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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 the cellular response to DNA damage. (a) Lentiviral knockdown was performed in HeLa cells using the indicated shRNAs, followed by Western blotting as indicated (top). Colony formation assay was then performed in quadruplicate using the knockdown cells. Y axis represents colony survival normalized to control. (b) U2OS-DR-GFP reporter cells were treated with the indicated shRNAs, and frequency of HR was determined by flow cytometry after transfection with control or I-SceI vector. Error bars represent ± SD.
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fig6: LSD1 promotes the cellular response to DNA damage. (a) Lentiviral knockdown was performed in HeLa cells using the indicated shRNAs, followed by Western blotting as indicated (top). Colony formation assay was then performed in quadruplicate using the knockdown cells. Y axis represents colony survival normalized to control. (b) U2OS-DR-GFP reporter cells were treated with the indicated shRNAs, and frequency of HR was determined by flow cytometry after transfection with control or I-SceI vector. Error bars represent ± SD.

Mentions: Cells lacking factors directly involved in the DDR, such as 53BP1 and MDC1, are hypersensitive to γ-IR (FitzGerald et al., 2009; Coster and Goldberg, 2010). Therefore, we tested whether this was the case with LSD1. LSD1 was knocked down using lentiviral-mediated shRNA, and knockdown of 53BP1 was also performed in parallel as a positive control (Fig. 6 a). These cells were then used in a colony formation assay, which showed that knockdown of LSD1 caused a moderate sensitivity to γ-IR (approximately two- to threefold at 5 Gy; Fig. 6 a). This degree of γ-IR hypersensitivity, although modest, is similar to what is observed for certain other chromatin-modifying factors that have recently been shown to play a role in the DDR, such as components of the NuRD–CHD4 chromatin remodeling complex (Chou et al., 2010; Larsen et al., 2010; Polo et al., 2010; Smeenk et al., 2010).


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 the cellular response to DNA damage. (a) Lentiviral knockdown was performed in HeLa cells using the indicated shRNAs, followed by Western blotting as indicated (top). Colony formation assay was then performed in quadruplicate using the knockdown cells. Y axis represents colony survival normalized to control. (b) U2OS-DR-GFP reporter cells were treated with the indicated shRNAs, and frequency of HR was determined by flow cytometry after transfection with control or I-SceI vector. Error bars represent ± SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824007&req=5

fig6: LSD1 promotes the cellular response to DNA damage. (a) Lentiviral knockdown was performed in HeLa cells using the indicated shRNAs, followed by Western blotting as indicated (top). Colony formation assay was then performed in quadruplicate using the knockdown cells. Y axis represents colony survival normalized to control. (b) U2OS-DR-GFP reporter cells were treated with the indicated shRNAs, and frequency of HR was determined by flow cytometry after transfection with control or I-SceI vector. Error bars represent ± SD.
Mentions: Cells lacking factors directly involved in the DDR, such as 53BP1 and MDC1, are hypersensitive to γ-IR (FitzGerald et al., 2009; Coster and Goldberg, 2010). Therefore, we tested whether this was the case with LSD1. LSD1 was knocked down using lentiviral-mediated shRNA, and knockdown of 53BP1 was also performed in parallel as a positive control (Fig. 6 a). These cells were then used in a colony formation assay, which showed that knockdown of LSD1 caused a moderate sensitivity to γ-IR (approximately two- to threefold at 5 Gy; Fig. 6 a). This degree of γ-IR hypersensitivity, although modest, is similar to what is observed for certain other chromatin-modifying factors that have recently been shown to play a role in the DDR, such as components of the NuRD–CHD4 chromatin remodeling complex (Chou et al., 2010; Larsen et al., 2010; Polo et al., 2010; Smeenk et al., 2010).

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