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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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DNA repair–deficient cell lines are unable to efficiently resolve HDAC4 foci. Cells deficient for ATM, DNA-PK, and Nibrin were exposed to 1 Gy of IR and fixed and stained for HDAC4 1 and 24 h after IR. The average percentage of each cell line showing IR-induced HDAC4 was determined and the data presented in a histogram. Representative images of the HDAC4 staining pattern in the respective cell lines 24 h after IR are presented in the right panels. (A) 1 h after IR (1 Gy), respectively 95 ± 4.3% and 89 ± 4.0% of ATM-deficient FT169A and ATM-restored Y25 cells showed induced foci, a difference that was statistically insignificant. In contrast, 24 h after IR, respectively 82 ± 3.1% and 33 ± 5.9% of FT169A and Y25 cells showed induced foci (P < 0.001). PEB cells, derived from FT169A cells via stably transfecting with the empty parental vector (and which remain deficient for ATM protein and radiosensitive) continued to show high levels of induced HDAC4 foci after IR. Representative images of (B) FT169A (ATM−), (C) Y25 (ATM+), and (D) PEB (ATM control) cell lines 24 h after IR. (E) 1 h after IR, respectively 93 ± 6.7% and 95 ± 3.4% of DNA-PK–deficient MO69J and DNA-PK–proficient MO59K cells showed induced foci, a difference that was not significant. In contrast, 24 h after IR, respectively 86 ± 10.5% and 40 ± 9.6% of MO59J and MO59K showed induced foci (P < 0.001). Representative images of (F) MO59J (DNAPK−) and (G) MO59K (DNAPK+) cells 24 h after IR. In the MO59K cells with persistent foci, the number of foci per cell was also consistently fewer than in the MO59J cells (average of 11 ± 3.1 vs. 45 ± 3.7 foci, respectively, per MO59K vs. MO59J cell; P < 0.001). (H) Nibrin-deficient cells (NBS−) showed high levels of HDAC4 foci at both 1 (95 ± 5.4% of cells) and 24 h (83 ± 2.6%) after IR. In contrast, respectively 89 ± 2.4% and 9 ± 3.1% of HeLa cells (HeLa) showed foci. TSA treatment of HeLa cells (HeLa + TSA) did not prevent foci formation, but inhibited their resolution (93 ± 4.5% and 31 ± 14.5% of HeLa cells pretreated with TSA showed foci at 1 and 24 h after IR). Representative images of (I) Nibrin-deficient (NBS−), (J) HeLa (HeLa), and (K) HeLa cells pretreated with TSA (HeLa + TSA) 24 h after IR. Bar, 5 μm. Data represent the average of three experiments. The nuclei are outlined in blue. Error bars indicate the SD.
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fig3: DNA repair–deficient cell lines are unable to efficiently resolve HDAC4 foci. Cells deficient for ATM, DNA-PK, and Nibrin were exposed to 1 Gy of IR and fixed and stained for HDAC4 1 and 24 h after IR. The average percentage of each cell line showing IR-induced HDAC4 was determined and the data presented in a histogram. Representative images of the HDAC4 staining pattern in the respective cell lines 24 h after IR are presented in the right panels. (A) 1 h after IR (1 Gy), respectively 95 ± 4.3% and 89 ± 4.0% of ATM-deficient FT169A and ATM-restored Y25 cells showed induced foci, a difference that was statistically insignificant. In contrast, 24 h after IR, respectively 82 ± 3.1% and 33 ± 5.9% of FT169A and Y25 cells showed induced foci (P < 0.001). PEB cells, derived from FT169A cells via stably transfecting with the empty parental vector (and which remain deficient for ATM protein and radiosensitive) continued to show high levels of induced HDAC4 foci after IR. Representative images of (B) FT169A (ATM−), (C) Y25 (ATM+), and (D) PEB (ATM control) cell lines 24 h after IR. (E) 1 h after IR, respectively 93 ± 6.7% and 95 ± 3.4% of DNA-PK–deficient MO69J and DNA-PK–proficient MO59K cells showed induced foci, a difference that was not significant. In contrast, 24 h after IR, respectively 86 ± 10.5% and 40 ± 9.6% of MO59J and MO59K showed induced foci (P < 0.001). Representative images of (F) MO59J (DNAPK−) and (G) MO59K (DNAPK+) cells 24 h after IR. In the MO59K cells with persistent foci, the number of foci per cell was also consistently fewer than in the MO59J cells (average of 11 ± 3.1 vs. 45 ± 3.7 foci, respectively, per MO59K vs. MO59J cell; P < 0.001). (H) Nibrin-deficient cells (NBS−) showed high levels of HDAC4 foci at both 1 (95 ± 5.4% of cells) and 24 h (83 ± 2.6%) after IR. In contrast, respectively 89 ± 2.4% and 9 ± 3.1% of HeLa cells (HeLa) showed foci. TSA treatment of HeLa cells (HeLa + TSA) did not prevent foci formation, but inhibited their resolution (93 ± 4.5% and 31 ± 14.5% of HeLa cells pretreated with TSA showed foci at 1 and 24 h after IR). Representative images of (I) Nibrin-deficient (NBS−), (J) HeLa (HeLa), and (K) HeLa cells pretreated with TSA (HeLa + TSA) 24 h after IR. Bar, 5 μm. Data represent the average of three experiments. The nuclei are outlined in blue. Error bars indicate the SD.

Mentions: We next examined in a range of human cell lines the genetic determinants that specified HDAC4 foci formation. We found that HDAC4 foci formation did not depend on DNA damage response genes, i.e., ATM (ataxia telangiectasia mutated), Nibrin, or DNA-PK, as cell lines defective for these genes formed foci with similar kinetics as HeLa cells (Fig. 3). However, the repair-deficient cell lines differed from HeLa (and other repair-proficient) cells in the persistence of HDAC4 foci after exposure to low doses of IR. For example, HDAC4 foci readily formed 1 h after radiation in the ATM-deficient FT169 cell line, as well as in its isogenic derivatives Y25 (in which ATM is restored by expression of a full-length cDNA) and PEB (expressing empty vector and hence remaining ATM deficient). 24 h after IR, HDAC4 foci were significantly reduced only in the ATM-positive Y25 cells (Fig. 3, A–D). A similar difference in the resolution of HDAC4 foci was observed between DNA-PK–deficient MO59J cells and DNA-PK–positive MO59K cells. Although MO59K cells did not completely resolve their HDAC4 foci, the average number of foci was less than in the MO59J cells (Fig. 3, E–G). We believe that MO59K cells did not efficiently resolve HDAC4 foci because of their inherent radiosensitivity relative to HeLa cells (Wang et al., 1997; unpublished data), which efficiently resolves foci at low doses of IR (Fig. 3 J). Lastly, we examined HDAC4 foci formation in the radiosensitive Nijmegen breakage syndrome (NBS) mutant cell lines and found that they too retained high levels of foci 24 h after IR (Fig. 3, H and I). We found that foci formation by HDAC4 in HeLa cells was unimpeded by TSA. However, the resolution of HDAC4 foci in HeLa cells was partially inhibited by TSA (Fig. 3, H and K).


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)

DNA repair–deficient cell lines are unable to efficiently resolve HDAC4 foci. Cells deficient for ATM, DNA-PK, and Nibrin were exposed to 1 Gy of IR and fixed and stained for HDAC4 1 and 24 h after IR. The average percentage of each cell line showing IR-induced HDAC4 was determined and the data presented in a histogram. Representative images of the HDAC4 staining pattern in the respective cell lines 24 h after IR are presented in the right panels. (A) 1 h after IR (1 Gy), respectively 95 ± 4.3% and 89 ± 4.0% of ATM-deficient FT169A and ATM-restored Y25 cells showed induced foci, a difference that was statistically insignificant. In contrast, 24 h after IR, respectively 82 ± 3.1% and 33 ± 5.9% of FT169A and Y25 cells showed induced foci (P < 0.001). PEB cells, derived from FT169A cells via stably transfecting with the empty parental vector (and which remain deficient for ATM protein and radiosensitive) continued to show high levels of induced HDAC4 foci after IR. Representative images of (B) FT169A (ATM−), (C) Y25 (ATM+), and (D) PEB (ATM control) cell lines 24 h after IR. (E) 1 h after IR, respectively 93 ± 6.7% and 95 ± 3.4% of DNA-PK–deficient MO69J and DNA-PK–proficient MO59K cells showed induced foci, a difference that was not significant. In contrast, 24 h after IR, respectively 86 ± 10.5% and 40 ± 9.6% of MO59J and MO59K showed induced foci (P < 0.001). Representative images of (F) MO59J (DNAPK−) and (G) MO59K (DNAPK+) cells 24 h after IR. In the MO59K cells with persistent foci, the number of foci per cell was also consistently fewer than in the MO59J cells (average of 11 ± 3.1 vs. 45 ± 3.7 foci, respectively, per MO59K vs. MO59J cell; P < 0.001). (H) Nibrin-deficient cells (NBS−) showed high levels of HDAC4 foci at both 1 (95 ± 5.4% of cells) and 24 h (83 ± 2.6%) after IR. In contrast, respectively 89 ± 2.4% and 9 ± 3.1% of HeLa cells (HeLa) showed foci. TSA treatment of HeLa cells (HeLa + TSA) did not prevent foci formation, but inhibited their resolution (93 ± 4.5% and 31 ± 14.5% of HeLa cells pretreated with TSA showed foci at 1 and 24 h after IR). Representative images of (I) Nibrin-deficient (NBS−), (J) HeLa (HeLa), and (K) HeLa cells pretreated with TSA (HeLa + TSA) 24 h after IR. Bar, 5 μm. Data represent the average of three experiments. The nuclei are outlined in blue. Error bars indicate the SD.
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fig3: DNA repair–deficient cell lines are unable to efficiently resolve HDAC4 foci. Cells deficient for ATM, DNA-PK, and Nibrin were exposed to 1 Gy of IR and fixed and stained for HDAC4 1 and 24 h after IR. The average percentage of each cell line showing IR-induced HDAC4 was determined and the data presented in a histogram. Representative images of the HDAC4 staining pattern in the respective cell lines 24 h after IR are presented in the right panels. (A) 1 h after IR (1 Gy), respectively 95 ± 4.3% and 89 ± 4.0% of ATM-deficient FT169A and ATM-restored Y25 cells showed induced foci, a difference that was statistically insignificant. In contrast, 24 h after IR, respectively 82 ± 3.1% and 33 ± 5.9% of FT169A and Y25 cells showed induced foci (P < 0.001). PEB cells, derived from FT169A cells via stably transfecting with the empty parental vector (and which remain deficient for ATM protein and radiosensitive) continued to show high levels of induced HDAC4 foci after IR. Representative images of (B) FT169A (ATM−), (C) Y25 (ATM+), and (D) PEB (ATM control) cell lines 24 h after IR. (E) 1 h after IR, respectively 93 ± 6.7% and 95 ± 3.4% of DNA-PK–deficient MO69J and DNA-PK–proficient MO59K cells showed induced foci, a difference that was not significant. In contrast, 24 h after IR, respectively 86 ± 10.5% and 40 ± 9.6% of MO59J and MO59K showed induced foci (P < 0.001). Representative images of (F) MO59J (DNAPK−) and (G) MO59K (DNAPK+) cells 24 h after IR. In the MO59K cells with persistent foci, the number of foci per cell was also consistently fewer than in the MO59J cells (average of 11 ± 3.1 vs. 45 ± 3.7 foci, respectively, per MO59K vs. MO59J cell; P < 0.001). (H) Nibrin-deficient cells (NBS−) showed high levels of HDAC4 foci at both 1 (95 ± 5.4% of cells) and 24 h (83 ± 2.6%) after IR. In contrast, respectively 89 ± 2.4% and 9 ± 3.1% of HeLa cells (HeLa) showed foci. TSA treatment of HeLa cells (HeLa + TSA) did not prevent foci formation, but inhibited their resolution (93 ± 4.5% and 31 ± 14.5% of HeLa cells pretreated with TSA showed foci at 1 and 24 h after IR). Representative images of (I) Nibrin-deficient (NBS−), (J) HeLa (HeLa), and (K) HeLa cells pretreated with TSA (HeLa + TSA) 24 h after IR. Bar, 5 μm. Data represent the average of three experiments. The nuclei are outlined in blue. Error bars indicate the SD.
Mentions: We next examined in a range of human cell lines the genetic determinants that specified HDAC4 foci formation. We found that HDAC4 foci formation did not depend on DNA damage response genes, i.e., ATM (ataxia telangiectasia mutated), Nibrin, or DNA-PK, as cell lines defective for these genes formed foci with similar kinetics as HeLa cells (Fig. 3). However, the repair-deficient cell lines differed from HeLa (and other repair-proficient) cells in the persistence of HDAC4 foci after exposure to low doses of IR. For example, HDAC4 foci readily formed 1 h after radiation in the ATM-deficient FT169 cell line, as well as in its isogenic derivatives Y25 (in which ATM is restored by expression of a full-length cDNA) and PEB (expressing empty vector and hence remaining ATM deficient). 24 h after IR, HDAC4 foci were significantly reduced only in the ATM-positive Y25 cells (Fig. 3, A–D). A similar difference in the resolution of HDAC4 foci was observed between DNA-PK–deficient MO59J cells and DNA-PK–positive MO59K cells. Although MO59K cells did not completely resolve their HDAC4 foci, the average number of foci was less than in the MO59J cells (Fig. 3, E–G). We believe that MO59K cells did not efficiently resolve HDAC4 foci because of their inherent radiosensitivity relative to HeLa cells (Wang et al., 1997; unpublished data), which efficiently resolves foci at low doses of IR (Fig. 3 J). Lastly, we examined HDAC4 foci formation in the radiosensitive Nijmegen breakage syndrome (NBS) mutant cell lines and found that they too retained high levels of foci 24 h after IR (Fig. 3, H and I). We found that foci formation by HDAC4 in HeLa cells was unimpeded by TSA. However, the resolution of HDAC4 foci in HeLa cells was partially inhibited by TSA (Fig. 3, H and K).

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