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HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response.

Lee YH, Kuo CY, Stark JM, Shih HM, Ann DK - Nucleic Acids Res. (2013)

Bottom Line: In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs.Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions.We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA.

ABSTRACT
The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G2/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.

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HP1 plays an important role in apoptosis and cell cycle checkpoint control. (A) U2OS cells or HP1-depleted U2OS cells were cultured in low serum medium for 18 h and irradiated at 4 Gy. The cells were cultured for another 3 h, stained with PI and 5 × 104 were analyzed by flow cytometry. (B) U2OS, HCC1937 (BRCA1-deficient or supplemented) cells and HP1-depleted MCF7 and HCC1937 cells were cultured in low serum medium for 18 h. Irradiated and sham-irradiated cells were cultured for 3 h before fixing, then stained with an anti-histone H3 phospho-serine 10 antibody (Millipore) and analyzed by FACS.
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gkt231-F6: HP1 plays an important role in apoptosis and cell cycle checkpoint control. (A) U2OS cells or HP1-depleted U2OS cells were cultured in low serum medium for 18 h and irradiated at 4 Gy. The cells were cultured for another 3 h, stained with PI and 5 × 104 were analyzed by flow cytometry. (B) U2OS, HCC1937 (BRCA1-deficient or supplemented) cells and HP1-depleted MCF7 and HCC1937 cells were cultured in low serum medium for 18 h. Irradiated and sham-irradiated cells were cultured for 3 h before fixing, then stained with an anti-histone H3 phospho-serine 10 antibody (Millipore) and analyzed by FACS.

Mentions: It seems likely that HP1 promoted HR repair by recruiting BRCA1 to the DSB sites. The tumor suppressor activity of BRCA1 can also be assayed through its regulatory role in the cell cycle checkpoint. Accordingly, we sought to define the role of HP1 in the control of the cell cycle checkpoint. U2OS cells or HP1-depleted U2OS cells were irradiated (4 Gy), then fixed and permeabilized. Fixed cells were then stained with PI and analyzed using flow cytometry. Depleting HP1 affected the overall U2OS cell cycle profile by increasing the sub-G1 population and decreasing the G1 population (Figure 6A). U2OS cells had ∼11% of cells in sub-G1 cell, which serves as a baseline control. Notably, the sub-G1 populations of cells depleted of HP1α, HP1β and HP1γ increased to 20, 23 and 30%, respectively, which is consistent with the elevated apoptosis levels observed in HP1-depleted cells after irradiation (Figure 2). However, the G1 population of U2OS cells (27%) decreased to 12.8, 21 and 6.4% in U2OS cells depleted for HP1α, HP1β and HP1γ, after irradiation. A similar cell cycle profile was obtained using HP1-depleted AsiSI-ER-U2OS cells after 4-OHT treatment (Supplementary Figure S8). Based on the observations of increased sub-G1 populations in HP1-depleted U2OS cells, we propose that HP1 could be involved in regulating the control of apoptosis and the cell cycle.Figure 6.


HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response.

Lee YH, Kuo CY, Stark JM, Shih HM, Ann DK - Nucleic Acids Res. (2013)

HP1 plays an important role in apoptosis and cell cycle checkpoint control. (A) U2OS cells or HP1-depleted U2OS cells were cultured in low serum medium for 18 h and irradiated at 4 Gy. The cells were cultured for another 3 h, stained with PI and 5 × 104 were analyzed by flow cytometry. (B) U2OS, HCC1937 (BRCA1-deficient or supplemented) cells and HP1-depleted MCF7 and HCC1937 cells were cultured in low serum medium for 18 h. Irradiated and sham-irradiated cells were cultured for 3 h before fixing, then stained with an anti-histone H3 phospho-serine 10 antibody (Millipore) and analyzed by FACS.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675466&req=5

gkt231-F6: HP1 plays an important role in apoptosis and cell cycle checkpoint control. (A) U2OS cells or HP1-depleted U2OS cells were cultured in low serum medium for 18 h and irradiated at 4 Gy. The cells were cultured for another 3 h, stained with PI and 5 × 104 were analyzed by flow cytometry. (B) U2OS, HCC1937 (BRCA1-deficient or supplemented) cells and HP1-depleted MCF7 and HCC1937 cells were cultured in low serum medium for 18 h. Irradiated and sham-irradiated cells were cultured for 3 h before fixing, then stained with an anti-histone H3 phospho-serine 10 antibody (Millipore) and analyzed by FACS.
Mentions: It seems likely that HP1 promoted HR repair by recruiting BRCA1 to the DSB sites. The tumor suppressor activity of BRCA1 can also be assayed through its regulatory role in the cell cycle checkpoint. Accordingly, we sought to define the role of HP1 in the control of the cell cycle checkpoint. U2OS cells or HP1-depleted U2OS cells were irradiated (4 Gy), then fixed and permeabilized. Fixed cells were then stained with PI and analyzed using flow cytometry. Depleting HP1 affected the overall U2OS cell cycle profile by increasing the sub-G1 population and decreasing the G1 population (Figure 6A). U2OS cells had ∼11% of cells in sub-G1 cell, which serves as a baseline control. Notably, the sub-G1 populations of cells depleted of HP1α, HP1β and HP1γ increased to 20, 23 and 30%, respectively, which is consistent with the elevated apoptosis levels observed in HP1-depleted cells after irradiation (Figure 2). However, the G1 population of U2OS cells (27%) decreased to 12.8, 21 and 6.4% in U2OS cells depleted for HP1α, HP1β and HP1γ, after irradiation. A similar cell cycle profile was obtained using HP1-depleted AsiSI-ER-U2OS cells after 4-OHT treatment (Supplementary Figure S8). Based on the observations of increased sub-G1 populations in HP1-depleted U2OS cells, we propose that HP1 could be involved in regulating the control of apoptosis and the cell cycle.Figure 6.

Bottom Line: In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs.Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions.We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA.

ABSTRACT
The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G2/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.

Show MeSH
Related in: MedlinePlus