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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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Transcription-dependent SMARCA5 (re)localization to UV-C damage. (A) Live cell images (left) before, 5 and 140 s after UV-C (266 nm) laser-induced local damage (arrows) of GFP-CSB (expressed in CSB-deficient CS1AN fibroblasts) and SMARCA5-GFP (expressed in U2OS cells). Scale bar is 5 μm. Graphs (right) show the normalized fluorescence intensities (n > 10 cells) that indicate recruitment to the damage center (blue), the damage periphery (orange) and outside the damaged area (red; mean ± standard error of the mean) of GFP-CSB (top) and SMARCA5-GFP. (B) Treatment with α-amanitin impairs the binding to DNA damage sites of GFP-CSB (P < 0.01 compared to control) and SMARCA5-GFP (peripheral recruitment, P = 0.018 compared to control)). (C) Line scans of GFP-CSB and SMARCA5-GFP intensity along the indicated line in the image (n = 5 cells). (D) GFP-CSB (P = 0.363 compared to control) and SMARCA5-GFP recruitment (center P = 0.682, periphery P = 0.36 compared to control) to DNA damage is unaffected upon PARP inhibition using olaparib (n > 10 cells, mean ± standard error of the mean). RF denotes ‘relative fluorescence’. All results were confirmed using independent, duplicate experiments.
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Figure 2: Transcription-dependent SMARCA5 (re)localization to UV-C damage. (A) Live cell images (left) before, 5 and 140 s after UV-C (266 nm) laser-induced local damage (arrows) of GFP-CSB (expressed in CSB-deficient CS1AN fibroblasts) and SMARCA5-GFP (expressed in U2OS cells). Scale bar is 5 μm. Graphs (right) show the normalized fluorescence intensities (n > 10 cells) that indicate recruitment to the damage center (blue), the damage periphery (orange) and outside the damaged area (red; mean ± standard error of the mean) of GFP-CSB (top) and SMARCA5-GFP. (B) Treatment with α-amanitin impairs the binding to DNA damage sites of GFP-CSB (P < 0.01 compared to control) and SMARCA5-GFP (peripheral recruitment, P = 0.018 compared to control)). (C) Line scans of GFP-CSB and SMARCA5-GFP intensity along the indicated line in the image (n = 5 cells). (D) GFP-CSB (P = 0.363 compared to control) and SMARCA5-GFP recruitment (center P = 0.682, periphery P = 0.36 compared to control) to DNA damage is unaffected upon PARP inhibition using olaparib (n > 10 cells, mean ± standard error of the mean). RF denotes ‘relative fluorescence’. All results were confirmed using independent, duplicate experiments.

Mentions: TC-NER factors such as CSB localize to DNA damage induced by a 266-nm UV-C laser, which specifically induces CPD and 6-4PP photolesions (Figure 2A and Supplementary Movie S1) (35,41). Stably expressed GFP-tagged SMARCA5 also rapidly accumulated at local UV-C damage, in both U2OS and MRC5 cells (Figure 2A, Supplementary Figure S2A and Supplementary Movie S2), in a dose-dependent manner (Supplementary Figure S2B). This was confirmed by local UV damage induction using a microporous filter (Supplementary Figure S2C) (43). The association of TC-NER factors with TC-NER complexes depends on stalling of RNApolII complexes and thus on active transcription (4,36). Inhibition of RNApolII activity using α-amanitin (44) indeed decreased the accumulation of both CSB and SMARCA5 at local damage (Figure 2B). SMARCA5 recruitment was also attenuated by the transcription elongation inhibitor DRB (Supplementary Figure S3A, in which both recruitment to the center and periphery of the damaged area are quantified as explained below) (45). These results confirm a function of SMARCA5 in TC-NER and suggest that this protein may localize to UV damage depending on RNApolII stalling.


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)

Transcription-dependent SMARCA5 (re)localization to UV-C damage. (A) Live cell images (left) before, 5 and 140 s after UV-C (266 nm) laser-induced local damage (arrows) of GFP-CSB (expressed in CSB-deficient CS1AN fibroblasts) and SMARCA5-GFP (expressed in U2OS cells). Scale bar is 5 μm. Graphs (right) show the normalized fluorescence intensities (n > 10 cells) that indicate recruitment to the damage center (blue), the damage periphery (orange) and outside the damaged area (red; mean ± standard error of the mean) of GFP-CSB (top) and SMARCA5-GFP. (B) Treatment with α-amanitin impairs the binding to DNA damage sites of GFP-CSB (P < 0.01 compared to control) and SMARCA5-GFP (peripheral recruitment, P = 0.018 compared to control)). (C) Line scans of GFP-CSB and SMARCA5-GFP intensity along the indicated line in the image (n = 5 cells). (D) GFP-CSB (P = 0.363 compared to control) and SMARCA5-GFP recruitment (center P = 0.682, periphery P = 0.36 compared to control) to DNA damage is unaffected upon PARP inhibition using olaparib (n > 10 cells, mean ± standard error of the mean). RF denotes ‘relative fluorescence’. All results were confirmed using independent, duplicate experiments.
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Figure 2: Transcription-dependent SMARCA5 (re)localization to UV-C damage. (A) Live cell images (left) before, 5 and 140 s after UV-C (266 nm) laser-induced local damage (arrows) of GFP-CSB (expressed in CSB-deficient CS1AN fibroblasts) and SMARCA5-GFP (expressed in U2OS cells). Scale bar is 5 μm. Graphs (right) show the normalized fluorescence intensities (n > 10 cells) that indicate recruitment to the damage center (blue), the damage periphery (orange) and outside the damaged area (red; mean ± standard error of the mean) of GFP-CSB (top) and SMARCA5-GFP. (B) Treatment with α-amanitin impairs the binding to DNA damage sites of GFP-CSB (P < 0.01 compared to control) and SMARCA5-GFP (peripheral recruitment, P = 0.018 compared to control)). (C) Line scans of GFP-CSB and SMARCA5-GFP intensity along the indicated line in the image (n = 5 cells). (D) GFP-CSB (P = 0.363 compared to control) and SMARCA5-GFP recruitment (center P = 0.682, periphery P = 0.36 compared to control) to DNA damage is unaffected upon PARP inhibition using olaparib (n > 10 cells, mean ± standard error of the mean). RF denotes ‘relative fluorescence’. All results were confirmed using independent, duplicate experiments.
Mentions: TC-NER factors such as CSB localize to DNA damage induced by a 266-nm UV-C laser, which specifically induces CPD and 6-4PP photolesions (Figure 2A and Supplementary Movie S1) (35,41). Stably expressed GFP-tagged SMARCA5 also rapidly accumulated at local UV-C damage, in both U2OS and MRC5 cells (Figure 2A, Supplementary Figure S2A and Supplementary Movie S2), in a dose-dependent manner (Supplementary Figure S2B). This was confirmed by local UV damage induction using a microporous filter (Supplementary Figure S2C) (43). The association of TC-NER factors with TC-NER complexes depends on stalling of RNApolII complexes and thus on active transcription (4,36). Inhibition of RNApolII activity using α-amanitin (44) indeed decreased the accumulation of both CSB and SMARCA5 at local damage (Figure 2B). SMARCA5 recruitment was also attenuated by the transcription elongation inhibitor DRB (Supplementary Figure S3A, in which both recruitment to the center and periphery of the damaged area are quantified as explained below) (45). These results confirm a function of SMARCA5 in TC-NER and suggest that this protein may localize to UV damage depending on RNApolII stalling.

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