Oxidative Modification of Guanine in a Potential Z-DNA-Forming Sequence of a Gene Promoter Impacts Gene Expression

Abstract

One response to oxidation of guanine (G) to 8-oxo-7,8-dihydroguanine (OG) in a gene promoter is regulation of mRNA expression suggesting an epigenetic-like role for OG. A proposed mechanism involves G oxidation within a potential G-quadruplex-forming sequence (PQS) in the promoter enabling a structural shift from B-DNA to a G-quadruplex fold (G4). When OG was located in the coding vs. template strand, base excision repair led to an on/off transcriptional switch. Herein, a G-rich, potential Z-DNA-forming sequence (PZS) comprised of a d(GC) n repeat was explored to determine whether oxidation in this motif was also a transcriptional switch. Bioinformatic analysis found 1650 PZSs of length >10 nts in the human genome that were overrepresented in promoters and 5′-UTRs. Studies in human cells transfected with a luciferase reporter plasmid in which OG was synthesized in a PZS context in the promoter found that a coding strand OG increased expression, and a template strand OG decreased expression. The initial base excision repair product of OG, an abasic site (AP), was also found to yield similar expression changes as OG. Biophysical studies on model Z-DNA strands found OG favored a shift in the equilibrium to Z-DNA from B-DNA, while an AP disrupted Z-DNA to favor a hairpin placing AP in the loop where it is a poor substrate for the endonuclease APE1. Overall, the impact of OG and AP in a PZS on gene expression was similar to that in a PQS but reduced in magnitude.

Regulation of GC box activity by 8-oxoguanine

“…The magnitude of the observed effect was higher when 8-oxodG was placed in the pyrimidine-rich strand; however, 8-oxodG in the purine-rich strand also caused a clear impairment of the promoter activity. Thus, in the minimal promoter consisting of a single GC box, the presence of 8-oxodG residues in either DNA strand invariantly led to a decreased promoter activity, in contrast to the alternating activation or repression reported by others in the context of more complex promoter structures [ 36 , 37 , 39 ].…”

“…An advantage of such a design is the possibility of inserting a synthetic 8-oxodG either into the purine-rich or the pyrimidine-rich DNA strand. This is different from previous studies of various GC-rich promoter elements, where the effects of 8-oxodG were only addressed within the G-runs of potential G-quadruplex (G4) forming sequences [ 18 , 36 , 39 ]. Next, we verified the GC box function in the pGCbox-W and pGCbox-C reporter vectors by transfection into HeLa cells.…”

“…The presence of 8-oxodG in DNA may affect transcription via multiple mechanisms [ 31 , 32 ]. The effects induced directly by 8-oxodG include interference with binding of transcription activators/repressors to the cognate DNA sequences [ [33] , [34] , [35] ] and modulation of DNA folding into non-B structures [ 36 , 37 ]. Also, the downstream molecular events and their outcomes for the gene transcription can vary strongly among different promoters.…”

“…In turn, further cleavage of the AP residue by APE1 can lead either to transcriptional activation [ 27 , 43 ] or repression [ 39 , 41 , 44 ], adding another complexity level to the mechanisms of transcriptional regulation by 8-oxodG. In GC-rich regulatory elements, some of the observed responses were assigned to the regulation of DNA folding into non-canonical structures by 8-oxodG, AP lesions and proteins bound to these modifications [ 36 , 37 , 39 , 45 ].…”

New perspectives in cancer biology from a study of canonical and non-canonical functions of base excision repair proteins with a focus on early steps

“…In contrast, the presence of 8-oxo-dG on the template strand activates the transcription-coupled nucleotide excision repair pathway, which attenuates transcription (101,119). Very recently, the same group reported a similar mechanism of action for the G-rich potential Z-DNA forming sequences (PZS) (123). This concept has also been shown for other promoters such as that of the endonuclease III-like protein 1 (NTHL1) (101), RAD17 (124), proliferating cell nuclear antigen (PCNA) (125) and NEIL3 (126), which expanded the generalisation of this model mechanism and emphasised the importance of G4-fold oxidation as a critical driver for gene activation.…”