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Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription.

Aydin ÖZ, Marteijn JA, Ribeiro-Silva C, Rodríguez López A, Wijgers N, Smeenk G, van Attikum H, Poot RA, Vermeulen W, Lans H - Nucleic Acids Res. (2014)

Bottom Line: Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription.After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain.Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Medical Genetics Cluster, Cancer Genomics Netherlands, Erasmus MC, Rotterdam, 3015 GE, The Netherlands.

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WSTF and ACF1 are recruited to UV damage to regulate CSB recruitment. ACF1-GFP (A) and GFP-WSTF (B) are recruited to DNA damage induced by UV-C (266 nm) laser. Graphs depict normalized fluorescence intensities indicating DNA damage recruitment in the damage center (blue), the damage periphery (orange) and outside the damaged area (red) (mean ± standard error of the mean; n > 10 cells). Representative images of the accumulation of ACF1-GFP and GFP-WSTF at sites of UV damage are shown below the graphs (scale bar is 5 μm). (C) GFP immunoprecipitation of GFP-tagged SMARCA5 (WT) and ATPase (ATP), HAND and SLIDE domain deletion mutants. CTRL is control. Only deletion of the SLIDE domain impairs the interaction of SMARCA5 with ACF1 and WSTF. (D,E) Graphs of the normalized fluorescence intensity indicating DNA damage recruitment of GFP-CSB in cells in which ACF1 [(D); P = 0.033 compared to control] or WSTF [(E); P = 0.117 compared to control] is depleted by siRNA (mean ± standard error of the mean; n > 10 cells). Results were confirmed using independent, duplicate experiments.
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Figure 7: WSTF and ACF1 are recruited to UV damage to regulate CSB recruitment. ACF1-GFP (A) and GFP-WSTF (B) are recruited to DNA damage induced by UV-C (266 nm) laser. Graphs depict normalized fluorescence intensities indicating DNA damage recruitment in the damage center (blue), the damage periphery (orange) and outside the damaged area (red) (mean ± standard error of the mean; n > 10 cells). Representative images of the accumulation of ACF1-GFP and GFP-WSTF at sites of UV damage are shown below the graphs (scale bar is 5 μm). (C) GFP immunoprecipitation of GFP-tagged SMARCA5 (WT) and ATPase (ATP), HAND and SLIDE domain deletion mutants. CTRL is control. Only deletion of the SLIDE domain impairs the interaction of SMARCA5 with ACF1 and WSTF. (D,E) Graphs of the normalized fluorescence intensity indicating DNA damage recruitment of GFP-CSB in cells in which ACF1 [(D); P = 0.033 compared to control] or WSTF [(E); P = 0.117 compared to control] is depleted by siRNA (mean ± standard error of the mean; n > 10 cells). Results were confirmed using independent, duplicate experiments.

Mentions: CSB (Figure 5C), ACF1 and WSTF (Supplementary Figure S5A and B) were immunoprecipitated using chromatin-enriched nuclear extracts from 10 14-cm culture dishes of GFP-CSB expressing CS1AN cells or five 14-cm culture dishes of U2OS cells expressing ACF1-GFP or GFP-WSTF. Cells were collected 20 min (CSB) or 5 min (ACF1/WSTF) after irradiation (20 J/m2) by scraping in 3 ml of phosphate buffered saline (PBS) containing protease inhibitor cocktail (Roche), centrifuged for 5 min at 1500 rpm and washed again with PBS. Cells were swollen in 5 x pellet volume of Hepes buffer (10-mM HEPES pH 7.6, 1.5-mM MgCl2, 10-mM KCl, 0.5-mM Dithiothreitol, protease inhibitor cocktail) for 10 min. Nuclei were isolated by douncing cells with a type A pestle and centrifugation at 3000 rpm for 10 min at 4°C. Nuclei were washed and resuspended in 1.5 x pellet volumes of Hepes buffer (100-mM HEPES pH 7.6, 1.5-mM MgCl2, 150-mM NaCl, 25% glycerol, protease inhibitor, 0.5-mM Dithiothreitol) and subsequently dounced using a pestle type B. Next, chromatin was digested with 25-U Microccocal nuclease (MNase; Sigma) for 1 h at 4°C. These conditions were chosen such that DNA was digested to mononucleosome size. The resulting chromatin-enriched nucleoplasmic fraction was cleared from insoluble nuclear material by centrifugation at 15 000 rpm for 15 min. For immunoprecipitation of SMARCA5 mutants (Figure 7C), extracts were prepared by scraping cells from a 14-cm dish in Radioimmunoprecipitation assay buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulphate; Roche protease inhibitor cocktail) followed by sonication (to obtain DNA fragments <800 bp) and 16 000 g centrifugation at 4°C for 10 min to remove insoluble material. Extracts were incubated with GFP-trap beads (Chromotek) for 2 h at 4°C. Subsequently, beads were washed four times in Hepes buffer and boiled in Laemmli sample buffer for analysis by western blotting.


Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription.

Aydin ÖZ, Marteijn JA, Ribeiro-Silva C, Rodríguez López A, Wijgers N, Smeenk G, van Attikum H, Poot RA, Vermeulen W, Lans H - Nucleic Acids Res. (2014)

WSTF and ACF1 are recruited to UV damage to regulate CSB recruitment. ACF1-GFP (A) and GFP-WSTF (B) are recruited to DNA damage induced by UV-C (266 nm) laser. Graphs depict normalized fluorescence intensities indicating DNA damage recruitment in the damage center (blue), the damage periphery (orange) and outside the damaged area (red) (mean ± standard error of the mean; n > 10 cells). Representative images of the accumulation of ACF1-GFP and GFP-WSTF at sites of UV damage are shown below the graphs (scale bar is 5 μm). (C) GFP immunoprecipitation of GFP-tagged SMARCA5 (WT) and ATPase (ATP), HAND and SLIDE domain deletion mutants. CTRL is control. Only deletion of the SLIDE domain impairs the interaction of SMARCA5 with ACF1 and WSTF. (D,E) Graphs of the normalized fluorescence intensity indicating DNA damage recruitment of GFP-CSB in cells in which ACF1 [(D); P = 0.033 compared to control] or WSTF [(E); P = 0.117 compared to control] is depleted by siRNA (mean ± standard error of the mean; n > 10 cells). Results were confirmed using independent, duplicate experiments.
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Figure 7: WSTF and ACF1 are recruited to UV damage to regulate CSB recruitment. ACF1-GFP (A) and GFP-WSTF (B) are recruited to DNA damage induced by UV-C (266 nm) laser. Graphs depict normalized fluorescence intensities indicating DNA damage recruitment in the damage center (blue), the damage periphery (orange) and outside the damaged area (red) (mean ± standard error of the mean; n > 10 cells). Representative images of the accumulation of ACF1-GFP and GFP-WSTF at sites of UV damage are shown below the graphs (scale bar is 5 μm). (C) GFP immunoprecipitation of GFP-tagged SMARCA5 (WT) and ATPase (ATP), HAND and SLIDE domain deletion mutants. CTRL is control. Only deletion of the SLIDE domain impairs the interaction of SMARCA5 with ACF1 and WSTF. (D,E) Graphs of the normalized fluorescence intensity indicating DNA damage recruitment of GFP-CSB in cells in which ACF1 [(D); P = 0.033 compared to control] or WSTF [(E); P = 0.117 compared to control] is depleted by siRNA (mean ± standard error of the mean; n > 10 cells). Results were confirmed using independent, duplicate experiments.
Mentions: CSB (Figure 5C), ACF1 and WSTF (Supplementary Figure S5A and B) were immunoprecipitated using chromatin-enriched nuclear extracts from 10 14-cm culture dishes of GFP-CSB expressing CS1AN cells or five 14-cm culture dishes of U2OS cells expressing ACF1-GFP or GFP-WSTF. Cells were collected 20 min (CSB) or 5 min (ACF1/WSTF) after irradiation (20 J/m2) by scraping in 3 ml of phosphate buffered saline (PBS) containing protease inhibitor cocktail (Roche), centrifuged for 5 min at 1500 rpm and washed again with PBS. Cells were swollen in 5 x pellet volume of Hepes buffer (10-mM HEPES pH 7.6, 1.5-mM MgCl2, 10-mM KCl, 0.5-mM Dithiothreitol, protease inhibitor cocktail) for 10 min. Nuclei were isolated by douncing cells with a type A pestle and centrifugation at 3000 rpm for 10 min at 4°C. Nuclei were washed and resuspended in 1.5 x pellet volumes of Hepes buffer (100-mM HEPES pH 7.6, 1.5-mM MgCl2, 150-mM NaCl, 25% glycerol, protease inhibitor, 0.5-mM Dithiothreitol) and subsequently dounced using a pestle type B. Next, chromatin was digested with 25-U Microccocal nuclease (MNase; Sigma) for 1 h at 4°C. These conditions were chosen such that DNA was digested to mononucleosome size. The resulting chromatin-enriched nucleoplasmic fraction was cleared from insoluble nuclear material by centrifugation at 15 000 rpm for 15 min. For immunoprecipitation of SMARCA5 mutants (Figure 7C), extracts were prepared by scraping cells from a 14-cm dish in Radioimmunoprecipitation assay buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulphate; Roche protease inhibitor cocktail) followed by sonication (to obtain DNA fragments <800 bp) and 16 000 g centrifugation at 4°C for 10 min to remove insoluble material. Extracts were incubated with GFP-trap beads (Chromotek) for 2 h at 4°C. Subsequently, beads were washed four times in Hepes buffer and boiled in Laemmli sample buffer for analysis by western blotting.

Bottom Line: Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription.After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain.Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Medical Genetics Cluster, Cancer Genomics Netherlands, Erasmus MC, Rotterdam, 3015 GE, The Netherlands.

Show MeSH
Related in: MedlinePlus