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The histone demethylase LSD1/KDM1A promotes the DNA damage response.

Mosammaparast N, Kim H, Laurent B, Zhao Y, Lim HJ, Majid MC, Dango S, Luo Y, Hempel K, Sowa ME, Gygi SP, Steen H, Harper JW, Yankner B, Shi Y - J. Cell Biol. (2013)

Bottom Line: Although loss of LSD1 did not affect the initial formation of pH2A.X foci, 53BP1 and BRCA1 complex recruitment were reduced upon LSD1 knockdown.Mechanistically, this was likely a result of compromised histone ubiquitylation preferentially in late S/G2.Consistent with a role in the DDR, knockdown of LSD1 resulted in moderate hypersensitivity to γ-irradiation and increased homologous recombination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, Washington University in St. Louis, St. Louis, MO 63110.

ABSTRACT
Histone demethylation is known to regulate transcription, but its role in other processes is largely unknown. We report a role for the histone demethylase LSD1/KDM1A in the DNA damage response (DDR). We show that LSD1 is recruited directly to sites of DNA damage. H3K4 dimethylation, a major substrate for LSD1, is reduced at sites of DNA damage in an LSD1-dependent manner. The E3 ubiquitin ligase RNF168 physically interacts with LSD1 and we find this interaction to be important for LSD1 recruitment to DNA damage sites. Although loss of LSD1 did not affect the initial formation of pH2A.X foci, 53BP1 and BRCA1 complex recruitment were reduced upon LSD1 knockdown. Mechanistically, this was likely a result of compromised histone ubiquitylation preferentially in late S/G2. Consistent with a role in the DDR, knockdown of LSD1 resulted in moderate hypersensitivity to γ-irradiation and increased homologous recombination. Our findings uncover a direct role for LSD1 in the DDR and place LSD1 downstream of RNF168 in the DDR pathway.

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LSD1 promotes 53BP1 IRIF formation in late S/G2 cells. (a) LSD1 affects 53BP1 foci formation in a subset of cells but does not affect pH2A.X or MDC1 foci formation. U2OS cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using the indicated antibodies. (b) U2OS-FUCCI cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using an antibody against 53BP1. Bars, 20 µm. (c) The experiment in panel b was performed in triplicate, and 53BP1 foci were quantified as shown. Error bars represent ± SD. *, P < 0.01; n.s., not significant.
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fig7: LSD1 promotes 53BP1 IRIF formation in late S/G2 cells. (a) LSD1 affects 53BP1 foci formation in a subset of cells but does not affect pH2A.X or MDC1 foci formation. U2OS cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using the indicated antibodies. (b) U2OS-FUCCI cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using an antibody against 53BP1. Bars, 20 µm. (c) The experiment in panel b was performed in triplicate, and 53BP1 foci were quantified as shown. Error bars represent ± SD. *, P < 0.01; n.s., not significant.

Mentions: Numerous studies have suggested that RNF168 plays an important role in the recruitment of 53BP1 to sites of DNA damage (Doil et al., 2009; Stewart et al., 2009). Because LSD1 can interact with RNF168, is recruited to sites of damage in an RNF168-dependent manner, and regulates HR like 53BP1, we investigated whether LSD1 plays a role in the recruitment of 53BP1 to IRIF. We used lentiviral shRNA constructs to knock down LSD1 in U2OS cells (Fig. S1 a). These cells were then treated with γ-IR (10 Gy) and IRIF was assessed by immunofluorescence microscopy. Knockdown of LSD1 did not affect induction of pH2A.X or MDC1 foci in response to γ-IR (Fig. 7 a, left), suggesting that the initial signaling events related to the DDR are intact and not LSD1 dependent. In contrast, knockdown of LSD1 with three different shRNAs, but not two control shRNAs, had a noticeable effect on the formation of 53BP1 IRIF in a subset of cells (Fig. 7 a and not depicted). Some LSD1 knockdown cells had significantly more diffuse nucleoplasmic 53BP1 signal, while other cells lacked 53BP1 foci. These data suggest that LSD1 plays a role in the recruitment of 53BP1 to IRIF downstream of MDC1 and pH2A.X.


The histone demethylase LSD1/KDM1A promotes the DNA damage response.

Mosammaparast N, Kim H, Laurent B, Zhao Y, Lim HJ, Majid MC, Dango S, Luo Y, Hempel K, Sowa ME, Gygi SP, Steen H, Harper JW, Yankner B, Shi Y - J. Cell Biol. (2013)

LSD1 promotes 53BP1 IRIF formation in late S/G2 cells. (a) LSD1 affects 53BP1 foci formation in a subset of cells but does not affect pH2A.X or MDC1 foci formation. U2OS cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using the indicated antibodies. (b) U2OS-FUCCI cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using an antibody against 53BP1. Bars, 20 µm. (c) The experiment in panel b was performed in triplicate, and 53BP1 foci were quantified as shown. Error bars represent ± SD. *, P < 0.01; n.s., not significant.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824007&req=5

fig7: LSD1 promotes 53BP1 IRIF formation in late S/G2 cells. (a) LSD1 affects 53BP1 foci formation in a subset of cells but does not affect pH2A.X or MDC1 foci formation. U2OS cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using the indicated antibodies. (b) U2OS-FUCCI cells were infected with the indicated lentiviral shRNAs, exposed to 10 Gy IR, incubated at 37°C for 1 h, and processed for immunofluorescence using an antibody against 53BP1. Bars, 20 µm. (c) The experiment in panel b was performed in triplicate, and 53BP1 foci were quantified as shown. Error bars represent ± SD. *, P < 0.01; n.s., not significant.
Mentions: Numerous studies have suggested that RNF168 plays an important role in the recruitment of 53BP1 to sites of DNA damage (Doil et al., 2009; Stewart et al., 2009). Because LSD1 can interact with RNF168, is recruited to sites of damage in an RNF168-dependent manner, and regulates HR like 53BP1, we investigated whether LSD1 plays a role in the recruitment of 53BP1 to IRIF. We used lentiviral shRNA constructs to knock down LSD1 in U2OS cells (Fig. S1 a). These cells were then treated with γ-IR (10 Gy) and IRIF was assessed by immunofluorescence microscopy. Knockdown of LSD1 did not affect induction of pH2A.X or MDC1 foci in response to γ-IR (Fig. 7 a, left), suggesting that the initial signaling events related to the DDR are intact and not LSD1 dependent. In contrast, knockdown of LSD1 with three different shRNAs, but not two control shRNAs, had a noticeable effect on the formation of 53BP1 IRIF in a subset of cells (Fig. 7 a and not depicted). Some LSD1 knockdown cells had significantly more diffuse nucleoplasmic 53BP1 signal, while other cells lacked 53BP1 foci. These data suggest that LSD1 plays a role in the recruitment of 53BP1 to IRIF downstream of MDC1 and pH2A.X.

Bottom Line: Although loss of LSD1 did not affect the initial formation of pH2A.X foci, 53BP1 and BRCA1 complex recruitment were reduced upon LSD1 knockdown.Mechanistically, this was likely a result of compromised histone ubiquitylation preferentially in late S/G2.Consistent with a role in the DDR, knockdown of LSD1 resulted in moderate hypersensitivity to γ-irradiation and increased homologous recombination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, Washington University in St. Louis, St. Louis, MO 63110.

ABSTRACT
Histone demethylation is known to regulate transcription, but its role in other processes is largely unknown. We report a role for the histone demethylase LSD1/KDM1A in the DNA damage response (DDR). We show that LSD1 is recruited directly to sites of DNA damage. H3K4 dimethylation, a major substrate for LSD1, is reduced at sites of DNA damage in an LSD1-dependent manner. The E3 ubiquitin ligase RNF168 physically interacts with LSD1 and we find this interaction to be important for LSD1 recruitment to DNA damage sites. Although loss of LSD1 did not affect the initial formation of pH2A.X foci, 53BP1 and BRCA1 complex recruitment were reduced upon LSD1 knockdown. Mechanistically, this was likely a result of compromised histone ubiquitylation preferentially in late S/G2. Consistent with a role in the DDR, knockdown of LSD1 resulted in moderate hypersensitivity to γ-irradiation and increased homologous recombination. Our findings uncover a direct role for LSD1 in the DDR and place LSD1 downstream of RNF168 in the DDR pathway.

Show MeSH
Related in: MedlinePlus