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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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Related in: MedlinePlus

Silencing of HDAC2 and HDAC6 does not abrogate the G2 checkpoint. Parallel plates of cells were treated with control, HDAC2, HDAC4, or HDAC6 siRNA. 36 h after siRNA treatment, cells were irradiated (5 Gy) and harvested 8 h later for immunoblotting (A) and cell cycle analysis by FACS® (B). (A) Immunoblot of cell lysates from cells treated with siRNA. Cell lysates were separated on 7.5% SDS-PAGE, transferred onto nitrocellulose, and the membranes were probed with anti-HDAC4, HDAC6, HDAC2, Mad1, or α-tubulin antibodies. These show efficient silencing of the target proteins by the HDAC2, 4, and 6 siRNA. Mad1 and α-tubulin are not affected by the specific siRNA treatments and serve as loading controls. (B) Cell cycle distribution of treated cells. Cell cycle analysis shows that the silencing of HDAC4 protein had the most conspicuous effect on abrogation of the G2 checkpoint after IR, relative to silencing of HDAC2 and HDAC6.
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fig7: Silencing of HDAC2 and HDAC6 does not abrogate the G2 checkpoint. Parallel plates of cells were treated with control, HDAC2, HDAC4, or HDAC6 siRNA. 36 h after siRNA treatment, cells were irradiated (5 Gy) and harvested 8 h later for immunoblotting (A) and cell cycle analysis by FACS® (B). (A) Immunoblot of cell lysates from cells treated with siRNA. Cell lysates were separated on 7.5% SDS-PAGE, transferred onto nitrocellulose, and the membranes were probed with anti-HDAC4, HDAC6, HDAC2, Mad1, or α-tubulin antibodies. These show efficient silencing of the target proteins by the HDAC2, 4, and 6 siRNA. Mad1 and α-tubulin are not affected by the specific siRNA treatments and serve as loading controls. (B) Cell cycle distribution of treated cells. Cell cycle analysis shows that the silencing of HDAC4 protein had the most conspicuous effect on abrogation of the G2 checkpoint after IR, relative to silencing of HDAC2 and HDAC6.

Mentions: Lastly, we examined whether abrogation of the G2 delay occurs when HDACs other than HDAC4 are silenced. Silencing of HDAC2 and HDAC6 did not interfere with the cell's ability to arrest in G2 in response to DNA damage (Fig. 7, A and B). Only silencing of HDAC4 appreciably diminished the proportion of cells delayed in G2 after IR.


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)

Silencing of HDAC2 and HDAC6 does not abrogate the G2 checkpoint. Parallel plates of cells were treated with control, HDAC2, HDAC4, or HDAC6 siRNA. 36 h after siRNA treatment, cells were irradiated (5 Gy) and harvested 8 h later for immunoblotting (A) and cell cycle analysis by FACS® (B). (A) Immunoblot of cell lysates from cells treated with siRNA. Cell lysates were separated on 7.5% SDS-PAGE, transferred onto nitrocellulose, and the membranes were probed with anti-HDAC4, HDAC6, HDAC2, Mad1, or α-tubulin antibodies. These show efficient silencing of the target proteins by the HDAC2, 4, and 6 siRNA. Mad1 and α-tubulin are not affected by the specific siRNA treatments and serve as loading controls. (B) Cell cycle distribution of treated cells. Cell cycle analysis shows that the silencing of HDAC4 protein had the most conspicuous effect on abrogation of the G2 checkpoint after IR, relative to silencing of HDAC2 and HDAC6.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172769&req=5

fig7: Silencing of HDAC2 and HDAC6 does not abrogate the G2 checkpoint. Parallel plates of cells were treated with control, HDAC2, HDAC4, or HDAC6 siRNA. 36 h after siRNA treatment, cells were irradiated (5 Gy) and harvested 8 h later for immunoblotting (A) and cell cycle analysis by FACS® (B). (A) Immunoblot of cell lysates from cells treated with siRNA. Cell lysates were separated on 7.5% SDS-PAGE, transferred onto nitrocellulose, and the membranes were probed with anti-HDAC4, HDAC6, HDAC2, Mad1, or α-tubulin antibodies. These show efficient silencing of the target proteins by the HDAC2, 4, and 6 siRNA. Mad1 and α-tubulin are not affected by the specific siRNA treatments and serve as loading controls. (B) Cell cycle distribution of treated cells. Cell cycle analysis shows that the silencing of HDAC4 protein had the most conspicuous effect on abrogation of the G2 checkpoint after IR, relative to silencing of HDAC2 and HDAC6.
Mentions: Lastly, we examined whether abrogation of the G2 delay occurs when HDACs other than HDAC4 are silenced. Silencing of HDAC2 and HDAC6 did not interfere with the cell's ability to arrest in G2 in response to DNA damage (Fig. 7, A and B). Only silencing of HDAC4 appreciably diminished the proportion of cells delayed in G2 after IR.

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