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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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Silencing of HDAC4 expression abrogates the DNA damage–induced G2 checkpoint and decreases cell viability. HeLa cells were transfected with control or HDAC4 siRNA, followed 36 h later by mock (No IR) or 5-Gy IR (IR). 8 h after IR, parallel plates of cells were harvested, and cell cycle distribution was analyzed via flow cytometry (A and B) or immunofluorescence (C and D). (A) Silencing of HDAC4 does not have a large effect on unirradiated cells, but it reduced the proportion of irradiated cells with G2/M DNA content. Cells with DNA content less than G1 cells (sub-G1) likely represent apoptotic cells/cell debris, and is especially apparent in cells silenced for HDAC4 and irradiated. (B) Histograms showing cell cycle distribution data from A, indicating the percentage of cells in each part of the cell cycle. The experiment was repeated three times with similar results. (C and D) To better distinguish the proportions of the total population of G2 cells, cells were stained for cyclin B1, CENP-F, and DAPI and analyzed via immunofluorescence. G2-phase cells show cytoplasmic cyclin B1 staining, diffuse nuclear CENP-F staining, and uncondensed chromatin (stained with DAPI) (Liao et al., 1995; Kao et al., 2001). (C) For each pair of images, the left panel shows cyclin B1 or CENP-F staining, and the right panel shows the same cells stained with DAPI. Cells treated with control siRNA show cytoplasmic cyclin B1 and nuclear CENP-F staining, indicative of the IR-induced G2 checkpoint. In contrast, few HDAC4 siRNA–treated cells were blocked in G2, but instead showed fragmented and condensed chromatin that retained cyclin B1 and CENP-F staining. (D) Histogram showing the distribution of CENP-F staining cells with uncondensed (G2 blocked) versus condensed or fragmented chromatin after IR of cells treated with control or HDAC4 siRNA. At least 300 cells were counted for each experiment. (E) Histogram showing plating efficiency of untreated controls or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were plated onto fresh dishes with fresh media 48 h after siRNA treatment, and the proportion of surviving cells capable of excluding Trypan blue was determined 12 h later. The experiment was repeated three times with similar results. Error bars indicate the SD. (F) Survival curves indicating the radiosensitivity of control cells or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were prepared as in E and irradiated with the doses indicated, and the proportion of surviving colonies was determined 2 wk later. All values were corrected for the plating efficiency. Error bars indicate the SD.
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fig6: Silencing of HDAC4 expression abrogates the DNA damage–induced G2 checkpoint and decreases cell viability. HeLa cells were transfected with control or HDAC4 siRNA, followed 36 h later by mock (No IR) or 5-Gy IR (IR). 8 h after IR, parallel plates of cells were harvested, and cell cycle distribution was analyzed via flow cytometry (A and B) or immunofluorescence (C and D). (A) Silencing of HDAC4 does not have a large effect on unirradiated cells, but it reduced the proportion of irradiated cells with G2/M DNA content. Cells with DNA content less than G1 cells (sub-G1) likely represent apoptotic cells/cell debris, and is especially apparent in cells silenced for HDAC4 and irradiated. (B) Histograms showing cell cycle distribution data from A, indicating the percentage of cells in each part of the cell cycle. The experiment was repeated three times with similar results. (C and D) To better distinguish the proportions of the total population of G2 cells, cells were stained for cyclin B1, CENP-F, and DAPI and analyzed via immunofluorescence. G2-phase cells show cytoplasmic cyclin B1 staining, diffuse nuclear CENP-F staining, and uncondensed chromatin (stained with DAPI) (Liao et al., 1995; Kao et al., 2001). (C) For each pair of images, the left panel shows cyclin B1 or CENP-F staining, and the right panel shows the same cells stained with DAPI. Cells treated with control siRNA show cytoplasmic cyclin B1 and nuclear CENP-F staining, indicative of the IR-induced G2 checkpoint. In contrast, few HDAC4 siRNA–treated cells were blocked in G2, but instead showed fragmented and condensed chromatin that retained cyclin B1 and CENP-F staining. (D) Histogram showing the distribution of CENP-F staining cells with uncondensed (G2 blocked) versus condensed or fragmented chromatin after IR of cells treated with control or HDAC4 siRNA. At least 300 cells were counted for each experiment. (E) Histogram showing plating efficiency of untreated controls or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were plated onto fresh dishes with fresh media 48 h after siRNA treatment, and the proportion of surviving cells capable of excluding Trypan blue was determined 12 h later. The experiment was repeated three times with similar results. Error bars indicate the SD. (F) Survival curves indicating the radiosensitivity of control cells or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were prepared as in E and irradiated with the doses indicated, and the proportion of surviving colonies was determined 2 wk later. All values were corrected for the plating efficiency. Error bars indicate the SD.

Mentions: We further studied the effects of silencing HDAC4 on cell cycle distribution and cellular viability after DNA damage. Treatment with HDAC4 siRNA did not appear to have a major effect in altering the cell cycle distribution of unirradiated HeLa cells, compared with cells treated with control siRNA (Fig. 6, A and B) or untreated cells (unpublished data). IR of HeLa cells treated with control siRNA resulted in the expected accumulation of cells in G2, characteristic of the IR-induced checkpoint. In contrast, treatment with HDAC4 siRNA markedly decreased the proportion of cells with G2/M DNA content but was accompanied by an increase in cells with DNA content less than 2N (sub-G1) (Fig. 6, A and B). This reduction in G2/M cells was not due to an S-phase delay because HDAC4 siRNA did not affect normal S-phase progression, as determined by FACS® analysis and BrdU incorporation (unpublished data). Flow cytometry based on DNA content alone does not distinguish between cells in G2 and mitosis. We therefore performed immunofluorescence on irradiated cells that were transfected with control and HDAC4 siRNA. Control cells were blocked in G2 after IR, as determined by high cyclin B1 levels in the cytoplasm, CENP-F distributed uniformly in the nucleus, and uncondensed chromatin (Fig. 6, C and D), as described previously (Liao et al., 1995; Kao et al., 2001). In contrast, few of the HDAC4 siRNA–treated cells appeared to be in G2. Although many of these cells still retained cyclin B1 and CENP-F staining, the cyclin B1 was often not uniformly cytoplasmic, the CENP-F staining of the nuclei was frequently uneven, and many of nuclei were condensed or fragmented. Many of these condensed cells (>70%), in fact, showed high levels of phosphohistone H3 staining, along with cyclin B1, suggesting progression into mitosis (see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200209065/DC1). These observations together suggest that cells silenced for HDAC4 do not maintain a uniform G2 delay after IR. One notes, however, that as 53BP1 has been established to mediate the damage-induced G2 checkpoint, the abrogation of this checkpoint by silencing HDAC4 may be mediated, at least in part, through decreasing 53BP1 protein (DiTullio et al., 2002; Fernandez-Capetillo et al., 2002; Wang et al., 2002).


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 HDAC4 expression abrogates the DNA damage–induced G2 checkpoint and decreases cell viability. HeLa cells were transfected with control or HDAC4 siRNA, followed 36 h later by mock (No IR) or 5-Gy IR (IR). 8 h after IR, parallel plates of cells were harvested, and cell cycle distribution was analyzed via flow cytometry (A and B) or immunofluorescence (C and D). (A) Silencing of HDAC4 does not have a large effect on unirradiated cells, but it reduced the proportion of irradiated cells with G2/M DNA content. Cells with DNA content less than G1 cells (sub-G1) likely represent apoptotic cells/cell debris, and is especially apparent in cells silenced for HDAC4 and irradiated. (B) Histograms showing cell cycle distribution data from A, indicating the percentage of cells in each part of the cell cycle. The experiment was repeated three times with similar results. (C and D) To better distinguish the proportions of the total population of G2 cells, cells were stained for cyclin B1, CENP-F, and DAPI and analyzed via immunofluorescence. G2-phase cells show cytoplasmic cyclin B1 staining, diffuse nuclear CENP-F staining, and uncondensed chromatin (stained with DAPI) (Liao et al., 1995; Kao et al., 2001). (C) For each pair of images, the left panel shows cyclin B1 or CENP-F staining, and the right panel shows the same cells stained with DAPI. Cells treated with control siRNA show cytoplasmic cyclin B1 and nuclear CENP-F staining, indicative of the IR-induced G2 checkpoint. In contrast, few HDAC4 siRNA–treated cells were blocked in G2, but instead showed fragmented and condensed chromatin that retained cyclin B1 and CENP-F staining. (D) Histogram showing the distribution of CENP-F staining cells with uncondensed (G2 blocked) versus condensed or fragmented chromatin after IR of cells treated with control or HDAC4 siRNA. At least 300 cells were counted for each experiment. (E) Histogram showing plating efficiency of untreated controls or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were plated onto fresh dishes with fresh media 48 h after siRNA treatment, and the proportion of surviving cells capable of excluding Trypan blue was determined 12 h later. The experiment was repeated three times with similar results. Error bars indicate the SD. (F) Survival curves indicating the radiosensitivity of control cells or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were prepared as in E and irradiated with the doses indicated, and the proportion of surviving colonies was determined 2 wk later. All values were corrected for the plating efficiency. Error bars indicate the SD.
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Related In: Results  -  Collection

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fig6: Silencing of HDAC4 expression abrogates the DNA damage–induced G2 checkpoint and decreases cell viability. HeLa cells were transfected with control or HDAC4 siRNA, followed 36 h later by mock (No IR) or 5-Gy IR (IR). 8 h after IR, parallel plates of cells were harvested, and cell cycle distribution was analyzed via flow cytometry (A and B) or immunofluorescence (C and D). (A) Silencing of HDAC4 does not have a large effect on unirradiated cells, but it reduced the proportion of irradiated cells with G2/M DNA content. Cells with DNA content less than G1 cells (sub-G1) likely represent apoptotic cells/cell debris, and is especially apparent in cells silenced for HDAC4 and irradiated. (B) Histograms showing cell cycle distribution data from A, indicating the percentage of cells in each part of the cell cycle. The experiment was repeated three times with similar results. (C and D) To better distinguish the proportions of the total population of G2 cells, cells were stained for cyclin B1, CENP-F, and DAPI and analyzed via immunofluorescence. G2-phase cells show cytoplasmic cyclin B1 staining, diffuse nuclear CENP-F staining, and uncondensed chromatin (stained with DAPI) (Liao et al., 1995; Kao et al., 2001). (C) For each pair of images, the left panel shows cyclin B1 or CENP-F staining, and the right panel shows the same cells stained with DAPI. Cells treated with control siRNA show cytoplasmic cyclin B1 and nuclear CENP-F staining, indicative of the IR-induced G2 checkpoint. In contrast, few HDAC4 siRNA–treated cells were blocked in G2, but instead showed fragmented and condensed chromatin that retained cyclin B1 and CENP-F staining. (D) Histogram showing the distribution of CENP-F staining cells with uncondensed (G2 blocked) versus condensed or fragmented chromatin after IR of cells treated with control or HDAC4 siRNA. At least 300 cells were counted for each experiment. (E) Histogram showing plating efficiency of untreated controls or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were plated onto fresh dishes with fresh media 48 h after siRNA treatment, and the proportion of surviving cells capable of excluding Trypan blue was determined 12 h later. The experiment was repeated three times with similar results. Error bars indicate the SD. (F) Survival curves indicating the radiosensitivity of control cells or cells treated with control, HDAC4, or 53BP1 siRNA. Cells were prepared as in E and irradiated with the doses indicated, and the proportion of surviving colonies was determined 2 wk later. All values were corrected for the plating efficiency. Error bars indicate the SD.
Mentions: We further studied the effects of silencing HDAC4 on cell cycle distribution and cellular viability after DNA damage. Treatment with HDAC4 siRNA did not appear to have a major effect in altering the cell cycle distribution of unirradiated HeLa cells, compared with cells treated with control siRNA (Fig. 6, A and B) or untreated cells (unpublished data). IR of HeLa cells treated with control siRNA resulted in the expected accumulation of cells in G2, characteristic of the IR-induced checkpoint. In contrast, treatment with HDAC4 siRNA markedly decreased the proportion of cells with G2/M DNA content but was accompanied by an increase in cells with DNA content less than 2N (sub-G1) (Fig. 6, A and B). This reduction in G2/M cells was not due to an S-phase delay because HDAC4 siRNA did not affect normal S-phase progression, as determined by FACS® analysis and BrdU incorporation (unpublished data). Flow cytometry based on DNA content alone does not distinguish between cells in G2 and mitosis. We therefore performed immunofluorescence on irradiated cells that were transfected with control and HDAC4 siRNA. Control cells were blocked in G2 after IR, as determined by high cyclin B1 levels in the cytoplasm, CENP-F distributed uniformly in the nucleus, and uncondensed chromatin (Fig. 6, C and D), as described previously (Liao et al., 1995; Kao et al., 2001). In contrast, few of the HDAC4 siRNA–treated cells appeared to be in G2. Although many of these cells still retained cyclin B1 and CENP-F staining, the cyclin B1 was often not uniformly cytoplasmic, the CENP-F staining of the nuclei was frequently uneven, and many of nuclei were condensed or fragmented. Many of these condensed cells (>70%), in fact, showed high levels of phosphohistone H3 staining, along with cyclin B1, suggesting progression into mitosis (see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200209065/DC1). These observations together suggest that cells silenced for HDAC4 do not maintain a uniform G2 delay after IR. One notes, however, that as 53BP1 has been established to mediate the damage-induced G2 checkpoint, the abrogation of this checkpoint by silencing HDAC4 may be mediated, at least in part, through decreasing 53BP1 protein (DiTullio et al., 2002; Fernandez-Capetillo et al., 2002; Wang et al., 2002).

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