OGG1-DNA interactions facilitate NF-κB binding to DNA targets

Abstract

DNA repair protein counteracting oxidative promoter lesions may modulate gene expression. Oxidative DNA bases modified by reactive oxygen species (ROS), primarily as 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair (BER) pathway. Because cellular response to oxidative challenge is accompanied by DNA damage repair, we tested whether the repair by OGG1 is compatible with transcription factor binding and gene expression. We performed electrophoretic mobility shift assay (EMSA) using wild-type sequence deriving from Cxcl2 gene promoter and the same sequence bearing a single synthetic 8-oxoG at defined 5' or 3' guanine in runs of guanines to mimic oxidative effects. We showed that DNA occupancy of NF-κB present in nuclear extracts from tumour necrosis factor alpha (TNFα) exposed cells is OGG1 and 8-oxoG position dependent, importantly, OGG1 counteracting 8-oxoG outside consensus motif had a profound influence on purified NF-κB binding to DNA. Furthermore, OGG1 is essential for NF-κB dependent gene expression, prior to 8-oxoG excised from DNA. These observations imply that pre-excision step(s) during OGG1 initiated BER evoked by ROS facilitates NF-κB DNA occupancy and gene expression.

Figure 1. Ogg1 depletion decreases Cxcl2 promoter driven luciferase activity.

(a,b) Luciferase expression driven by Cxcl2 promoter is dependent on Ogg1 in MLE-12 (a), and MEF cells (b). All cells were exposed to TNFα for six hours. Insert: Ogg1-siRNA mediated Ogg1 knockdown in MLE-12 cells (WB analysis). (c,d) Decreased luciferase mRNA levels in Ogg1-siRNA transfected MLE-12 cells (c) and Ogg1^−/− MEF cells (d) upon TNFα exposure. Whole RNAs were isolated at 1 h post-exposure and luciferase mRNA levels were determined by RT-qPCR. (e) TNFα-induced promoter activation is inhibited by antioxidant pre-treatment in MLE-12 cells. RLU, relative luciferase unit. **p < 0.01, ***p < 0.001 (n = 3).

Figure 2. OGG1 is essential for NF-κB directed promoter activation.

(a) Graphical depiction of Cxcl2 promoter constructs: a wild-type and three mutated Cxcl2 promoter fragments were cloned to drive luciferase expression as described in Materials and Methods. (b,c) Luciferase expression driven by Wt and mutated Cxcl2 promoter. Constructs were transfected into MLE-12 (b) and MEF (c) cells. TNFα treated for 6 h and dual luciferase assays were performed as described in Materials and Methods. (d,e) Effect of Ogg1 on mutated Luc expression. κB2-Luc and κB1-Luc constructs were transfected into Ogg1 expression and siRNA-mediated knockdown MLE-12 cells. TNFα treated for 6 h and dual luciferase assays were performed as described above. RLU, relative luciferase unit. ***p < 0.001 (n = 3).

Figure 3. Effect of oxidative DNA damage on NF-κB binding present in nuclear extracts.

(a,b) Ogg1 depletion decreases levels of NF-κB-DNA association. MLE-12 cells were transfected either with negative control (NC) or Ogg1 siRNA treated without (N) or with (T) TNFα for 30 min, and NE were prepared. NE (1 μg) were incubated with annealed oligonucleotides at RT for 5 min, and EMSA was carried out using κB2 oligoes (a) and κB1 oligoes (b). In some cases, binding was analysed with 100-fold unlabelled wild-type canonical κB oligonucleotide competitors. Arrowhead denote non-specific bands, * and ** indicates unknown bands. (c,d) Histogram of gel data shown in a (lanes 2–15) and b (lanes 2–9). The binding site occupancy of NF-κB on individual probes were calculated as a fold change in bands intensity compared to that of Wt-oligo using Image J software. (e) Independent nuclear translocation of p65. MLE-12 cells were transfected either with control or Ogg1 siRNA. After 48 hour transfection, cells were exposed to TNFα for 30 min and fractionated into cytosolic and nuclear extracts. Western blot analysis with the indicated antibodies, Histone H1 was a nuclear fraction control and GAPDH was a cytosolic fraction control.

Figure 4. OGG1 enhances NF-κB-DNA association.

(a) Kinetic NF-κB homo- and heterodimer binding to κB2-Wt oligo. EMSA shows purified p65 (3.75 ng/sample) and p50 (2.75 ng/sample) proteins binding to κB2-Wt probe over a 10 min time course. (b,c) Effects of OGG1 encountering 8-oxoG on NF-κB home- and heterodimer binding to κB2-oligoes (b) and κB1-oligoes (c). Purified p65 (3.75 ng/sample) and p50 (2.75 ng/sample) were pre-incubated and oligoes were added with or without purified OGG1 (4 ng/sample) after 10 min incubation. (d) The effect of OGG1 on NF-κB subunit binding to κB2-G6 oligo. Lanes 1–3, recombinant p65 (3.75 ng/sample) and p50 (2.75 ng/sample) were incubated with increasing amounts of OGG1 (1, 2 and 4 ng), and then mixed with oligo at RT for 10 min. Lanes 4–6, increasing amounts of OGG1 (1, 2 and 4 ng) binding to κB2-G6 oligo. Lanes 7–15, pre-incubated p65 (3.75, 7.5 and 15 ng respectively) were mixed alone or with 2 or 4 ng of purified OGG1, followed by adding κB2-G6 oligo. Lanes 16–19, pre-incubated p50 (2.75 ng/sample) were mixed with increasing amounts of OGG1 (1, 2 and 4 ng respectively), followed by an EMSA with κB2-G6 oligo. <denotes p65/p50 heterodimer-DNA complex, *denotes p50/p50 homodimer-DNA complex, triangle indicates OGG1-DNA complex, NS denotes non-specific bands. (e) Purified OGG1 binds to 8-oxoG containing DNA. EMSA was performed using increasing concentrations of purified OGG1 (0, 2, 4 and 8 ng respectively) incubated with indicated 8-oxoG containing oligoes at RT for 10 min.

Figure 5. OGG1 is required for a subset of pro-inflammatory gene expression upon TNFα stimulation.

(a) Changes in global 8-oxoG levels assayed by dot blot analysis. Genomic DNA was isolated at times indicated and immunoblotted as described in Materials and Methods. (b) Changes in OGG1 substrate levels in Cxcl2, Tnf and Il-1β proximal promoter regions after TNFα challenge. DNAs were isolated at 0 and 30 min after TNFα exposure and promoter regions were amplified by RT-qPCR after mock (−) and OGG1 digestion (+). (c) Time course of gene expression upon TNFα exposure of MLE-12 cells. RNAs were isolated at times indicated and mRNA levels were determined by RT-qPCR. (d,e) Ogg1 depletion decreases gene expression in MLE12 (d) and MEF (e) cells. Parallel cultures of MLE12 cells were transfected with control or Ogg1-siRNA, after which TNFα treated for 30 min. RNAs were isolated at times indicated and mRNA levels were determined by RT-qPCR. (f) TNFα-induced gene activation is inhibited by antioxidant pre-treatment. ***p < 0.001 (n = 3).