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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 is recruited to sites of DNA damage. (a) UV laser microirradiation was performed on U2OS cells, incubated at 37°C for 10 min, stained for endogenous LSD1, and analyzed by confocal microscopy. (b) U2OS cells were microirradiated as in panel a, and then stained for CoREST or BHC80 and pH2A.X as indicated. Bar, 10 µm. (c) PCR was performed using rDNA or GAPDH primers with LSD1 ChIP material after DSBs were induced with retroviral I-PpoI in U2OS cells. The indicated time points represent the length of 4-OH-tamoxifen (4HT) treatment to induce DSBs. Shown below is the change of LSD1 rDNA ChIP quantified and normalized to input. (d) U2OS cells transduced with control or I-PpoI–expressing virus and were treated with 4HT for 4 h. ChIP was then performed using antibodies against 53BP1 or LSD1, and real-time qPCR was done on the ChIP material using rDNA primers.
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fig1: LSD1 is recruited to sites of DNA damage. (a) UV laser microirradiation was performed on U2OS cells, incubated at 37°C for 10 min, stained for endogenous LSD1, and analyzed by confocal microscopy. (b) U2OS cells were microirradiated as in panel a, and then stained for CoREST or BHC80 and pH2A.X as indicated. Bar, 10 µm. (c) PCR was performed using rDNA or GAPDH primers with LSD1 ChIP material after DSBs were induced with retroviral I-PpoI in U2OS cells. The indicated time points represent the length of 4-OH-tamoxifen (4HT) treatment to induce DSBs. Shown below is the change of LSD1 rDNA ChIP quantified and normalized to input. (d) U2OS cells transduced with control or I-PpoI–expressing virus and were treated with 4HT for 4 h. ChIP was then performed using antibodies against 53BP1 or LSD1, and real-time qPCR was done on the ChIP material using rDNA primers.

Mentions: Recent studies have suggested that H3K4 methylation plays a role in controlling the DDR and recombination events (Cheung et al., 2010; Daniel and Nussenzweig, 2012). Specifically, H3K4 methylation appears to be tightly associated with regions of increased HR and crossovers during meiosis (Borde et al., 2009; Sommermeyer et al., 2013). We therefore wished to determine whether LSD1, an H3K4 demethylase, plays a role in the DDR by being recruited to sites of DNA damage. We used immunostaining to determine whether LSD1 concentrates at sites of DNA damage induced by UV laser microirradiation. Strikingly, 10 min after irradiation, endogenous LSD1 was found at the laser path, colocalizing with pH2A.X (Fig. 1 a and Fig. S1). Knockdown of LSD1 by shRNA resulted in the loss of LSD1 signal at the laser stripe, demonstrating specificity (Fig. S1, a and b). We also found two LSD1-associated proteins, CoREST and BHC80 (Lee et al., 2005; Shi et al., 2005), to colocalize with pH2A.X under the same conditions (Fig. 1 b). Importantly, neither the histone H3K9/K36 demethylase JMJD2A nor the H3K27 demethylase JMJD3 were colocalized with pH2A.X using laser microirradiation (Fig. S1 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 is recruited to sites of DNA damage. (a) UV laser microirradiation was performed on U2OS cells, incubated at 37°C for 10 min, stained for endogenous LSD1, and analyzed by confocal microscopy. (b) U2OS cells were microirradiated as in panel a, and then stained for CoREST or BHC80 and pH2A.X as indicated. Bar, 10 µm. (c) PCR was performed using rDNA or GAPDH primers with LSD1 ChIP material after DSBs were induced with retroviral I-PpoI in U2OS cells. The indicated time points represent the length of 4-OH-tamoxifen (4HT) treatment to induce DSBs. Shown below is the change of LSD1 rDNA ChIP quantified and normalized to input. (d) U2OS cells transduced with control or I-PpoI–expressing virus and were treated with 4HT for 4 h. ChIP was then performed using antibodies against 53BP1 or LSD1, and real-time qPCR was done on the ChIP material using rDNA primers.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: LSD1 is recruited to sites of DNA damage. (a) UV laser microirradiation was performed on U2OS cells, incubated at 37°C for 10 min, stained for endogenous LSD1, and analyzed by confocal microscopy. (b) U2OS cells were microirradiated as in panel a, and then stained for CoREST or BHC80 and pH2A.X as indicated. Bar, 10 µm. (c) PCR was performed using rDNA or GAPDH primers with LSD1 ChIP material after DSBs were induced with retroviral I-PpoI in U2OS cells. The indicated time points represent the length of 4-OH-tamoxifen (4HT) treatment to induce DSBs. Shown below is the change of LSD1 rDNA ChIP quantified and normalized to input. (d) U2OS cells transduced with control or I-PpoI–expressing virus and were treated with 4HT for 4 h. ChIP was then performed using antibodies against 53BP1 or LSD1, and real-time qPCR was done on the ChIP material using rDNA primers.
Mentions: Recent studies have suggested that H3K4 methylation plays a role in controlling the DDR and recombination events (Cheung et al., 2010; Daniel and Nussenzweig, 2012). Specifically, H3K4 methylation appears to be tightly associated with regions of increased HR and crossovers during meiosis (Borde et al., 2009; Sommermeyer et al., 2013). We therefore wished to determine whether LSD1, an H3K4 demethylase, plays a role in the DDR by being recruited to sites of DNA damage. We used immunostaining to determine whether LSD1 concentrates at sites of DNA damage induced by UV laser microirradiation. Strikingly, 10 min after irradiation, endogenous LSD1 was found at the laser path, colocalizing with pH2A.X (Fig. 1 a and Fig. S1). Knockdown of LSD1 by shRNA resulted in the loss of LSD1 signal at the laser stripe, demonstrating specificity (Fig. S1, a and b). We also found two LSD1-associated proteins, CoREST and BHC80 (Lee et al., 2005; Shi et al., 2005), to colocalize with pH2A.X under the same conditions (Fig. 1 b). Importantly, neither the histone H3K9/K36 demethylase JMJD2A nor the H3K27 demethylase JMJD3 were colocalized with pH2A.X using laser microirradiation (Fig. S1 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