenn.edu

ABSTRACT
Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.

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Dose and time dependency of DNA damage–induced HDAC4 foci. (A) HDAC4-associated deacetylase activity is not appreciably changed by IR. HDAC4 or Ncor was immunoprecipitated from HeLa lysates and assayed for deacetylase activity. Ncor and HDAC4 treated with TSA serve as positive and negative controls, respectively, for the deacetylase assays. (B) HDAC4 levels do not change after IR. Equal amounts of total protein (25 μg) from HeLa cells were separated via 4–20% gradient SDS-PAGE, transferred to nitrocellulose, and probed with rabbit preimmune serum (left), anti-HDAC4 (right), or anti–α-tubulin antibody (loading control) (bottom). Mock-irradiated (−IR) or irradiated (+IR) cells were harvested 1 h after 3 Gy. (C) HDAC4 foci formation is dependent on the dose of IR. HeLa cells exposed to increasing doses of IR were fixed after 1 h and stained for HDAC4 foci. At least 100 cells were counted for each dose. Error bars represent the SD, and each point represents the average of three experiments. (D) Kinetics of HDAC4 foci formation. HeLa cells irradiated with 1 and 8 Gy were fixed at various times, and the average number of cells showing induced HDAC4 foci was counted. Time is depicted on a log scale. (E) Comparison of the kinetics of foci formation amongst HDAC4, 53BP1, and Rad51. HeLa cells were irradiated (3 Gy), fixed at the indicated times, and separately stained for HDAC4, 53BP1, and Rad51 foci. Error bars represent the SD, and each point represents the average of three experiments. (F) Coimmunoprecipitation of HDAC4 and 53BP1. HeLa cells were lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) and, for each sample, 50 μg of lysate from unirradiated (IR−) or irradiated cells (IR+) was incubated with the indicated antibodies or preimmune serum (IP). The immunoprecipitates were separated on 7.5% SDS-PAGE gels and probed for 53BP1 (top) and HDAC4 (bottom) (IB). Total cell lysate (25 μg, Whole cell) served as a positive control for the immunoblots. (G) Colocalization of HDAC4 foci relative to 53BP1 and Rad51 foci. HeLa cells were irradiated with 3 Gy and 1 h later costained with (a) rabbit anti-HDAC4 and (b) rat anti-53BP1. (c) Images from A and B were merged to reveal that most foci were coincident. The nuclei are outlined in blue. Bar, 5 μm.
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fig2: Dose and time dependency of DNA damage–induced HDAC4 foci. (A) HDAC4-associated deacetylase activity is not appreciably changed by IR. HDAC4 or Ncor was immunoprecipitated from HeLa lysates and assayed for deacetylase activity. Ncor and HDAC4 treated with TSA serve as positive and negative controls, respectively, for the deacetylase assays. (B) HDAC4 levels do not change after IR. Equal amounts of total protein (25 μg) from HeLa cells were separated via 4–20% gradient SDS-PAGE, transferred to nitrocellulose, and probed with rabbit preimmune serum (left), anti-HDAC4 (right), or anti–α-tubulin antibody (loading control) (bottom). Mock-irradiated (−IR) or irradiated (+IR) cells were harvested 1 h after 3 Gy. (C) HDAC4 foci formation is dependent on the dose of IR. HeLa cells exposed to increasing doses of IR were fixed after 1 h and stained for HDAC4 foci. At least 100 cells were counted for each dose. Error bars represent the SD, and each point represents the average of three experiments. (D) Kinetics of HDAC4 foci formation. HeLa cells irradiated with 1 and 8 Gy were fixed at various times, and the average number of cells showing induced HDAC4 foci was counted. Time is depicted on a log scale. (E) Comparison of the kinetics of foci formation amongst HDAC4, 53BP1, and Rad51. HeLa cells were irradiated (3 Gy), fixed at the indicated times, and separately stained for HDAC4, 53BP1, and Rad51 foci. Error bars represent the SD, and each point represents the average of three experiments. (F) Coimmunoprecipitation of HDAC4 and 53BP1. HeLa cells were lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) and, for each sample, 50 μg of lysate from unirradiated (IR−) or irradiated cells (IR+) was incubated with the indicated antibodies or preimmune serum (IP). The immunoprecipitates were separated on 7.5% SDS-PAGE gels and probed for 53BP1 (top) and HDAC4 (bottom) (IB). Total cell lysate (25 μg, Whole cell) served as a positive control for the immunoblots. (G) Colocalization of HDAC4 foci relative to 53BP1 and Rad51 foci. HeLa cells were irradiated with 3 Gy and 1 h later costained with (a) rabbit anti-HDAC4 and (b) rat anti-53BP1. (c) Images from A and B were merged to reveal that most foci were coincident. The nuclei are outlined in blue. Bar, 5 μm.

Mentions: The formation of HDAC4 foci after exposure to DNA damage was not accompanied by significant changes in HDAC4 deacetylase activity or protein levels (Fig. 2, A and B). We noted however a dose-dependent increase in the average number of HDAC4 foci per cell (Fig. 2 C). IR resulted in approximately 30 foci per Gy up to 3 Gy, beyond which the increase in the number of foci with increasing dose was difficult to discern. Kinetics of foci formation was the same between cells exposed to nonlethal (1 Gy) and lethal (8 Gy) doses (Fig. 2 D). HDAC4 foci were evident within 5 min after IR and reached a maximum by 1 h. Interestingly, the number of foci in cells irradiated with 1 Gy progressively decreased to background levels by 24 h after IR. In contrast, the majority (87 ± 7%) of HeLa cells irradiated with 8 Gy retained their HDAC4 foci as long as 48 h after IR. Foci were evident beyond 48 h when there was a high level of cell death (unpublished data). The persistence of HDAC4 foci therefore appears to correlate with lethal doses of IR, which presumably result in accumulation of unrepaired DNA.


Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response.

Kao GD, McKenna WG, Guenther MG, Muschel RJ, Lazar MA, Yen TJ - J. Cell Biol. (2003)

Dose and time dependency of DNA damage–induced HDAC4 foci. (A) HDAC4-associated deacetylase activity is not appreciably changed by IR. HDAC4 or Ncor was immunoprecipitated from HeLa lysates and assayed for deacetylase activity. Ncor and HDAC4 treated with TSA serve as positive and negative controls, respectively, for the deacetylase assays. (B) HDAC4 levels do not change after IR. Equal amounts of total protein (25 μg) from HeLa cells were separated via 4–20% gradient SDS-PAGE, transferred to nitrocellulose, and probed with rabbit preimmune serum (left), anti-HDAC4 (right), or anti–α-tubulin antibody (loading control) (bottom). Mock-irradiated (−IR) or irradiated (+IR) cells were harvested 1 h after 3 Gy. (C) HDAC4 foci formation is dependent on the dose of IR. HeLa cells exposed to increasing doses of IR were fixed after 1 h and stained for HDAC4 foci. At least 100 cells were counted for each dose. Error bars represent the SD, and each point represents the average of three experiments. (D) Kinetics of HDAC4 foci formation. HeLa cells irradiated with 1 and 8 Gy were fixed at various times, and the average number of cells showing induced HDAC4 foci was counted. Time is depicted on a log scale. (E) Comparison of the kinetics of foci formation amongst HDAC4, 53BP1, and Rad51. HeLa cells were irradiated (3 Gy), fixed at the indicated times, and separately stained for HDAC4, 53BP1, and Rad51 foci. Error bars represent the SD, and each point represents the average of three experiments. (F) Coimmunoprecipitation of HDAC4 and 53BP1. HeLa cells were lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) and, for each sample, 50 μg of lysate from unirradiated (IR−) or irradiated cells (IR+) was incubated with the indicated antibodies or preimmune serum (IP). The immunoprecipitates were separated on 7.5% SDS-PAGE gels and probed for 53BP1 (top) and HDAC4 (bottom) (IB). Total cell lysate (25 μg, Whole cell) served as a positive control for the immunoblots. (G) Colocalization of HDAC4 foci relative to 53BP1 and Rad51 foci. HeLa cells were irradiated with 3 Gy and 1 h later costained with (a) rabbit anti-HDAC4 and (b) rat anti-53BP1. (c) Images from A and B were merged to reveal that most foci were coincident. The nuclei are outlined in blue. Bar, 5 μm.
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fig2: Dose and time dependency of DNA damage–induced HDAC4 foci. (A) HDAC4-associated deacetylase activity is not appreciably changed by IR. HDAC4 or Ncor was immunoprecipitated from HeLa lysates and assayed for deacetylase activity. Ncor and HDAC4 treated with TSA serve as positive and negative controls, respectively, for the deacetylase assays. (B) HDAC4 levels do not change after IR. Equal amounts of total protein (25 μg) from HeLa cells were separated via 4–20% gradient SDS-PAGE, transferred to nitrocellulose, and probed with rabbit preimmune serum (left), anti-HDAC4 (right), or anti–α-tubulin antibody (loading control) (bottom). Mock-irradiated (−IR) or irradiated (+IR) cells were harvested 1 h after 3 Gy. (C) HDAC4 foci formation is dependent on the dose of IR. HeLa cells exposed to increasing doses of IR were fixed after 1 h and stained for HDAC4 foci. At least 100 cells were counted for each dose. Error bars represent the SD, and each point represents the average of three experiments. (D) Kinetics of HDAC4 foci formation. HeLa cells irradiated with 1 and 8 Gy were fixed at various times, and the average number of cells showing induced HDAC4 foci was counted. Time is depicted on a log scale. (E) Comparison of the kinetics of foci formation amongst HDAC4, 53BP1, and Rad51. HeLa cells were irradiated (3 Gy), fixed at the indicated times, and separately stained for HDAC4, 53BP1, and Rad51 foci. Error bars represent the SD, and each point represents the average of three experiments. (F) Coimmunoprecipitation of HDAC4 and 53BP1. HeLa cells were lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) and, for each sample, 50 μg of lysate from unirradiated (IR−) or irradiated cells (IR+) was incubated with the indicated antibodies or preimmune serum (IP). The immunoprecipitates were separated on 7.5% SDS-PAGE gels and probed for 53BP1 (top) and HDAC4 (bottom) (IB). Total cell lysate (25 μg, Whole cell) served as a positive control for the immunoblots. (G) Colocalization of HDAC4 foci relative to 53BP1 and Rad51 foci. HeLa cells were irradiated with 3 Gy and 1 h later costained with (a) rabbit anti-HDAC4 and (b) rat anti-53BP1. (c) Images from A and B were merged to reveal that most foci were coincident. The nuclei are outlined in blue. Bar, 5 μm.
Mentions: The formation of HDAC4 foci after exposure to DNA damage was not accompanied by significant changes in HDAC4 deacetylase activity or protein levels (Fig. 2, A and B). We noted however a dose-dependent increase in the average number of HDAC4 foci per cell (Fig. 2 C). IR resulted in approximately 30 foci per Gy up to 3 Gy, beyond which the increase in the number of foci with increasing dose was difficult to discern. Kinetics of foci formation was the same between cells exposed to nonlethal (1 Gy) and lethal (8 Gy) doses (Fig. 2 D). HDAC4 foci were evident within 5 min after IR and reached a maximum by 1 h. Interestingly, the number of foci in cells irradiated with 1 Gy progressively decreased to background levels by 24 h after IR. In contrast, the majority (87 ± 7%) of HeLa cells irradiated with 8 Gy retained their HDAC4 foci as long as 48 h after IR. Foci were evident beyond 48 h when there was a high level of cell death (unpublished data). The persistence of HDAC4 foci therefore appears to correlate with lethal doses of IR, which presumably result in accumulation of unrepaired DNA.

Bottom Line: Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear.Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells.Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. kao@xrt.upenn.edu

ABSTRACT
Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.

Show MeSH
Related in: MedlinePlus