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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 H2A/H2A.X ubiquitylation upon DNA damage. (a) 293T cells were infected with control or LSD1-specific shRNA, and then Western blotted with the indicated antibodies. (b and c) Cells from panel a were synchronized in late G2 using thymidine-nocodazole and were harvested 4 h after the indicated doses of γ-IR. Whole cell extracts were analyzed by Western blot using the indicated antibodies. (d) After knockdown and selection, U2OS cells were irradiated and processed for immunofluorescence using antibodies against MDC1 and ubiquitylated H2A. (e) Quantitation of panel d. (f) U2OS cells treated with control or LSD1-specific shRNA were laser microirradiated and stained with the indicated antibodies. (g) Quantitation of panel f. Bars, 20 µm.
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fig8: LSD1 promotes H2A/H2A.X ubiquitylation upon DNA damage. (a) 293T cells were infected with control or LSD1-specific shRNA, and then Western blotted with the indicated antibodies. (b and c) Cells from panel a were synchronized in late G2 using thymidine-nocodazole and were harvested 4 h after the indicated doses of γ-IR. Whole cell extracts were analyzed by Western blot using the indicated antibodies. (d) After knockdown and selection, U2OS cells were irradiated and processed for immunofluorescence using antibodies against MDC1 and ubiquitylated H2A. (e) Quantitation of panel d. (f) U2OS cells treated with control or LSD1-specific shRNA were laser microirradiated and stained with the indicated antibodies. (g) Quantitation of panel f. Bars, 20 µm.

Mentions: RNF168 is an E3 ligase that promotes H2A/H2A.X ubiquitylation, and this activity is important for the recruitment of 53BP1 to sites of damage (Doil et al., 2009; Stewart et al., 2009). Our data suggested that LSD1 is associated with RNF168 and promotes 53BP1 recruitment. Therefore, we next determined whether IR-induced H2A/H2A.X ubiquitylation is affected in the absence by LSD1 late in the cell cycle. Cells were synchronized in late G2 using sequential thymidine-nocodazole block after transduction with control or LSD1-specific shRNA, followed by γ-IR treatment (Fig. 8, a and b). LSD1 knockdown did not significantly alter IR-induced pH2A.X, consistent with the lack of observable difference in pH2A.X foci formation (Fig. 7 a). However, there was a reduced signal corresponding to ubiquitylated H2A.X and ubiquitylated pH2A.X, particularly upon γ-IR (Fig. 8 b). Global levels of H2A ubiquitylation appeared to be significantly affected only with γ-IR (Fig. 8 b, bottom, compare lanes 1 and 4), suggesting that LSD1 does not play a significant role in H2A ubiquitylation in the absence of DNA damage. 53BP1 recruitment may also be affected by dimethylation of H4K20, but we did not observe a difference in the global methylation status of H4K20me2 before or after γ-IR (Fig. 8 c).


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 H2A/H2A.X ubiquitylation upon DNA damage. (a) 293T cells were infected with control or LSD1-specific shRNA, and then Western blotted with the indicated antibodies. (b and c) Cells from panel a were synchronized in late G2 using thymidine-nocodazole and were harvested 4 h after the indicated doses of γ-IR. Whole cell extracts were analyzed by Western blot using the indicated antibodies. (d) After knockdown and selection, U2OS cells were irradiated and processed for immunofluorescence using antibodies against MDC1 and ubiquitylated H2A. (e) Quantitation of panel d. (f) U2OS cells treated with control or LSD1-specific shRNA were laser microirradiated and stained with the indicated antibodies. (g) Quantitation of panel f. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: LSD1 promotes H2A/H2A.X ubiquitylation upon DNA damage. (a) 293T cells were infected with control or LSD1-specific shRNA, and then Western blotted with the indicated antibodies. (b and c) Cells from panel a were synchronized in late G2 using thymidine-nocodazole and were harvested 4 h after the indicated doses of γ-IR. Whole cell extracts were analyzed by Western blot using the indicated antibodies. (d) After knockdown and selection, U2OS cells were irradiated and processed for immunofluorescence using antibodies against MDC1 and ubiquitylated H2A. (e) Quantitation of panel d. (f) U2OS cells treated with control or LSD1-specific shRNA were laser microirradiated and stained with the indicated antibodies. (g) Quantitation of panel f. Bars, 20 µm.
Mentions: RNF168 is an E3 ligase that promotes H2A/H2A.X ubiquitylation, and this activity is important for the recruitment of 53BP1 to sites of damage (Doil et al., 2009; Stewart et al., 2009). Our data suggested that LSD1 is associated with RNF168 and promotes 53BP1 recruitment. Therefore, we next determined whether IR-induced H2A/H2A.X ubiquitylation is affected in the absence by LSD1 late in the cell cycle. Cells were synchronized in late G2 using sequential thymidine-nocodazole block after transduction with control or LSD1-specific shRNA, followed by γ-IR treatment (Fig. 8, a and b). LSD1 knockdown did not significantly alter IR-induced pH2A.X, consistent with the lack of observable difference in pH2A.X foci formation (Fig. 7 a). However, there was a reduced signal corresponding to ubiquitylated H2A.X and ubiquitylated pH2A.X, particularly upon γ-IR (Fig. 8 b). Global levels of H2A ubiquitylation appeared to be significantly affected only with γ-IR (Fig. 8 b, bottom, compare lanes 1 and 4), suggesting that LSD1 does not play a significant role in H2A ubiquitylation in the absence of DNA damage. 53BP1 recruitment may also be affected by dimethylation of H4K20, but we did not observe a difference in the global methylation status of H4K20me2 before or after γ-IR (Fig. 8 c).

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