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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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The roles of HP1 for BRCA1 function and the choice between HR and NHEJ repair. HP1 is a chromatin-associated protein, but the distribution of HP1 on chromatin is not even. Irradiation partially removes HP1 from chromatin, but the remaining HP1 molecules have active roles in recruiting BRCA1 to the damaged DNA sites. HP1 facilitates BRCA1 recruitment to chromatin after DNA damage, thereby activating G2/M cell cycle arrest and error-free HR repair. However, if the chromatin is HP1- or BRCA1-deficient, 53BP1 should be recruited resulting in error-prone NHEJ repair of the DNA damage. The loss of both the G2/M checkpoint control and HR repair pathway may increase the likelihood of apoptosis.
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gkt231-F8: The roles of HP1 for BRCA1 function and the choice between HR and NHEJ repair. HP1 is a chromatin-associated protein, but the distribution of HP1 on chromatin is not even. Irradiation partially removes HP1 from chromatin, but the remaining HP1 molecules have active roles in recruiting BRCA1 to the damaged DNA sites. HP1 facilitates BRCA1 recruitment to chromatin after DNA damage, thereby activating G2/M cell cycle arrest and error-free HR repair. However, if the chromatin is HP1- or BRCA1-deficient, 53BP1 should be recruited resulting in error-prone NHEJ repair of the DNA damage. The loss of both the G2/M checkpoint control and HR repair pathway may increase the likelihood of apoptosis.

Mentions: Here, we also showed that HP1 was required for HR DNA repair, but not NHEJ repair. This result implies that HP1 may be a factor that influences the choice between the HR and NHEJ repair pathways for DSBs. Mechanisms for cells to choose between the two DNA repair pathways have not been clear until now. Published literature indicates that the repair choice could depend on cell cycle status, chromatin structure and other factors (50). HR repair takes place during the S and G2 phases, whereas NHEJ repair is more prevalent in cells at the G1 stage of the cell cycle. Recent reports suggest that BRCA1 is involved in the HR DNA repair pathway, and BRCA1 associates more with heterochromatic regions (42,51,52). However, HR repair by BRCA1 is inhibited by the DDR factor 53BP1, and 53BP1 may enhance the activity of the NHEJ DNA repair pathway (51,52). Our results show, unequivocally, that HP1 promoted HR and recruits BRCA1 to form foci. In contrast, depleting HP1 increased 53BP1 association with DSB sites and 53BP1 foci formation. It is plausible that the local concentration of HP1 could determine the occupancy of BRCA1 or 53BP1 on chromatin, supporting the hypothesis that HP1 is a critical determinant involved in the choice between HR and NHEJ repair. Our study suggests that different local concentrations of HP1 on chromatin could influence the outcome of the DDR (Figure 8). Irradiating cells caused the HP1 protein to partially dissociate from chromatin but the remaining HP1 facilitated the recruitment of BRCA1 to the damaged DNA sites (Figures 4D and 8). DNA lesions in HP1 abundant chromatin are repaired during G2/M cell cycle arrest by BRCA1-mediated HR repair. However, more 53BP1 was clearly recruited to DSB sites in HP1-depleted and BRCA1-deficient cells, which used NHEJ for repair or apoptosed. Very recently, Soria and Almouzni (53) reported differential contribution of HP1 subtypes to HR, during the time our manuscript was under review. Notably, results from their sister chromatid exchange (SCE) assay that HP1 depletion reduced SCE activities have independently validated our observations reported herein. However, there is a discrepancy between Soria’s and our data with respect to the role of HP1γ in HR repair as determined by reporter assays. Nonetheless, it does not affect our main conclusion on that HP1 promotes HR but not NHEJ repair. Therefore, we propose that differential local concentrations of HP1 could play a critical role in determining which DSB repair pathway is used (e.g. HR versus NHEJ).Figure 8.


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)

The roles of HP1 for BRCA1 function and the choice between HR and NHEJ repair. HP1 is a chromatin-associated protein, but the distribution of HP1 on chromatin is not even. Irradiation partially removes HP1 from chromatin, but the remaining HP1 molecules have active roles in recruiting BRCA1 to the damaged DNA sites. HP1 facilitates BRCA1 recruitment to chromatin after DNA damage, thereby activating G2/M cell cycle arrest and error-free HR repair. However, if the chromatin is HP1- or BRCA1-deficient, 53BP1 should be recruited resulting in error-prone NHEJ repair of the DNA damage. The loss of both the G2/M checkpoint control and HR repair pathway may increase the likelihood of apoptosis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt231-F8: The roles of HP1 for BRCA1 function and the choice between HR and NHEJ repair. HP1 is a chromatin-associated protein, but the distribution of HP1 on chromatin is not even. Irradiation partially removes HP1 from chromatin, but the remaining HP1 molecules have active roles in recruiting BRCA1 to the damaged DNA sites. HP1 facilitates BRCA1 recruitment to chromatin after DNA damage, thereby activating G2/M cell cycle arrest and error-free HR repair. However, if the chromatin is HP1- or BRCA1-deficient, 53BP1 should be recruited resulting in error-prone NHEJ repair of the DNA damage. The loss of both the G2/M checkpoint control and HR repair pathway may increase the likelihood of apoptosis.
Mentions: Here, we also showed that HP1 was required for HR DNA repair, but not NHEJ repair. This result implies that HP1 may be a factor that influences the choice between the HR and NHEJ repair pathways for DSBs. Mechanisms for cells to choose between the two DNA repair pathways have not been clear until now. Published literature indicates that the repair choice could depend on cell cycle status, chromatin structure and other factors (50). HR repair takes place during the S and G2 phases, whereas NHEJ repair is more prevalent in cells at the G1 stage of the cell cycle. Recent reports suggest that BRCA1 is involved in the HR DNA repair pathway, and BRCA1 associates more with heterochromatic regions (42,51,52). However, HR repair by BRCA1 is inhibited by the DDR factor 53BP1, and 53BP1 may enhance the activity of the NHEJ DNA repair pathway (51,52). Our results show, unequivocally, that HP1 promoted HR and recruits BRCA1 to form foci. In contrast, depleting HP1 increased 53BP1 association with DSB sites and 53BP1 foci formation. It is plausible that the local concentration of HP1 could determine the occupancy of BRCA1 or 53BP1 on chromatin, supporting the hypothesis that HP1 is a critical determinant involved in the choice between HR and NHEJ repair. Our study suggests that different local concentrations of HP1 on chromatin could influence the outcome of the DDR (Figure 8). Irradiating cells caused the HP1 protein to partially dissociate from chromatin but the remaining HP1 facilitated the recruitment of BRCA1 to the damaged DNA sites (Figures 4D and 8). DNA lesions in HP1 abundant chromatin are repaired during G2/M cell cycle arrest by BRCA1-mediated HR repair. However, more 53BP1 was clearly recruited to DSB sites in HP1-depleted and BRCA1-deficient cells, which used NHEJ for repair or apoptosed. Very recently, Soria and Almouzni (53) reported differential contribution of HP1 subtypes to HR, during the time our manuscript was under review. Notably, results from their sister chromatid exchange (SCE) assay that HP1 depletion reduced SCE activities have independently validated our observations reported herein. However, there is a discrepancy between Soria’s and our data with respect to the role of HP1γ in HR repair as determined by reporter assays. Nonetheless, it does not affect our main conclusion on that HP1 promotes HR but not NHEJ repair. Therefore, we propose that differential local concentrations of HP1 could play a critical role in determining which DSB repair pathway is used (e.g. HR versus NHEJ).Figure 8.

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