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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-dependent recruitment of BRCA1 to DSB sites. AsiSI-ER-U2OS cells and HP1-depleted AsiSI-ER-U2OS cells were cultured and treated with 4-OHT (4-hydroxytamoxifen; 100 µM) for 4 h. Soluble chromatins were prepared from each culture and immunoprecipitated with specific antibodies (A: BRCA1, B: 53BP1, C: Rad51 and D: HP1). Immunoprecipitated chromatins were analyzed by quantitative PCR using specific primer pairs located at Chr 1: chr1_89231183, Chr 6: chr6_90404906, Chr 21: distal region of chr21_21292316. The results shown are relative signals from the individual precipitated DNA compared with input chromatin. siHP1 is siHP1 α, β and γ combined. Figure 4B includes two insets with enlarged scales showing the increased 53BP1 occupancy at DSB sites of Chr 1 and Chr 6 on 4-OHT treatment.
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gkt231-F4: HP1-dependent recruitment of BRCA1 to DSB sites. AsiSI-ER-U2OS cells and HP1-depleted AsiSI-ER-U2OS cells were cultured and treated with 4-OHT (4-hydroxytamoxifen; 100 µM) for 4 h. Soluble chromatins were prepared from each culture and immunoprecipitated with specific antibodies (A: BRCA1, B: 53BP1, C: Rad51 and D: HP1). Immunoprecipitated chromatins were analyzed by quantitative PCR using specific primer pairs located at Chr 1: chr1_89231183, Chr 6: chr6_90404906, Chr 21: distal region of chr21_21292316. The results shown are relative signals from the individual precipitated DNA compared with input chromatin. siHP1 is siHP1 α, β and γ combined. Figure 4B includes two insets with enlarged scales showing the increased 53BP1 occupancy at DSB sites of Chr 1 and Chr 6 on 4-OHT treatment.

Mentions: To define the mechanism of HP1 regulation of BRCA1 and 53BP1 recruitment at the molecular level, we performed ChIP assays on U2OS cells containing the 4-OHT-induced AsiSI-dependent DNA damage system (25,26). Because the restriction enzyme AsiSI is fused to the estrogen receptor ligand-binding domain (ER LBD), the restriction enzyme activity of AsiSI-ER can be induced by 4-OHT treatment. Sheared chromatin was prepared from 4-OHT- or vehicle-treated AsiSI-ER-U2OS cells. The chromatin was immunoprecipitated with an anti-BRCA1 or anti-53BP1 antibody. Specific primer pairs for two DNA breaks sites created by AsiSI [two proximal regions of Chr 1 (chr1_89231183) and Chr 6 (chr6_90404906) and a distal region (chr21_21292316) of Chr 21] were used to amplify the immunoprecipitated chromatin. Both BRCA1 and 53BP1 were recruited to AsiSI-restricted DSB sites (Figure 4A and B). Notably, occupancy of BRCA1 at DSB sites located at Chr 1 and Chr 6 was clearly induced (Figure 4A). In addition, the recruitment of 53BP1 to Chr 1 and Chr 6 DSB sites was also detected (Figure 4B, insets), supporting increased occupancy of BRCA1 and 53BP1 at DSB sites. To determine the role played by HP1 in BRCA1 and 53BP1 recruitment, specific siRNAs against the three HP1 isoforms were transfected into AsiSI-ER-U2OS cells 48 h before 4-OHT-treatment. The depletion of HP1 had marked effects on the recruitment of BRCA1 and 53BP1 to the DSB sites. BRCA1 was no longer recruited to damaged chromatin (Chr 1 and Chr 6) in HP1-depleted cells, even after 4-OHT treatment (Figure 4A). Instead, 53BP1 recruitment to Chr 1 and Chr 6 was drastically enhanced (Figure 4B). In contrast, the recruitment of BRCA1 and 53BP1 to the DSB site in the distal region of Chr 21 was only mildly affected by depleting HP1 on DSB induction (Figure 4A and B), serving a negative control. The nature of BRCA1 and 53BP1 binding to distal Chr 21, although at a lower level, is not yet known, but it might suggest other functional roles for BRCA1, such as transcription regulation in response to DSB induction (37). Consistent with our foci formation assay data (Figure 3), HP1 played a critical role in regulating the association of the BRCA1 and 53BP1 DDR proteins with chromatin. HP1 facilitated BRCA1 recruitment to the DSB sites, but impedes the recruitment of 53BP1.Figure 4.


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-dependent recruitment of BRCA1 to DSB sites. AsiSI-ER-U2OS cells and HP1-depleted AsiSI-ER-U2OS cells were cultured and treated with 4-OHT (4-hydroxytamoxifen; 100 µM) for 4 h. Soluble chromatins were prepared from each culture and immunoprecipitated with specific antibodies (A: BRCA1, B: 53BP1, C: Rad51 and D: HP1). Immunoprecipitated chromatins were analyzed by quantitative PCR using specific primer pairs located at Chr 1: chr1_89231183, Chr 6: chr6_90404906, Chr 21: distal region of chr21_21292316. The results shown are relative signals from the individual precipitated DNA compared with input chromatin. siHP1 is siHP1 α, β and γ combined. Figure 4B includes two insets with enlarged scales showing the increased 53BP1 occupancy at DSB sites of Chr 1 and Chr 6 on 4-OHT treatment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt231-F4: HP1-dependent recruitment of BRCA1 to DSB sites. AsiSI-ER-U2OS cells and HP1-depleted AsiSI-ER-U2OS cells were cultured and treated with 4-OHT (4-hydroxytamoxifen; 100 µM) for 4 h. Soluble chromatins were prepared from each culture and immunoprecipitated with specific antibodies (A: BRCA1, B: 53BP1, C: Rad51 and D: HP1). Immunoprecipitated chromatins were analyzed by quantitative PCR using specific primer pairs located at Chr 1: chr1_89231183, Chr 6: chr6_90404906, Chr 21: distal region of chr21_21292316. The results shown are relative signals from the individual precipitated DNA compared with input chromatin. siHP1 is siHP1 α, β and γ combined. Figure 4B includes two insets with enlarged scales showing the increased 53BP1 occupancy at DSB sites of Chr 1 and Chr 6 on 4-OHT treatment.
Mentions: To define the mechanism of HP1 regulation of BRCA1 and 53BP1 recruitment at the molecular level, we performed ChIP assays on U2OS cells containing the 4-OHT-induced AsiSI-dependent DNA damage system (25,26). Because the restriction enzyme AsiSI is fused to the estrogen receptor ligand-binding domain (ER LBD), the restriction enzyme activity of AsiSI-ER can be induced by 4-OHT treatment. Sheared chromatin was prepared from 4-OHT- or vehicle-treated AsiSI-ER-U2OS cells. The chromatin was immunoprecipitated with an anti-BRCA1 or anti-53BP1 antibody. Specific primer pairs for two DNA breaks sites created by AsiSI [two proximal regions of Chr 1 (chr1_89231183) and Chr 6 (chr6_90404906) and a distal region (chr21_21292316) of Chr 21] were used to amplify the immunoprecipitated chromatin. Both BRCA1 and 53BP1 were recruited to AsiSI-restricted DSB sites (Figure 4A and B). Notably, occupancy of BRCA1 at DSB sites located at Chr 1 and Chr 6 was clearly induced (Figure 4A). In addition, the recruitment of 53BP1 to Chr 1 and Chr 6 DSB sites was also detected (Figure 4B, insets), supporting increased occupancy of BRCA1 and 53BP1 at DSB sites. To determine the role played by HP1 in BRCA1 and 53BP1 recruitment, specific siRNAs against the three HP1 isoforms were transfected into AsiSI-ER-U2OS cells 48 h before 4-OHT-treatment. The depletion of HP1 had marked effects on the recruitment of BRCA1 and 53BP1 to the DSB sites. BRCA1 was no longer recruited to damaged chromatin (Chr 1 and Chr 6) in HP1-depleted cells, even after 4-OHT treatment (Figure 4A). Instead, 53BP1 recruitment to Chr 1 and Chr 6 was drastically enhanced (Figure 4B). In contrast, the recruitment of BRCA1 and 53BP1 to the DSB site in the distal region of Chr 21 was only mildly affected by depleting HP1 on DSB induction (Figure 4A and B), serving a negative control. The nature of BRCA1 and 53BP1 binding to distal Chr 21, although at a lower level, is not yet known, but it might suggest other functional roles for BRCA1, such as transcription regulation in response to DSB induction (37). Consistent with our foci formation assay data (Figure 3), HP1 played a critical role in regulating the association of the BRCA1 and 53BP1 DDR proteins with chromatin. HP1 facilitated BRCA1 recruitment to the DSB sites, but impedes the recruitment of 53BP1.Figure 4.

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