Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

Abstract

DNA damage can significantly modulate expression of the affected genes either by direct structural interference with transcription components or as a collateral outcome of cellular repair attempts. Thus, DNA glycosylases of the base excision repair (BER) pathway have been implicated in negative transcriptional response to several spontaneously generated DNA base modifications, including a common oxidative DNA base modification 8-oxoguanine (8-oxoG). Here, we report that single 8-oxoG situated in the non-transcribed DNA strand of a reporter gene has a pronounced negative effect on transcription, driven by promoters of various strength and with different structural properties, including viral, human, and artificial promoters. We further show that the magnitude of the negative effect on the gene expression correlates with excision of the modified base by OGG1 in all promoter constructs tested. Moreover, by using expression vectors with nuclease resistant backbone modifications, we demonstrate that OGG1 does not catalyse DNA strand cleavage in vivo. Rather, cleavage of the phosphate bond 5' to 8-oxodG (catalysed by APE1) is essential and universally required for the onset of transcriptional silencing, regardless of the promoter structure. Hence, induction of transcriptional silencing emerges as a ubiquitous mode of biological response to 8-oxoG in DNA.

Figure 1.

Construction of plasmid vectors containing the indicated DNA base (8-oxoG) and backbone (phosphorothioate) modifications in the non-transcribed strand of the reporter EGFP gene. (A) Out of scale scheme of the pZA expression vector showing the EGFP coding sequence (signed open arrow), transcription start (broken arrow) and tandem Nt.Bpu10I nicking sites used for site-specific insertion of synthetic oligonucleotides. (B) Overview of synthetic oligonucleotides and the contained modifications. (C) Verification of the incorporation of the indicated synthetic DNA strands into vector DNA. Inhibition of ligation reaction in the absence of polynucleotidekinase (PNK) is an indicator of the successful strand exchange reaction, as described previously (31). The presence of 8-oxoG is confirmed by DNA strand scission by bacterial Fpg. Positions of covalently closed (cc) and the nicked circular (nc) forms are shown. (D) Incision of plasmid vectors containing the specified DNA modifications with 0.5 units human OGG1.

Figure 2.

Effects of 5′ and 3′ phosphorothioate bonds on the magnitude of inhibition of the EGFP gene expression by single synthetic 8-oxodG in HeLa cells. (A) Representative EGFP fluorescence distribution plots of cells transfected with vectors containing 8-oxodG in combination with the specified DNA backbone modifications (amber lines). Cells in the reference sample (overlaid blue line) were transfected with a control vector containing the corresponding synthetic oligonucleotide without 8-oxoG. (B) EGFP expression in cells transfected with the specified constructs, each normalised relative to the reference construct without 8-oxoG, transfected in parallel. Summary of 6 independent experiments (mean ± SD). P-values: Student's two-tailed t-test. (C) EGFP expression in the OGG1 knockdown (OGG1-sh) HeLa cells and the isogenic control cell line (No sh) analysed at 24 h post-transfection, as described in (A). Error bars show data range (n = 2). (D) DNA strand cleavage activities in the OGG1-overexpressing cell extracts towards plasmid substrates containing the specified modifications. Agarose gel and quantification of the nicked fraction.

Figure 3.

Inhibitory effect of single 8-oxoG on the expression of the inducible EGFP gene. (A) Scheme of the tetracycline-regulated (tet-on) EGFP expression vector. Two TetR binding motifs (TetO₂ ×2) were introduced into the 5′-untranslated region without affecting the protein-coding sequence. Tandem Nt.Bpu10I nicking sites were retained and used for incorporation of synthetic oligonucleotides, as above. (B) Verification of the incorporation of 8-oxoG into the tet-on vector. DNA strand scission analysis of constructs produced with unmodified synthetic oligonucleotide (G) or the oligonucleotide containing single 8-oxoG (8oG). (C) Representative fluorescence distribution plots of T-REx™-Hela cells 24 h post-transfection with the expression constructs containing synthetic oligonucleotides with or without 8-oxoG. Cells were incubated in the absence (- tet) or in the presence of tetracycline (+ tet). (D) Relative EGFP expression calculated at the uninduced (- tet) and induced (+ tet) conditions (n = 6, ± SD).

Figure 4.

Deletion mutants of the CMV-IE promoter are susceptible to the inhibition of the reporter gene expression by single 8-oxoG. (A) List of vectors containing the modified CMV_1111 promoter or its truncated versions together with the map of the introduced deletions. Batons show the canonical CRE sequences. (B) Fluorescence distribution plots and quantification of EGFP expression in HeLa cells 24 h post-transfection with the specified promoter constructs without artificially introduced base modifications. (C) Overlaid EGFP fluorescence distribution plots of cells transfected with vectors containing synthetic DNA strand with one 8-oxoG (amber) and those containing the respective unmodified oligonucleotide (blue). (D) Impact of single 8-oxoG on the gene expression as a function of promoter strength. Data of three independent experiments and the best-fit linear regression lines. EGFP expression was measured at 6 and 24 h post-transfection and calculated relative to expression of the matched construct without 8-oxoG.

Figure 5.

Effect of 8-oxoG on expression controlled by the artificial GR-TK promoter. (A) Elements of the glucocorticoid-regulated EGFP expression vector: minimal TK promoter with transcription start (grey box with broken arrow); EGFP coding sequence (open arrow); two Nt.Bpu10I nicking sites in the NTS, used for the insertion of synthetic oligonucleotides; and glucocorticoid receptor synthetic response element (GRE). (B) Expression of vector constructs containing ‘G’ and ‘8-oxoG’ (overlaid) at the uninduced (- DEX) and induced (+ DEX) conditions. Representative fluorescence distribution plots of cells transfected with the indicated constructs and the median fluorescence values (bar graphs below). (C) EGFP expression under the conditions of co-transfectional (0 h) and delayed (8 h) induction. Representative fluorescence distribution plots and mean EGFP expression, relative to the ‘G’ construct (n = 3, ±SD).

Figure 6.

Effect of 8-oxoG on expression controlled by human promoters. (A) Mean EGFP expression in HeLa cells transfected with the specified constructs (±SD, Student's two-tailed t-test). (B) EGFP expression in the OGG1 knockdown (OGG1-sh) HeLa cells and the isogenic control cell line (No sh) measured at 48 h post-transfections. Representative fluorescent distribution plots and mean relative expression (bar charts below, n = 4, ±SD). (C) Effect of 5′ phosphorothioate on the expression of the pACTB and pRASSF1 constructs containing single 8-oxodG (8oG, s-8oG). Blue colour shows the control ‘G’ construct. Result representative of three independent experiments.

Figure 7.

Reversal of the 8-oxoG-induced transcriptional repression by Trichostatin A (TSA). Fluorescence distribution plots of HeLa cells incubated 24 h in the presence of the specified concentrations of TSA. Amber lines show EGFP expression in cells transfected with the specified pZAJ expression constructs containing 8-oxodG flanked by phosphorothioate linkages, as explained in Figure 1. Overlaid blue line shows the result of parallel transfection with the control construct devoid of 8-oxoG. Bar graph below shows quantification of the median EGFP fluorescence. For each TSA concentration, values are normalised relative to the reference construct without 8-oxoG (‘G’). Representative result of at least three (constructs with phosphorothioates) or more independent experiments.

Figure 8.

Inhibition of the EGFP gene expression by single apurinic site (AP). (A) Vectors used for site-specific insertion of synthetic oligonucleotides containing a single AP lesion (tetrahydrofuran) on the place of dG in the transcribed (TS) or the non-transcribed (NTS) strand of the EGFP gene. (B) Activity of APE1 toward plasmid substrates containing single synthetic AP lesion and the effects of the specified phosphorothioate linkages. (C) Fluorescence distribution plots of HeLa cells 24 h post-transfection with the expression constructs containing the indicated modifications in the specified DNA strand (TS or NTS), compared to the ‘G’ construct (overlaid blue line). Analogous plots for cells incubated in parallel with PARP inhibitors are shown in Supplementary Figure S5. (D) Mean relative EGFP expression of the specified constructs in the absence (DMSO) and in the presence of the indicated PARP inhibitors (n = 3, ±SD).