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GCN5 and E2F1 stimulate nucleotide excision repair by promoting H3K9 acetylation at sites of damage.

Guo R, Chen J, Mitchell DL, Johnson DG - Nucleic Acids Res. (2010)

Bottom Line: However, the molecular mechanism by which UV radiation induces histone acetylation to allow for efficient NER is not completely understood.UV radiation induces the acetylation of histone H3 lysine 9 (H3K9) and this requires both GCN5 and E2F1.These findings demonstrate a direct role for GCN5 and E2F1 in NER involving H3K9 acetylation and increased accessibility to the NER machinery.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Carcinogenesis, UT MD Anderson Cancer Center, Science Park-Research Division, 1808 Park Road 1C, PO Box 389, Smithville, TX 78957, USA.

ABSTRACT
Chromatin structure is known to be a barrier to DNA repair and a large number of studies have now identified various factors that modify histones and remodel nucleosomes to facilitate repair. In response to ultraviolet (UV) radiation several histones are acetylated and this enhances the repair of DNA photoproducts by the nucleotide excision repair (NER) pathway. However, the molecular mechanism by which UV radiation induces histone acetylation to allow for efficient NER is not completely understood. We recently discovered that the E2F1 transcription factor accumulates at sites of UV-induced DNA damage and directly stimulates NER through a non-transcriptional mechanism. Here we demonstrate that E2F1 associates with the GCN5 acetyltransferase in response to UV radiation and recruits GCN5 to sites of damage. UV radiation induces the acetylation of histone H3 lysine 9 (H3K9) and this requires both GCN5 and E2F1. Moreover, as previously observed for E2F1, knock down of GCN5 results in impaired recruitment of NER factors to sites of damage and inefficient DNA repair. These findings demonstrate a direct role for GCN5 and E2F1 in NER involving H3K9 acetylation and increased accessibility to the NER machinery.

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E2F1 is required for GCN5 localization to sites of UV damage. (A) NHFs were transfected with siRNA to E2F1 or control siRNA and 48 h later exposed to 500 J/m2 of UVB 30 min prior to harvest. Western blot analysis was performed using whole-cell lysates and antibodies to E2F1 and GCN5. (B) NHFs transfected with control siRNA (upper panels) or siRNA to E2F1 (lower panels) were irradiated with 50 J/m2 of UVC through a filter. Thirty minute post-irradiation, cells were fluorescently stained for CPD (red) or GCN5 (green) and counter-stained with DAPI. (C) Co-localization of GCN5 with CPD were scored from three independent experiments as described. Asterisk indicates statistically significant difference (P < 0.05). (D) NHFs were exposed to 500 J/m2 of UVB 30 min prior to harvest (lanes 1 and 3) or untreated (lane 2) as indicated. Immunoprecipitation was performed using control IgG (lane 1) or antibody to E2F1 (lanes 2 and 3) and the precipitate was subjected to western blot analysis for E2F1 (top panel) and GCN5 (bottom panel). An input control western blot is shown at right.
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Figure 2: E2F1 is required for GCN5 localization to sites of UV damage. (A) NHFs were transfected with siRNA to E2F1 or control siRNA and 48 h later exposed to 500 J/m2 of UVB 30 min prior to harvest. Western blot analysis was performed using whole-cell lysates and antibodies to E2F1 and GCN5. (B) NHFs transfected with control siRNA (upper panels) or siRNA to E2F1 (lower panels) were irradiated with 50 J/m2 of UVC through a filter. Thirty minute post-irradiation, cells were fluorescently stained for CPD (red) or GCN5 (green) and counter-stained with DAPI. (C) Co-localization of GCN5 with CPD were scored from three independent experiments as described. Asterisk indicates statistically significant difference (P < 0.05). (D) NHFs were exposed to 500 J/m2 of UVB 30 min prior to harvest (lanes 1 and 3) or untreated (lane 2) as indicated. Immunoprecipitation was performed using control IgG (lane 1) or antibody to E2F1 (lanes 2 and 3) and the precipitate was subjected to western blot analysis for E2F1 (top panel) and GCN5 (bottom panel). An input control western blot is shown at right.

Mentions: We recently discovered that the E2F1 transcription factor accumulates at sites of UV-induced DNA damage and functions to stimulate NER through a non-transcriptional mechanism (19). Given that GCN5 can partner with E2F factors in the context of transcription (33), we asked whether E2F1 might be involved in the localization of GCN5 to sites of UV damage. Depletion of E2F1 with siRNA did not affect the expression of GCN5 but did significantly decrease the co-localization of GCN5 with sites of damage (Figure 2A–C). Moreover, UV radiation induced a stable association between the endogenous GCN5 and E2F1 proteins (Figure 2D).Figure 2.


GCN5 and E2F1 stimulate nucleotide excision repair by promoting H3K9 acetylation at sites of damage.

Guo R, Chen J, Mitchell DL, Johnson DG - Nucleic Acids Res. (2010)

E2F1 is required for GCN5 localization to sites of UV damage. (A) NHFs were transfected with siRNA to E2F1 or control siRNA and 48 h later exposed to 500 J/m2 of UVB 30 min prior to harvest. Western blot analysis was performed using whole-cell lysates and antibodies to E2F1 and GCN5. (B) NHFs transfected with control siRNA (upper panels) or siRNA to E2F1 (lower panels) were irradiated with 50 J/m2 of UVC through a filter. Thirty minute post-irradiation, cells were fluorescently stained for CPD (red) or GCN5 (green) and counter-stained with DAPI. (C) Co-localization of GCN5 with CPD were scored from three independent experiments as described. Asterisk indicates statistically significant difference (P < 0.05). (D) NHFs were exposed to 500 J/m2 of UVB 30 min prior to harvest (lanes 1 and 3) or untreated (lane 2) as indicated. Immunoprecipitation was performed using control IgG (lane 1) or antibody to E2F1 (lanes 2 and 3) and the precipitate was subjected to western blot analysis for E2F1 (top panel) and GCN5 (bottom panel). An input control western blot is shown at right.
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Figure 2: E2F1 is required for GCN5 localization to sites of UV damage. (A) NHFs were transfected with siRNA to E2F1 or control siRNA and 48 h later exposed to 500 J/m2 of UVB 30 min prior to harvest. Western blot analysis was performed using whole-cell lysates and antibodies to E2F1 and GCN5. (B) NHFs transfected with control siRNA (upper panels) or siRNA to E2F1 (lower panels) were irradiated with 50 J/m2 of UVC through a filter. Thirty minute post-irradiation, cells were fluorescently stained for CPD (red) or GCN5 (green) and counter-stained with DAPI. (C) Co-localization of GCN5 with CPD were scored from three independent experiments as described. Asterisk indicates statistically significant difference (P < 0.05). (D) NHFs were exposed to 500 J/m2 of UVB 30 min prior to harvest (lanes 1 and 3) or untreated (lane 2) as indicated. Immunoprecipitation was performed using control IgG (lane 1) or antibody to E2F1 (lanes 2 and 3) and the precipitate was subjected to western blot analysis for E2F1 (top panel) and GCN5 (bottom panel). An input control western blot is shown at right.
Mentions: We recently discovered that the E2F1 transcription factor accumulates at sites of UV-induced DNA damage and functions to stimulate NER through a non-transcriptional mechanism (19). Given that GCN5 can partner with E2F factors in the context of transcription (33), we asked whether E2F1 might be involved in the localization of GCN5 to sites of UV damage. Depletion of E2F1 with siRNA did not affect the expression of GCN5 but did significantly decrease the co-localization of GCN5 with sites of damage (Figure 2A–C). Moreover, UV radiation induced a stable association between the endogenous GCN5 and E2F1 proteins (Figure 2D).Figure 2.

Bottom Line: However, the molecular mechanism by which UV radiation induces histone acetylation to allow for efficient NER is not completely understood.UV radiation induces the acetylation of histone H3 lysine 9 (H3K9) and this requires both GCN5 and E2F1.These findings demonstrate a direct role for GCN5 and E2F1 in NER involving H3K9 acetylation and increased accessibility to the NER machinery.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Carcinogenesis, UT MD Anderson Cancer Center, Science Park-Research Division, 1808 Park Road 1C, PO Box 389, Smithville, TX 78957, USA.

ABSTRACT
Chromatin structure is known to be a barrier to DNA repair and a large number of studies have now identified various factors that modify histones and remodel nucleosomes to facilitate repair. In response to ultraviolet (UV) radiation several histones are acetylated and this enhances the repair of DNA photoproducts by the nucleotide excision repair (NER) pathway. However, the molecular mechanism by which UV radiation induces histone acetylation to allow for efficient NER is not completely understood. We recently discovered that the E2F1 transcription factor accumulates at sites of UV-induced DNA damage and directly stimulates NER through a non-transcriptional mechanism. Here we demonstrate that E2F1 associates with the GCN5 acetyltransferase in response to UV radiation and recruits GCN5 to sites of damage. UV radiation induces the acetylation of histone H3 lysine 9 (H3K9) and this requires both GCN5 and E2F1. Moreover, as previously observed for E2F1, knock down of GCN5 results in impaired recruitment of NER factors to sites of damage and inefficient DNA repair. These findings demonstrate a direct role for GCN5 and E2F1 in NER involving H3K9 acetylation and increased accessibility to the NER machinery.

Show MeSH
Related in: MedlinePlus