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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.

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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 recruitment to sites of DNA damage is dependent on RNF168. (a) Flag-HA-LSD1 was stably expressed in U2OS cells, and then treated with the ATM inhibitor KU55933 (15 µM) or DMSO 1 h before laser microirradiation. The cells were then stained for HA and pH2A.X. (b) Wild-type (H2A.X+/+) or H2A.X-deficient (H2A.X−/−) MEFs stably expressing HA-tagged LSD1 were laser microirradiated and stained as in panel a. (c) Quantitation of a and b, with error bars representing the SD of duplicate experiments. (d) Wild-type or 53BP1-deficient (53BP1−/−) MEFs stably expressing Flag-HA–tagged LSD1 were laser microirradiated and stained as in panel a. (e) U2OS cells were treated with the indicated shRNAs, microirradiated, and stained for endogenous LSD1 and pH2A.X. (f) Quantitation of d and e, with error bars representing the SD of duplicate experiments. Bars, 20 µm.
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fig3: LSD1 recruitment to sites of DNA damage is dependent on RNF168. (a) Flag-HA-LSD1 was stably expressed in U2OS cells, and then treated with the ATM inhibitor KU55933 (15 µM) or DMSO 1 h before laser microirradiation. The cells were then stained for HA and pH2A.X. (b) Wild-type (H2A.X+/+) or H2A.X-deficient (H2A.X−/−) MEFs stably expressing HA-tagged LSD1 were laser microirradiated and stained as in panel a. (c) Quantitation of a and b, with error bars representing the SD of duplicate experiments. (d) Wild-type or 53BP1-deficient (53BP1−/−) MEFs stably expressing Flag-HA–tagged LSD1 were laser microirradiated and stained as in panel a. (e) U2OS cells were treated with the indicated shRNAs, microirradiated, and stained for endogenous LSD1 and pH2A.X. (f) Quantitation of d and e, with error bars representing the SD of duplicate experiments. Bars, 20 µm.

Mentions: We next wished to determine the mechanism by which LSD1 is recruited to sites of DNA damage. We first considered whether LSD1 recruitment is dependent on the ATM or ataxia telangiectasia and Rad3 related (ATR) kinase pathways. Comparable numbers of cells treated with either the ATM inhibitor KU55933 or DMSO control displayed visible HA-LSD1 stripes overlapping with the pH2AX signal upon DNA damage (Fig. 3, a and c). There was also no change in LSD1 recruitment using an ATR inhibitor (Toledo et al., 2011; Fig. S1 g). At the concentrations used, both inhibitors were indeed functional (Fig. S2 a). Similarly, the loss of H2A.X did not affect LSD1 recruitment (Fig. 3, b and c). We considered the possibility that LSD1 recruitment may be PARP dependent because certain chromatin-modifying factors that are rapidly recruited to sites of damage are also PARP dependent (Chou et al., 2010; Polo et al., 2010). However, inhibition of PARP also did not affect LSD1 recruitment (Fig. S2, a–c). Interestingly, certain chromatin-associated factors, such as 53BP1, have rapid recruitment to sites of DNA damage even in the absence of H2A.X, but are not retained without H2A.X (Celeste et al., 2003). We then asked whether 53BP1 or a key factor responsible for its recruitment, RNF168, are important for LSD1 recruitment. Laser microirradiation experiments did not reveal a difference in the recruitment of LSD1 in 53BP1−/− MEFs (Fig. 3, d and f). However, knockdown of RNF168 (Fig. S2 d) caused a significant reduction in the recruitment of LSD1 to sites of DNA damage (Fig. 3 e). Quantitation revealed only ∼40% of pH2A.X-positive stripes were positive for LSD1 stripes upon RNF168 knockdown, as opposed to >90% in control knockdown cells (Fig. 3 f). RNF168 knockdown also reduced recruitment of HA-tagged LSD1 to microirradiation sites (unpublished data).


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 recruitment to sites of DNA damage is dependent on RNF168. (a) Flag-HA-LSD1 was stably expressed in U2OS cells, and then treated with the ATM inhibitor KU55933 (15 µM) or DMSO 1 h before laser microirradiation. The cells were then stained for HA and pH2A.X. (b) Wild-type (H2A.X+/+) or H2A.X-deficient (H2A.X−/−) MEFs stably expressing HA-tagged LSD1 were laser microirradiated and stained as in panel a. (c) Quantitation of a and b, with error bars representing the SD of duplicate experiments. (d) Wild-type or 53BP1-deficient (53BP1−/−) MEFs stably expressing Flag-HA–tagged LSD1 were laser microirradiated and stained as in panel a. (e) U2OS cells were treated with the indicated shRNAs, microirradiated, and stained for endogenous LSD1 and pH2A.X. (f) Quantitation of d and e, with error bars representing the SD of duplicate experiments. Bars, 20 µm.
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fig3: LSD1 recruitment to sites of DNA damage is dependent on RNF168. (a) Flag-HA-LSD1 was stably expressed in U2OS cells, and then treated with the ATM inhibitor KU55933 (15 µM) or DMSO 1 h before laser microirradiation. The cells were then stained for HA and pH2A.X. (b) Wild-type (H2A.X+/+) or H2A.X-deficient (H2A.X−/−) MEFs stably expressing HA-tagged LSD1 were laser microirradiated and stained as in panel a. (c) Quantitation of a and b, with error bars representing the SD of duplicate experiments. (d) Wild-type or 53BP1-deficient (53BP1−/−) MEFs stably expressing Flag-HA–tagged LSD1 were laser microirradiated and stained as in panel a. (e) U2OS cells were treated with the indicated shRNAs, microirradiated, and stained for endogenous LSD1 and pH2A.X. (f) Quantitation of d and e, with error bars representing the SD of duplicate experiments. Bars, 20 µm.
Mentions: We next wished to determine the mechanism by which LSD1 is recruited to sites of DNA damage. We first considered whether LSD1 recruitment is dependent on the ATM or ataxia telangiectasia and Rad3 related (ATR) kinase pathways. Comparable numbers of cells treated with either the ATM inhibitor KU55933 or DMSO control displayed visible HA-LSD1 stripes overlapping with the pH2AX signal upon DNA damage (Fig. 3, a and c). There was also no change in LSD1 recruitment using an ATR inhibitor (Toledo et al., 2011; Fig. S1 g). At the concentrations used, both inhibitors were indeed functional (Fig. S2 a). Similarly, the loss of H2A.X did not affect LSD1 recruitment (Fig. 3, b and c). We considered the possibility that LSD1 recruitment may be PARP dependent because certain chromatin-modifying factors that are rapidly recruited to sites of damage are also PARP dependent (Chou et al., 2010; Polo et al., 2010). However, inhibition of PARP also did not affect LSD1 recruitment (Fig. S2, a–c). Interestingly, certain chromatin-associated factors, such as 53BP1, have rapid recruitment to sites of DNA damage even in the absence of H2A.X, but are not retained without H2A.X (Celeste et al., 2003). We then asked whether 53BP1 or a key factor responsible for its recruitment, RNF168, are important for LSD1 recruitment. Laser microirradiation experiments did not reveal a difference in the recruitment of LSD1 in 53BP1−/− MEFs (Fig. 3, d and f). However, knockdown of RNF168 (Fig. S2 d) caused a significant reduction in the recruitment of LSD1 to sites of DNA damage (Fig. 3 e). Quantitation revealed only ∼40% of pH2A.X-positive stripes were positive for LSD1 stripes upon RNF168 knockdown, as opposed to >90% in control knockdown cells (Fig. 3 f). RNF168 knockdown also reduced recruitment of HA-tagged LSD1 to microirradiation sites (unpublished data).

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