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Regulation of 53BP1 protein stability by RNF8 and RNF168 is important for efficient DNA double-strand break repair.

Hu Y, Wang C, Huang K, Xia F, Parvin JD, Mondal N - PLoS ONE (2014)

Bottom Line: In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168.Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form.Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
53BP1 regulates DNA double-strand break (DSB) repair. In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168. We observed that 53BP1 protein was diffusely abundant in nuclei, and upon ionizing radiation, 53BP1 was everywhere degraded except at DNA damage sites. Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form. Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished. Our data suggest a novel mechanism for responding to DSB that upon ionizing radiation, 53BP1 was divided into two populations, ensuring functional DSB repair: damage site-bound 53BP1 whose binding signal is known to be generated by RNF8 and RNF168; and unbound bulk 53BP1 whose ensuing degradation is regulated by RNF8 and RNF168.

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53BP1 protein abundance decreases upon irradiation.A. HeLa cells were subjected to 10 Gy X-rays and total cell lysates were prepared in 2% SDS containing buffer at the indicated time points. Immunoblots of indicated protein were shown. H4 was a loading control. The positions of the molecular mass markers in kDa (K) are indicated at the left. B. HA-53BP1 plasmid was transfected into 293T cells, and 48 h post-transfection, cells were irradiated and extracted in SDS containing buffer; NT indicates no transfection (lanes 1–4). Retinal pigment epithelia cells (RPE; lanes 5, 6) and normal mammary epithelial cells (MCF10A; lanes 7, 8) were exposed to 10 Gy X-rays and total cell lysates were prepared in SDS containing buffer 4 h after irradiation. HeLa cells were exposed to 10 Gy irradiation similarly and total cell lysates were extracted 4 h post-IR in buffer containing 2% SDS and 8 M urea (lanes 9, 10). Immunoblots were developed using anti-HA antibody to detect HA-tagged 53BP1 protein in 293T cells (lane 1–4), and antibody specific for 53BP1 to recognize endogenous 53BP1 in RPE, MCF10A and HeLa cells (lane 5–10). C. HeLa cells were subjected to immunofluorescence microscopy 4 h post-irradiation (10 Gy). Cells were stained for 53BP1 (green; top) and merged with DAPI stain of DNA (blue; bottom). D. Similar to the immunofluorescence microscopy experiment in panel C, RPE and MCF10A cells were stained for 53BP1 protein before and after irradiation. For the immunofluorescence images, 100 nuclei were analyzed in HeLa cells, and 30 nuclei were analyzed in RPE or MCF10A cells using ImageJ software. Mean intensity of 53BP1 signal at foci regions in each nucleus in irradiation sample were scored and normalized according to the mean intensity of diffuse 53BP1 signal in no irradiation sample. Results (mean +/− SEM) were shown for three cell lines. E. The distribution of pixel intensities of 53BP1 nuclear stain in HeLa cells, as in panel C, was plotted for No IR (blue) and IR (red). Results were shown from 80–100 nuclei in each sample. Pixel intensities within the range of 850–1400 were shown in the graph inset.
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pone-0110522-g002: 53BP1 protein abundance decreases upon irradiation.A. HeLa cells were subjected to 10 Gy X-rays and total cell lysates were prepared in 2% SDS containing buffer at the indicated time points. Immunoblots of indicated protein were shown. H4 was a loading control. The positions of the molecular mass markers in kDa (K) are indicated at the left. B. HA-53BP1 plasmid was transfected into 293T cells, and 48 h post-transfection, cells were irradiated and extracted in SDS containing buffer; NT indicates no transfection (lanes 1–4). Retinal pigment epithelia cells (RPE; lanes 5, 6) and normal mammary epithelial cells (MCF10A; lanes 7, 8) were exposed to 10 Gy X-rays and total cell lysates were prepared in SDS containing buffer 4 h after irradiation. HeLa cells were exposed to 10 Gy irradiation similarly and total cell lysates were extracted 4 h post-IR in buffer containing 2% SDS and 8 M urea (lanes 9, 10). Immunoblots were developed using anti-HA antibody to detect HA-tagged 53BP1 protein in 293T cells (lane 1–4), and antibody specific for 53BP1 to recognize endogenous 53BP1 in RPE, MCF10A and HeLa cells (lane 5–10). C. HeLa cells were subjected to immunofluorescence microscopy 4 h post-irradiation (10 Gy). Cells were stained for 53BP1 (green; top) and merged with DAPI stain of DNA (blue; bottom). D. Similar to the immunofluorescence microscopy experiment in panel C, RPE and MCF10A cells were stained for 53BP1 protein before and after irradiation. For the immunofluorescence images, 100 nuclei were analyzed in HeLa cells, and 30 nuclei were analyzed in RPE or MCF10A cells using ImageJ software. Mean intensity of 53BP1 signal at foci regions in each nucleus in irradiation sample were scored and normalized according to the mean intensity of diffuse 53BP1 signal in no irradiation sample. Results (mean +/− SEM) were shown for three cell lines. E. The distribution of pixel intensities of 53BP1 nuclear stain in HeLa cells, as in panel C, was plotted for No IR (blue) and IR (red). Results were shown from 80–100 nuclei in each sample. Pixel intensities within the range of 850–1400 were shown in the graph inset.

Mentions: 53BP1 forms ionizing radiation induced foci (IRIF) in response to DNA damage, and the protein has highest abundance during G1 phase, a stage in the cell cycle associated with NHEJ activity [44]. To investigate 53BP1 protein dynamics in response to irradiation-induced DNA damage, we evaluated changes in 53BP1 protein bulk level four hours post-irradiation (10 Gy). Surprisingly, the 53BP1 protein level decreased markedly compared to the non-irradiated control (Figure 2A, lanes 1–6). In this experiment we were careful to extract all of the 53BP1 protein in the HeLa cells by including 2% SDS in the lysis solution followed by a thorough sonication and heating at 100°C. Thus, the absence of 53BP1 protein in the irradiated cell lysates was not due to compartmentalization of the protein into an insoluble fraction. In contrast to 53BP1, the protein abundance of the homologous recombination factor RAD51 did not change upon irradiation. The DSB damage signal sensor γ-H2AX was used as a positive control and histone H4 served as the loading control. We found that 53BP1 protein levels decreased to very low concentration as early as 15 minutes following ionizing radiation and were restored after 24 hours (Figure 2A, lanes 2–7). In order to rule out that the detection of degradation of 53BP1 protein was specific for the antibody, we also tested for degradation when using HA-tagged 53BP1 protein expressed from a transiently transfected plasmid. Upon irradiation of 293T cells transfected with a HA-53BP1 expression plasmid and lysis in SDS containing buffer, detection of the HA-53BP1 via its epitope tag also revealed a substantial decrease in protein levels 4 h post-irradiation (Figure 2B, lanes 1, 2). Detection of the HA-53BP1 protein by the anti-HA antibody was specific for transfected samples (Figure 2B, lane 3, 4). Furthermore, in two non-cancer cell lines, human retinal pigment epithelium (RPE) cells and normal mammary epithelium MCF10A cells, the endogenous 53BP1 protein levels in 2% SDS lysates were diminished 4 h post-IR (Figure 2B, lane 6, 8), consistent with the results from the HeLa and 293T cell lines. To rule out any potential artifact due to the incomplete extraction of 53BP1 protein from chromatin, urea containing buffer (8 M urea and 2% SDS in phosphate-buffered saline) was used as a reliable means for the complete extraction of the chromatin-bound protein followed by a thorough sonication and heating at 100°C. 53BP1 protein extracted by this method showed a greatly diminished level 4 h post-IR in HeLa cells, compared to no irradiation (Figure 2B, lane 9, 10), further confirming the above results. The level of repair protein RAD51 did not change upon irradiation. γ-H2AX was a positive damage sensor and histone H4 was used as a loading control in 293T, RPE, MCF10A and HeLa cell lysates (Figure 2B).


Regulation of 53BP1 protein stability by RNF8 and RNF168 is important for efficient DNA double-strand break repair.

Hu Y, Wang C, Huang K, Xia F, Parvin JD, Mondal N - PLoS ONE (2014)

53BP1 protein abundance decreases upon irradiation.A. HeLa cells were subjected to 10 Gy X-rays and total cell lysates were prepared in 2% SDS containing buffer at the indicated time points. Immunoblots of indicated protein were shown. H4 was a loading control. The positions of the molecular mass markers in kDa (K) are indicated at the left. B. HA-53BP1 plasmid was transfected into 293T cells, and 48 h post-transfection, cells were irradiated and extracted in SDS containing buffer; NT indicates no transfection (lanes 1–4). Retinal pigment epithelia cells (RPE; lanes 5, 6) and normal mammary epithelial cells (MCF10A; lanes 7, 8) were exposed to 10 Gy X-rays and total cell lysates were prepared in SDS containing buffer 4 h after irradiation. HeLa cells were exposed to 10 Gy irradiation similarly and total cell lysates were extracted 4 h post-IR in buffer containing 2% SDS and 8 M urea (lanes 9, 10). Immunoblots were developed using anti-HA antibody to detect HA-tagged 53BP1 protein in 293T cells (lane 1–4), and antibody specific for 53BP1 to recognize endogenous 53BP1 in RPE, MCF10A and HeLa cells (lane 5–10). C. HeLa cells were subjected to immunofluorescence microscopy 4 h post-irradiation (10 Gy). Cells were stained for 53BP1 (green; top) and merged with DAPI stain of DNA (blue; bottom). D. Similar to the immunofluorescence microscopy experiment in panel C, RPE and MCF10A cells were stained for 53BP1 protein before and after irradiation. For the immunofluorescence images, 100 nuclei were analyzed in HeLa cells, and 30 nuclei were analyzed in RPE or MCF10A cells using ImageJ software. Mean intensity of 53BP1 signal at foci regions in each nucleus in irradiation sample were scored and normalized according to the mean intensity of diffuse 53BP1 signal in no irradiation sample. Results (mean +/− SEM) were shown for three cell lines. E. The distribution of pixel intensities of 53BP1 nuclear stain in HeLa cells, as in panel C, was plotted for No IR (blue) and IR (red). Results were shown from 80–100 nuclei in each sample. Pixel intensities within the range of 850–1400 were shown in the graph inset.
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Related In: Results  -  Collection

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Show All Figures
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pone-0110522-g002: 53BP1 protein abundance decreases upon irradiation.A. HeLa cells were subjected to 10 Gy X-rays and total cell lysates were prepared in 2% SDS containing buffer at the indicated time points. Immunoblots of indicated protein were shown. H4 was a loading control. The positions of the molecular mass markers in kDa (K) are indicated at the left. B. HA-53BP1 plasmid was transfected into 293T cells, and 48 h post-transfection, cells were irradiated and extracted in SDS containing buffer; NT indicates no transfection (lanes 1–4). Retinal pigment epithelia cells (RPE; lanes 5, 6) and normal mammary epithelial cells (MCF10A; lanes 7, 8) were exposed to 10 Gy X-rays and total cell lysates were prepared in SDS containing buffer 4 h after irradiation. HeLa cells were exposed to 10 Gy irradiation similarly and total cell lysates were extracted 4 h post-IR in buffer containing 2% SDS and 8 M urea (lanes 9, 10). Immunoblots were developed using anti-HA antibody to detect HA-tagged 53BP1 protein in 293T cells (lane 1–4), and antibody specific for 53BP1 to recognize endogenous 53BP1 in RPE, MCF10A and HeLa cells (lane 5–10). C. HeLa cells were subjected to immunofluorescence microscopy 4 h post-irradiation (10 Gy). Cells were stained for 53BP1 (green; top) and merged with DAPI stain of DNA (blue; bottom). D. Similar to the immunofluorescence microscopy experiment in panel C, RPE and MCF10A cells were stained for 53BP1 protein before and after irradiation. For the immunofluorescence images, 100 nuclei were analyzed in HeLa cells, and 30 nuclei were analyzed in RPE or MCF10A cells using ImageJ software. Mean intensity of 53BP1 signal at foci regions in each nucleus in irradiation sample were scored and normalized according to the mean intensity of diffuse 53BP1 signal in no irradiation sample. Results (mean +/− SEM) were shown for three cell lines. E. The distribution of pixel intensities of 53BP1 nuclear stain in HeLa cells, as in panel C, was plotted for No IR (blue) and IR (red). Results were shown from 80–100 nuclei in each sample. Pixel intensities within the range of 850–1400 were shown in the graph inset.
Mentions: 53BP1 forms ionizing radiation induced foci (IRIF) in response to DNA damage, and the protein has highest abundance during G1 phase, a stage in the cell cycle associated with NHEJ activity [44]. To investigate 53BP1 protein dynamics in response to irradiation-induced DNA damage, we evaluated changes in 53BP1 protein bulk level four hours post-irradiation (10 Gy). Surprisingly, the 53BP1 protein level decreased markedly compared to the non-irradiated control (Figure 2A, lanes 1–6). In this experiment we were careful to extract all of the 53BP1 protein in the HeLa cells by including 2% SDS in the lysis solution followed by a thorough sonication and heating at 100°C. Thus, the absence of 53BP1 protein in the irradiated cell lysates was not due to compartmentalization of the protein into an insoluble fraction. In contrast to 53BP1, the protein abundance of the homologous recombination factor RAD51 did not change upon irradiation. The DSB damage signal sensor γ-H2AX was used as a positive control and histone H4 served as the loading control. We found that 53BP1 protein levels decreased to very low concentration as early as 15 minutes following ionizing radiation and were restored after 24 hours (Figure 2A, lanes 2–7). In order to rule out that the detection of degradation of 53BP1 protein was specific for the antibody, we also tested for degradation when using HA-tagged 53BP1 protein expressed from a transiently transfected plasmid. Upon irradiation of 293T cells transfected with a HA-53BP1 expression plasmid and lysis in SDS containing buffer, detection of the HA-53BP1 via its epitope tag also revealed a substantial decrease in protein levels 4 h post-irradiation (Figure 2B, lanes 1, 2). Detection of the HA-53BP1 protein by the anti-HA antibody was specific for transfected samples (Figure 2B, lane 3, 4). Furthermore, in two non-cancer cell lines, human retinal pigment epithelium (RPE) cells and normal mammary epithelium MCF10A cells, the endogenous 53BP1 protein levels in 2% SDS lysates were diminished 4 h post-IR (Figure 2B, lane 6, 8), consistent with the results from the HeLa and 293T cell lines. To rule out any potential artifact due to the incomplete extraction of 53BP1 protein from chromatin, urea containing buffer (8 M urea and 2% SDS in phosphate-buffered saline) was used as a reliable means for the complete extraction of the chromatin-bound protein followed by a thorough sonication and heating at 100°C. 53BP1 protein extracted by this method showed a greatly diminished level 4 h post-IR in HeLa cells, compared to no irradiation (Figure 2B, lane 9, 10), further confirming the above results. The level of repair protein RAD51 did not change upon irradiation. γ-H2AX was a positive damage sensor and histone H4 was used as a loading control in 293T, RPE, MCF10A and HeLa cell lysates (Figure 2B).

Bottom Line: In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168.Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form.Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
53BP1 regulates DNA double-strand break (DSB) repair. In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168. We observed that 53BP1 protein was diffusely abundant in nuclei, and upon ionizing radiation, 53BP1 was everywhere degraded except at DNA damage sites. Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form. Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished. Our data suggest a novel mechanism for responding to DSB that upon ionizing radiation, 53BP1 was divided into two populations, ensuring functional DSB repair: damage site-bound 53BP1 whose binding signal is known to be generated by RNF8 and RNF168; and unbound bulk 53BP1 whose ensuing degradation is regulated by RNF8 and RNF168.

Show MeSH
Related in: MedlinePlus