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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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RNAi efficiently silences HDAC4 and 53BP1 protein expression. (A) HeLa cells were transfected with siRNA targeting two different sequences in HDAC4 or control siRNA, harvested 36 h later, and immunoblotted for HDAC4 and α-tubulin (loading control). Whole cell lysates from untransfected cells serve as a positive control. (B) Time course of HDAC4 siRNA–mediated silencing of protein expression. Parallel plates of HeLa cells were treated with HDAC4 siRNA and harvested at the times indicated. All lysates were separated on 7.5% SDS-PAGE and immunoblotted for HDAC4 or α-tubulin, indicating maximal silencing of the targeted HDAC4 protein by 36 h. (C and D) HDAC4 siRNA diminishes foci formation after IR. HeLa cells were treated with oligofectamine only, control siRNA, or HDAC4 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed and immunofluorescence for HDAC4 was performed. Representative cells are shown in C. The number of HDAC4 foci found in each cell was counted. At least 300 cells were counted for each treatment group. (D) The average number of HDAC4 foci per cell after IR for each treatment group is displayed in the histograms. (E) HDAC4 and 53BP1 siRNA silence expression of their targeted protein, as well as each other. HeLa cells were transfected with HDAC4 or 53BP1 siRNA and harvested 48 h later. Lysates were separated on SDS-PAGE and immunoblotted for 53BP1, HDAC4, and α-tubulin. (F) siRNA-mediated silencing of 53BP1 and HDAC4 protein expression is achieved by 24 h after treatment. Parallel plates of HeLa cells were transfected with 53BP1 siRNA and harvested at the indicated times after transfection. Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-53BP1, HDAC4, and α-tubulin antibodies. (G) HDAC4 and 53BP1 siRNA reduce only their target mRNA. RT-PCR was performed on total mRNA extracted from HeLa cells 48 h after treatment with control, HDAC4, or 53BP1 siRNA. RT-PCR was performed in parallel under identical conditions using primer pairs targeting 53BP1, HDAC4, or GADPH (as control), and the final product was separated via electrophoresis in ethidium bromide–labeled agarose and photographed under UV illumination. Each RT-PCR reaction yielded a single band as shown. (H and I) HDAC4 siRNA diminishes 53BP1 foci formation after IR. HeLa cells were transfected with oligofectamine control, HDAC4 siRNA, control siRNA, or 53BP1 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed, immunofluorescence for 53BP1 was performed, and the average number of foci per cell was determined and displayed in the histograms in H. Representative cells are shown in I.
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fig5: RNAi efficiently silences HDAC4 and 53BP1 protein expression. (A) HeLa cells were transfected with siRNA targeting two different sequences in HDAC4 or control siRNA, harvested 36 h later, and immunoblotted for HDAC4 and α-tubulin (loading control). Whole cell lysates from untransfected cells serve as a positive control. (B) Time course of HDAC4 siRNA–mediated silencing of protein expression. Parallel plates of HeLa cells were treated with HDAC4 siRNA and harvested at the times indicated. All lysates were separated on 7.5% SDS-PAGE and immunoblotted for HDAC4 or α-tubulin, indicating maximal silencing of the targeted HDAC4 protein by 36 h. (C and D) HDAC4 siRNA diminishes foci formation after IR. HeLa cells were treated with oligofectamine only, control siRNA, or HDAC4 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed and immunofluorescence for HDAC4 was performed. Representative cells are shown in C. The number of HDAC4 foci found in each cell was counted. At least 300 cells were counted for each treatment group. (D) The average number of HDAC4 foci per cell after IR for each treatment group is displayed in the histograms. (E) HDAC4 and 53BP1 siRNA silence expression of their targeted protein, as well as each other. HeLa cells were transfected with HDAC4 or 53BP1 siRNA and harvested 48 h later. Lysates were separated on SDS-PAGE and immunoblotted for 53BP1, HDAC4, and α-tubulin. (F) siRNA-mediated silencing of 53BP1 and HDAC4 protein expression is achieved by 24 h after treatment. Parallel plates of HeLa cells were transfected with 53BP1 siRNA and harvested at the indicated times after transfection. Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-53BP1, HDAC4, and α-tubulin antibodies. (G) HDAC4 and 53BP1 siRNA reduce only their target mRNA. RT-PCR was performed on total mRNA extracted from HeLa cells 48 h after treatment with control, HDAC4, or 53BP1 siRNA. RT-PCR was performed in parallel under identical conditions using primer pairs targeting 53BP1, HDAC4, or GADPH (as control), and the final product was separated via electrophoresis in ethidium bromide–labeled agarose and photographed under UV illumination. Each RT-PCR reaction yielded a single band as shown. (H and I) HDAC4 siRNA diminishes 53BP1 foci formation after IR. HeLa cells were transfected with oligofectamine control, HDAC4 siRNA, control siRNA, or 53BP1 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed, immunofluorescence for 53BP1 was performed, and the average number of foci per cell was determined and displayed in the histograms in H. Representative cells are shown in I.

Mentions: To assess further the functions of HDAC4, we silenced its expression by RNA interference (RNAi) (Elbashir et al., 2001). We found that short interfering RNA (siRNA) directed against two regions of HDAC4 efficiently repressed levels of HDAC4 protein at 48 h after transfection. A control siRNA that was unrelated to HDAC4 had no effect (Fig. 5 A). We conducted a time course experiment to determine the optimal time when HDAC4 expression was at its lowest after siRNA treatment. Considerable amounts of HDAC4 protein were detectable 12 h after transfection, but the level of protein progressively decreased such that very little HDAC4 protein was detectable by 36 h, indicating that it is optimal to assess silencing after 24 h (Fig. 5 B). We next examined the effect of the HDAC4 siRNA on foci formation after IR. As expected, HDAC4 foci formation after IR was substantially reduced with both HDAC4 siRNAs, whereas the control siRNA had no effect (Fig. 5, C and D).


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)

RNAi efficiently silences HDAC4 and 53BP1 protein expression. (A) HeLa cells were transfected with siRNA targeting two different sequences in HDAC4 or control siRNA, harvested 36 h later, and immunoblotted for HDAC4 and α-tubulin (loading control). Whole cell lysates from untransfected cells serve as a positive control. (B) Time course of HDAC4 siRNA–mediated silencing of protein expression. Parallel plates of HeLa cells were treated with HDAC4 siRNA and harvested at the times indicated. All lysates were separated on 7.5% SDS-PAGE and immunoblotted for HDAC4 or α-tubulin, indicating maximal silencing of the targeted HDAC4 protein by 36 h. (C and D) HDAC4 siRNA diminishes foci formation after IR. HeLa cells were treated with oligofectamine only, control siRNA, or HDAC4 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed and immunofluorescence for HDAC4 was performed. Representative cells are shown in C. The number of HDAC4 foci found in each cell was counted. At least 300 cells were counted for each treatment group. (D) The average number of HDAC4 foci per cell after IR for each treatment group is displayed in the histograms. (E) HDAC4 and 53BP1 siRNA silence expression of their targeted protein, as well as each other. HeLa cells were transfected with HDAC4 or 53BP1 siRNA and harvested 48 h later. Lysates were separated on SDS-PAGE and immunoblotted for 53BP1, HDAC4, and α-tubulin. (F) siRNA-mediated silencing of 53BP1 and HDAC4 protein expression is achieved by 24 h after treatment. Parallel plates of HeLa cells were transfected with 53BP1 siRNA and harvested at the indicated times after transfection. Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-53BP1, HDAC4, and α-tubulin antibodies. (G) HDAC4 and 53BP1 siRNA reduce only their target mRNA. RT-PCR was performed on total mRNA extracted from HeLa cells 48 h after treatment with control, HDAC4, or 53BP1 siRNA. RT-PCR was performed in parallel under identical conditions using primer pairs targeting 53BP1, HDAC4, or GADPH (as control), and the final product was separated via electrophoresis in ethidium bromide–labeled agarose and photographed under UV illumination. Each RT-PCR reaction yielded a single band as shown. (H and I) HDAC4 siRNA diminishes 53BP1 foci formation after IR. HeLa cells were transfected with oligofectamine control, HDAC4 siRNA, control siRNA, or 53BP1 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed, immunofluorescence for 53BP1 was performed, and the average number of foci per cell was determined and displayed in the histograms in H. Representative cells are shown in I.
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fig5: RNAi efficiently silences HDAC4 and 53BP1 protein expression. (A) HeLa cells were transfected with siRNA targeting two different sequences in HDAC4 or control siRNA, harvested 36 h later, and immunoblotted for HDAC4 and α-tubulin (loading control). Whole cell lysates from untransfected cells serve as a positive control. (B) Time course of HDAC4 siRNA–mediated silencing of protein expression. Parallel plates of HeLa cells were treated with HDAC4 siRNA and harvested at the times indicated. All lysates were separated on 7.5% SDS-PAGE and immunoblotted for HDAC4 or α-tubulin, indicating maximal silencing of the targeted HDAC4 protein by 36 h. (C and D) HDAC4 siRNA diminishes foci formation after IR. HeLa cells were treated with oligofectamine only, control siRNA, or HDAC4 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed and immunofluorescence for HDAC4 was performed. Representative cells are shown in C. The number of HDAC4 foci found in each cell was counted. At least 300 cells were counted for each treatment group. (D) The average number of HDAC4 foci per cell after IR for each treatment group is displayed in the histograms. (E) HDAC4 and 53BP1 siRNA silence expression of their targeted protein, as well as each other. HeLa cells were transfected with HDAC4 or 53BP1 siRNA and harvested 48 h later. Lysates were separated on SDS-PAGE and immunoblotted for 53BP1, HDAC4, and α-tubulin. (F) siRNA-mediated silencing of 53BP1 and HDAC4 protein expression is achieved by 24 h after treatment. Parallel plates of HeLa cells were transfected with 53BP1 siRNA and harvested at the indicated times after transfection. Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-53BP1, HDAC4, and α-tubulin antibodies. (G) HDAC4 and 53BP1 siRNA reduce only their target mRNA. RT-PCR was performed on total mRNA extracted from HeLa cells 48 h after treatment with control, HDAC4, or 53BP1 siRNA. RT-PCR was performed in parallel under identical conditions using primer pairs targeting 53BP1, HDAC4, or GADPH (as control), and the final product was separated via electrophoresis in ethidium bromide–labeled agarose and photographed under UV illumination. Each RT-PCR reaction yielded a single band as shown. (H and I) HDAC4 siRNA diminishes 53BP1 foci formation after IR. HeLa cells were transfected with oligofectamine control, HDAC4 siRNA, control siRNA, or 53BP1 siRNA and irradiated 36 h later with 2 Gy. 1 h after IR, the cells were fixed, immunofluorescence for 53BP1 was performed, and the average number of foci per cell was determined and displayed in the histograms in H. Representative cells are shown in I.
Mentions: To assess further the functions of HDAC4, we silenced its expression by RNA interference (RNAi) (Elbashir et al., 2001). We found that short interfering RNA (siRNA) directed against two regions of HDAC4 efficiently repressed levels of HDAC4 protein at 48 h after transfection. A control siRNA that was unrelated to HDAC4 had no effect (Fig. 5 A). We conducted a time course experiment to determine the optimal time when HDAC4 expression was at its lowest after siRNA treatment. Considerable amounts of HDAC4 protein were detectable 12 h after transfection, but the level of protein progressively decreased such that very little HDAC4 protein was detectable by 36 h, indicating that it is optimal to assess silencing after 24 h (Fig. 5 B). We next examined the effect of the HDAC4 siRNA on foci formation after IR. As expected, HDAC4 foci formation after IR was substantially reduced with both HDAC4 siRNAs, whereas the control siRNA had no effect (Fig. 5, C and D).

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