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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 is required for HR repair. U2OS-NHEJ cells and U2OS-HR cells were transfected by individual siRNAs targeting the three respective HP1 subtypes on day one. The I-SecI expression construct was transfected at 24 h after siRNA transfection, and cells were incubated for additional 48 h. The numbers of GFP-positive cells were determined by fluorescence-activated cell sorting (FACS). GFP- and DSred-expression constructs were co-transfected to normalize for transfection efficiency in the FACS assays. The assays were repeated at least four times (n = 5), and the average relative numbers are presented as the mean ± SD.
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gkt231-F5: HP1 is required for HR repair. U2OS-NHEJ cells and U2OS-HR cells were transfected by individual siRNAs targeting the three respective HP1 subtypes on day one. The I-SecI expression construct was transfected at 24 h after siRNA transfection, and cells were incubated for additional 48 h. The numbers of GFP-positive cells were determined by fluorescence-activated cell sorting (FACS). GFP- and DSred-expression constructs were co-transfected to normalize for transfection efficiency in the FACS assays. The assays were repeated at least four times (n = 5), and the average relative numbers are presented as the mean ± SD.

Mentions: The aforementioned findings indicate that HP1α, HP1β and HP1γ were required to promote BRCA1 recruitment to the sites of DNA damage. Given that BRCA1 is important for the HR repair pathway, we tested whether HP1 promoted HR. We used I-SceI-based, GFP U2OS-HR and U2OS-NHEJ reporter systems to determine the respective roles of HP1 in the HR and NHEJ repair pathways (23). I-SceI is a rare-cutting restriction endonuclease that recognizes a specific 18-bp sequence and leaves a 4-bp 5′-overhang on I-SceI-mediated cutting. The individual reporters were engineered to produce GFP-positive cells only if HR or NHEJ could repair the I-SceI-induced DSBs. Transfection of the I-SceI expression constructs into the reporter cell lines produced significant numbers of GFP-positive cells, which provided the baseline frequencies for HR and NHEJ (Figure 5). Transfection of the cells with siRNA targeting each HP1 subtypes (Supplementary Figure S2, right panels) led to a significant (P < 0.002) reduction in the number of GFP positive cells in the HR reporter cells for all HP1 subtypes (Figure 5, left panel). In contrast, depleting HP1 did not significantly affect the number of GFP-positive cells in U2OS–NHEJ reporter cells (Figure 5, right panel). These results indicate that all three HP1 subtypes were important for HR-dependent DNA repair, but they were not required for NHEJ repair. Because BRCA1 is an important factor in HR repair (39), we suggest that depleting HP1 dramatically reduced BRCA1 recruitment and foci formation, thereby impairing HR repair.Figure 5.


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 is required for HR repair. U2OS-NHEJ cells and U2OS-HR cells were transfected by individual siRNAs targeting the three respective HP1 subtypes on day one. The I-SecI expression construct was transfected at 24 h after siRNA transfection, and cells were incubated for additional 48 h. The numbers of GFP-positive cells were determined by fluorescence-activated cell sorting (FACS). GFP- and DSred-expression constructs were co-transfected to normalize for transfection efficiency in the FACS assays. The assays were repeated at least four times (n = 5), and the average relative numbers are presented as the mean ± SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt231-F5: HP1 is required for HR repair. U2OS-NHEJ cells and U2OS-HR cells were transfected by individual siRNAs targeting the three respective HP1 subtypes on day one. The I-SecI expression construct was transfected at 24 h after siRNA transfection, and cells were incubated for additional 48 h. The numbers of GFP-positive cells were determined by fluorescence-activated cell sorting (FACS). GFP- and DSred-expression constructs were co-transfected to normalize for transfection efficiency in the FACS assays. The assays were repeated at least four times (n = 5), and the average relative numbers are presented as the mean ± SD.
Mentions: The aforementioned findings indicate that HP1α, HP1β and HP1γ were required to promote BRCA1 recruitment to the sites of DNA damage. Given that BRCA1 is important for the HR repair pathway, we tested whether HP1 promoted HR. We used I-SceI-based, GFP U2OS-HR and U2OS-NHEJ reporter systems to determine the respective roles of HP1 in the HR and NHEJ repair pathways (23). I-SceI is a rare-cutting restriction endonuclease that recognizes a specific 18-bp sequence and leaves a 4-bp 5′-overhang on I-SceI-mediated cutting. The individual reporters were engineered to produce GFP-positive cells only if HR or NHEJ could repair the I-SceI-induced DSBs. Transfection of the I-SceI expression constructs into the reporter cell lines produced significant numbers of GFP-positive cells, which provided the baseline frequencies for HR and NHEJ (Figure 5). Transfection of the cells with siRNA targeting each HP1 subtypes (Supplementary Figure S2, right panels) led to a significant (P < 0.002) reduction in the number of GFP positive cells in the HR reporter cells for all HP1 subtypes (Figure 5, left panel). In contrast, depleting HP1 did not significantly affect the number of GFP-positive cells in U2OS–NHEJ reporter cells (Figure 5, right panel). These results indicate that all three HP1 subtypes were important for HR-dependent DNA repair, but they were not required for NHEJ repair. Because BRCA1 is an important factor in HR repair (39), we suggest that depleting HP1 dramatically reduced BRCA1 recruitment and foci formation, thereby impairing HR repair.Figure 5.

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