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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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RNF168 interacts with LSD1. (a) RNF168 was immunoprecipitated using Flag or control antibody from nuclear extract prepared from 293T cells stably expressing Flag-HA-RNF168, with or without irradiation as indicated. Western blotting was then performed against Flag or endogenous LSD1. Arrow indicates the Flag-HA-RNF168, which is expressed at relatively low levels. (b) Myc-RNF168 and HA-LSD1 were coexpressed, and LSD1 was immunoprecipitated using an HA antibody, followed by Western blotting as indicated. (c) Immunprecipitation was performed as in panel a from nocodazole-arrested 293T cells mock infected or stably expressing Flag-HA-RNF8 or Flag-HA-RNF168 as indicated. (d) Tagged wild-type LSD1 or NΔ-171 LSD1 were stably expressed in U2OS cells, microirradiated, incubated for 15 min, and then stained for HA and pH2A.X. Bar, 20 µm. (e) Immunprecipitation was performed from 293T cells expressing the indicated vectors, followed by Western blotting as indicated.
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fig5: RNF168 interacts with LSD1. (a) RNF168 was immunoprecipitated using Flag or control antibody from nuclear extract prepared from 293T cells stably expressing Flag-HA-RNF168, with or without irradiation as indicated. Western blotting was then performed against Flag or endogenous LSD1. Arrow indicates the Flag-HA-RNF168, which is expressed at relatively low levels. (b) Myc-RNF168 and HA-LSD1 were coexpressed, and LSD1 was immunoprecipitated using an HA antibody, followed by Western blotting as indicated. (c) Immunprecipitation was performed as in panel a from nocodazole-arrested 293T cells mock infected or stably expressing Flag-HA-RNF8 or Flag-HA-RNF168 as indicated. (d) Tagged wild-type LSD1 or NΔ-171 LSD1 were stably expressed in U2OS cells, microirradiated, incubated for 15 min, and then stained for HA and pH2A.X. Bar, 20 µm. (e) Immunprecipitation was performed from 293T cells expressing the indicated vectors, followed by Western blotting as indicated.

Mentions: Reminiscent of CHD4 and its dependency on physical interaction with RNF8 for recruitment to damage sites (Luijsterburg et al., 2012), it was possible that the recruitment of LSD1 to sites of DNA damage was dependent on physical interaction with RNF168. To test this, we first took an unbiased approach by generating a stable cell line expressing Flag-tagged RNF168 and immunoprecipitating the tagged RNF168 and its associated proteins from irradiated and nonirradiated cells for mass spectrometry analysis. Indeed, LSD1 was present in both samples, along with many other interactors (Table S1). We then confirmed the presence of LSD1 in the Flag-purified RNF168 complex by Western blotting (Fig. 5 a). We further demonstrated by reciprocal immunoprecipitation that HA-LSD1 effectively coimmunoprecipitated myc-tagged RNF168 (Fig. 5 b). Although immunoprecipitation of Flag–RNF168 from asynchronous cells demonstrated its association with LSD1, this interaction seems to be enhanced in nocodazole-arrested cells, at least partly because there was greater RNF168 present (Fig. S2 e). Whether the endogenous, untagged RNF168 is similarly enriched at the G2 phase of the cell cycle remains to be determined. As a control, we failed to identify an association between LSD1 and the other damage-associated E3 ligase RNF8 under the same conditions (Fig. 5 c). Using coimmunoprecipitation, we also confirmed the physical interaction between endogenous LSD1 and RNF168 (Fig. S2 f).


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)

RNF168 interacts with LSD1. (a) RNF168 was immunoprecipitated using Flag or control antibody from nuclear extract prepared from 293T cells stably expressing Flag-HA-RNF168, with or without irradiation as indicated. Western blotting was then performed against Flag or endogenous LSD1. Arrow indicates the Flag-HA-RNF168, which is expressed at relatively low levels. (b) Myc-RNF168 and HA-LSD1 were coexpressed, and LSD1 was immunoprecipitated using an HA antibody, followed by Western blotting as indicated. (c) Immunprecipitation was performed as in panel a from nocodazole-arrested 293T cells mock infected or stably expressing Flag-HA-RNF8 or Flag-HA-RNF168 as indicated. (d) Tagged wild-type LSD1 or NΔ-171 LSD1 were stably expressed in U2OS cells, microirradiated, incubated for 15 min, and then stained for HA and pH2A.X. Bar, 20 µm. (e) Immunprecipitation was performed from 293T cells expressing the indicated vectors, followed by Western blotting as indicated.
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fig5: RNF168 interacts with LSD1. (a) RNF168 was immunoprecipitated using Flag or control antibody from nuclear extract prepared from 293T cells stably expressing Flag-HA-RNF168, with or without irradiation as indicated. Western blotting was then performed against Flag or endogenous LSD1. Arrow indicates the Flag-HA-RNF168, which is expressed at relatively low levels. (b) Myc-RNF168 and HA-LSD1 were coexpressed, and LSD1 was immunoprecipitated using an HA antibody, followed by Western blotting as indicated. (c) Immunprecipitation was performed as in panel a from nocodazole-arrested 293T cells mock infected or stably expressing Flag-HA-RNF8 or Flag-HA-RNF168 as indicated. (d) Tagged wild-type LSD1 or NΔ-171 LSD1 were stably expressed in U2OS cells, microirradiated, incubated for 15 min, and then stained for HA and pH2A.X. Bar, 20 µm. (e) Immunprecipitation was performed from 293T cells expressing the indicated vectors, followed by Western blotting as indicated.
Mentions: Reminiscent of CHD4 and its dependency on physical interaction with RNF8 for recruitment to damage sites (Luijsterburg et al., 2012), it was possible that the recruitment of LSD1 to sites of DNA damage was dependent on physical interaction with RNF168. To test this, we first took an unbiased approach by generating a stable cell line expressing Flag-tagged RNF168 and immunoprecipitating the tagged RNF168 and its associated proteins from irradiated and nonirradiated cells for mass spectrometry analysis. Indeed, LSD1 was present in both samples, along with many other interactors (Table S1). We then confirmed the presence of LSD1 in the Flag-purified RNF168 complex by Western blotting (Fig. 5 a). We further demonstrated by reciprocal immunoprecipitation that HA-LSD1 effectively coimmunoprecipitated myc-tagged RNF168 (Fig. 5 b). Although immunoprecipitation of Flag–RNF168 from asynchronous cells demonstrated its association with LSD1, this interaction seems to be enhanced in nocodazole-arrested cells, at least partly because there was greater RNF168 present (Fig. S2 e). Whether the endogenous, untagged RNF168 is similarly enriched at the G2 phase of the cell cycle remains to be determined. As a control, we failed to identify an association between LSD1 and the other damage-associated E3 ligase RNF8 under the same conditions (Fig. 5 c). Using coimmunoprecipitation, we also confirmed the physical interaction between endogenous LSD1 and RNF168 (Fig. S2 f).

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