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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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HDAC4 foci are induced by IR in interphase cells. (A) Asynchronous HeLa cells were irradiated with 2 Gy IR and 1 h later were fixed and stained for HDAC4, cyclin E, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin E and DAPI. HDAC4 foci are apparent in cells in late G1/early S phase (G1–S) as well as in those not expressing cyclin E and presumably in G2 phase (G2). (B) Cells synchronized and mock irradiated or irradiated (2 Gy) in late S phase were fixed and stained 3 h later for HDAC4, cyclin B1, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin B1 and DAPI. After mock IR (No IR), S-phase cells have not yet expressed cyclin B1 (S), whereas cyclin B1 is cytoplasmic in G2 cells (G2) and coincides with the condensed chromatin in mitotic cells (M) (Kao et al., 1997; Hagting et al., 1999). After IR (IR, 3 h), HDAC4 foci are seen in most cells, including S phase (S) and those that have progressed to and blocked in G2 phase (G2). (C) Synchronized cells were irradiated in late S phase as in B, but fixed 5 h after IR (IR, 5 h). Most cells remain in G2, as indicated by cytoplasmic cyclin B1. However, a few cells have progressed into prophase, as indicated by the entry of cyclin B1 into the nucleus (arrowhead); in these cells, the HDAC4 foci are fewer and less distinct. Bar, 5 μm. (D) Average number of IR-induced HDAC4 foci at each position of the cell cycle. At least 100 cells were counted for each data point, and the experiment was repeated three times with similar results. Error bars represent the SD.
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fig4: HDAC4 foci are induced by IR in interphase cells. (A) Asynchronous HeLa cells were irradiated with 2 Gy IR and 1 h later were fixed and stained for HDAC4, cyclin E, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin E and DAPI. HDAC4 foci are apparent in cells in late G1/early S phase (G1–S) as well as in those not expressing cyclin E and presumably in G2 phase (G2). (B) Cells synchronized and mock irradiated or irradiated (2 Gy) in late S phase were fixed and stained 3 h later for HDAC4, cyclin B1, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin B1 and DAPI. After mock IR (No IR), S-phase cells have not yet expressed cyclin B1 (S), whereas cyclin B1 is cytoplasmic in G2 cells (G2) and coincides with the condensed chromatin in mitotic cells (M) (Kao et al., 1997; Hagting et al., 1999). After IR (IR, 3 h), HDAC4 foci are seen in most cells, including S phase (S) and those that have progressed to and blocked in G2 phase (G2). (C) Synchronized cells were irradiated in late S phase as in B, but fixed 5 h after IR (IR, 5 h). Most cells remain in G2, as indicated by cytoplasmic cyclin B1. However, a few cells have progressed into prophase, as indicated by the entry of cyclin B1 into the nucleus (arrowhead); in these cells, the HDAC4 foci are fewer and less distinct. Bar, 5 μm. (D) Average number of IR-induced HDAC4 foci at each position of the cell cycle. At least 100 cells were counted for each data point, and the experiment was repeated three times with similar results. Error bars represent the SD.

Mentions: HDAC4 foci were induced by DNA damage in nearly all cells, suggesting it was not limited to a specific phase of the cell cycle. We sought to confirm this in both asynchronous and synchronized HeLa cells (Fig. 4 A). Using cyclin E expression to distinguish G1–S from G2 cells, we found IR-induced HDAC4 foci in both. Next, we irradiated synchronized cells in S phase and then harvested when cells were beginning to progress into G2 (Fig. 4 B) or mitosis (Fig. 4 C). The average number of foci per cell was not decreased in the G2 cells but, interestingly, was decreased in prophase cells and not evident in mitotic cells (Fig. 4 D). These observations, however, do not permit us to distinguish whether HDAC4 foci form less efficiently in mitotic cells or whether the foci resolve as cells enter mitosis.


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)

HDAC4 foci are induced by IR in interphase cells. (A) Asynchronous HeLa cells were irradiated with 2 Gy IR and 1 h later were fixed and stained for HDAC4, cyclin E, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin E and DAPI. HDAC4 foci are apparent in cells in late G1/early S phase (G1–S) as well as in those not expressing cyclin E and presumably in G2 phase (G2). (B) Cells synchronized and mock irradiated or irradiated (2 Gy) in late S phase were fixed and stained 3 h later for HDAC4, cyclin B1, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin B1 and DAPI. After mock IR (No IR), S-phase cells have not yet expressed cyclin B1 (S), whereas cyclin B1 is cytoplasmic in G2 cells (G2) and coincides with the condensed chromatin in mitotic cells (M) (Kao et al., 1997; Hagting et al., 1999). After IR (IR, 3 h), HDAC4 foci are seen in most cells, including S phase (S) and those that have progressed to and blocked in G2 phase (G2). (C) Synchronized cells were irradiated in late S phase as in B, but fixed 5 h after IR (IR, 5 h). Most cells remain in G2, as indicated by cytoplasmic cyclin B1. However, a few cells have progressed into prophase, as indicated by the entry of cyclin B1 into the nucleus (arrowhead); in these cells, the HDAC4 foci are fewer and less distinct. Bar, 5 μm. (D) Average number of IR-induced HDAC4 foci at each position of the cell cycle. At least 100 cells were counted for each data point, and the experiment was repeated three times with similar results. Error bars represent the SD.
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fig4: HDAC4 foci are induced by IR in interphase cells. (A) Asynchronous HeLa cells were irradiated with 2 Gy IR and 1 h later were fixed and stained for HDAC4, cyclin E, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin E and DAPI. HDAC4 foci are apparent in cells in late G1/early S phase (G1–S) as well as in those not expressing cyclin E and presumably in G2 phase (G2). (B) Cells synchronized and mock irradiated or irradiated (2 Gy) in late S phase were fixed and stained 3 h later for HDAC4, cyclin B1, and DAPI. The top panels show HDAC4 staining, and the bottom panels show HDAC4 merged with cyclin B1 and DAPI. After mock IR (No IR), S-phase cells have not yet expressed cyclin B1 (S), whereas cyclin B1 is cytoplasmic in G2 cells (G2) and coincides with the condensed chromatin in mitotic cells (M) (Kao et al., 1997; Hagting et al., 1999). After IR (IR, 3 h), HDAC4 foci are seen in most cells, including S phase (S) and those that have progressed to and blocked in G2 phase (G2). (C) Synchronized cells were irradiated in late S phase as in B, but fixed 5 h after IR (IR, 5 h). Most cells remain in G2, as indicated by cytoplasmic cyclin B1. However, a few cells have progressed into prophase, as indicated by the entry of cyclin B1 into the nucleus (arrowhead); in these cells, the HDAC4 foci are fewer and less distinct. Bar, 5 μm. (D) Average number of IR-induced HDAC4 foci at each position of the cell cycle. At least 100 cells were counted for each data point, and the experiment was repeated three times with similar results. Error bars represent the SD.
Mentions: HDAC4 foci were induced by DNA damage in nearly all cells, suggesting it was not limited to a specific phase of the cell cycle. We sought to confirm this in both asynchronous and synchronized HeLa cells (Fig. 4 A). Using cyclin E expression to distinguish G1–S from G2 cells, we found IR-induced HDAC4 foci in both. Next, we irradiated synchronized cells in S phase and then harvested when cells were beginning to progress into G2 (Fig. 4 B) or mitosis (Fig. 4 C). The average number of foci per cell was not decreased in the G2 cells but, interestingly, was decreased in prophase cells and not evident in mitotic cells (Fig. 4 D). These observations, however, do not permit us to distinguish whether HDAC4 foci form less efficiently in mitotic cells or whether the foci resolve as cells enter mitosis.

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