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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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H3K4me2 demethylation marks sites of DNA damage and is cell cycle and LSD1 dependent. (a) UV laser microirradiation was performed on U2OS cells, and the cells were stained with antibodies against the indicated histone modifications. Arrows indicate nuclei with apparent loss of H3K4me2 signal. (b) Magnified view of the cell from top right of panel a, showing H3K4me2 or merged H3K4me2/pH2A.X signals. Intensity profiles of H3K4me2 (red) and pH2A.X (green) signals through the four indicated lines (2–5) are shown on the right. (c) I-PpoI ChIP/qPCR was performed using the indicated histone modification-specific antibodies and primers specific for rDNA. Error bars represent ± standard error. (d) Schematic of FUCCI cells. FUCCI cells coexpress a fragment of Cdt1 linked to the fluorescent protein mK02 (monomeric Kusabira orange 2), as well as a fragment of Geminin linked to the fluorescent protein mAG (monomeric Azami green). (e) UV laser microirradiation was performed on U2OS-FUCCI cells and subsequently stained for pH2A.X and H3K4me2. (f) UV laser microirradiation was performed on wild-type (WT) and LSD1−/− MEFs and subsequently stained for the indicated histone modifications. Bars, 10 µm.
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fig2: H3K4me2 demethylation marks sites of DNA damage and is cell cycle and LSD1 dependent. (a) UV laser microirradiation was performed on U2OS cells, and the cells were stained with antibodies against the indicated histone modifications. Arrows indicate nuclei with apparent loss of H3K4me2 signal. (b) Magnified view of the cell from top right of panel a, showing H3K4me2 or merged H3K4me2/pH2A.X signals. Intensity profiles of H3K4me2 (red) and pH2A.X (green) signals through the four indicated lines (2–5) are shown on the right. (c) I-PpoI ChIP/qPCR was performed using the indicated histone modification-specific antibodies and primers specific for rDNA. Error bars represent ± standard error. (d) Schematic of FUCCI cells. FUCCI cells coexpress a fragment of Cdt1 linked to the fluorescent protein mK02 (monomeric Kusabira orange 2), as well as a fragment of Geminin linked to the fluorescent protein mAG (monomeric Azami green). (e) UV laser microirradiation was performed on U2OS-FUCCI cells and subsequently stained for pH2A.X and H3K4me2. (f) UV laser microirradiation was performed on wild-type (WT) and LSD1−/− MEFs and subsequently stained for the indicated histone modifications. Bars, 10 µm.

Mentions: The fact that LSD1 is specifically recruited to sites of DNA damage suggested that a reduction of H3K4 methylation may be observed at sites of DNA damage. Consistent with this prediction, we found that H3K4me2 is lost in a significant population of cells upon laser microirradiation (Fig. 2 a, top). Profiling of the immunofluorescent signal confirmed an inverse correlation between pH2A.X and H3K4me2 (Fig. 2 b). We termed this loss of H3K4me2 at laser stripes as H3K4me2 antistripes. LSD1 has also been reported to demethylate H3K9me2 (Metzger et al., 2005), but we did not observe loss of H3K9me2 signal at pH2A.X stripes (Fig. 2 a, bottom). Furthermore, we did not observe loss of total H3 upon microirradiation (unpublished data). Collectively, these findings suggest that LSD1 may be responsible for the H3K4me2 reduction at DNA damage sites.


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)

H3K4me2 demethylation marks sites of DNA damage and is cell cycle and LSD1 dependent. (a) UV laser microirradiation was performed on U2OS cells, and the cells were stained with antibodies against the indicated histone modifications. Arrows indicate nuclei with apparent loss of H3K4me2 signal. (b) Magnified view of the cell from top right of panel a, showing H3K4me2 or merged H3K4me2/pH2A.X signals. Intensity profiles of H3K4me2 (red) and pH2A.X (green) signals through the four indicated lines (2–5) are shown on the right. (c) I-PpoI ChIP/qPCR was performed using the indicated histone modification-specific antibodies and primers specific for rDNA. Error bars represent ± standard error. (d) Schematic of FUCCI cells. FUCCI cells coexpress a fragment of Cdt1 linked to the fluorescent protein mK02 (monomeric Kusabira orange 2), as well as a fragment of Geminin linked to the fluorescent protein mAG (monomeric Azami green). (e) UV laser microirradiation was performed on U2OS-FUCCI cells and subsequently stained for pH2A.X and H3K4me2. (f) UV laser microirradiation was performed on wild-type (WT) and LSD1−/− MEFs and subsequently stained for the indicated histone modifications. Bars, 10 µm.
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fig2: H3K4me2 demethylation marks sites of DNA damage and is cell cycle and LSD1 dependent. (a) UV laser microirradiation was performed on U2OS cells, and the cells were stained with antibodies against the indicated histone modifications. Arrows indicate nuclei with apparent loss of H3K4me2 signal. (b) Magnified view of the cell from top right of panel a, showing H3K4me2 or merged H3K4me2/pH2A.X signals. Intensity profiles of H3K4me2 (red) and pH2A.X (green) signals through the four indicated lines (2–5) are shown on the right. (c) I-PpoI ChIP/qPCR was performed using the indicated histone modification-specific antibodies and primers specific for rDNA. Error bars represent ± standard error. (d) Schematic of FUCCI cells. FUCCI cells coexpress a fragment of Cdt1 linked to the fluorescent protein mK02 (monomeric Kusabira orange 2), as well as a fragment of Geminin linked to the fluorescent protein mAG (monomeric Azami green). (e) UV laser microirradiation was performed on U2OS-FUCCI cells and subsequently stained for pH2A.X and H3K4me2. (f) UV laser microirradiation was performed on wild-type (WT) and LSD1−/− MEFs and subsequently stained for the indicated histone modifications. Bars, 10 µm.
Mentions: The fact that LSD1 is specifically recruited to sites of DNA damage suggested that a reduction of H3K4 methylation may be observed at sites of DNA damage. Consistent with this prediction, we found that H3K4me2 is lost in a significant population of cells upon laser microirradiation (Fig. 2 a, top). Profiling of the immunofluorescent signal confirmed an inverse correlation between pH2A.X and H3K4me2 (Fig. 2 b). We termed this loss of H3K4me2 at laser stripes as H3K4me2 antistripes. LSD1 has also been reported to demethylate H3K9me2 (Metzger et al., 2005), but we did not observe loss of H3K9me2 signal at pH2A.X stripes (Fig. 2 a, bottom). Furthermore, we did not observe loss of total H3 upon microirradiation (unpublished data). Collectively, these findings suggest that LSD1 may be responsible for the H3K4me2 reduction at DNA damage sites.

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