Regulatory regions responsive to oxidative stress in the promoter of the human DNA glycosylase gene NEIL2

Abstract

Reactive oxygen species (ROS) generated endogenously or from exogenous sources produce mutagenic DNA lesions. If not repaired, these lesions could lead to genomic instability and, potentially, to cancer development. NEIL2 (EC 4.2.99.18), a mammalian base excision repair (BER) protein and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Perturbation in NEIL2 expression may, therefore, significantly impact BER capacity and promote genomic instability. To characterize the genetic and environmental factors regulating NEIL2 gene expression, we mapped the human NEIL2 transcriptional start site and partially characterized the promoter region of the gene using a luciferase reporter assay. We identified a strong positive regulatory region from nucleotide -206 to +90 and found that expression from this region was contingent on its being isolated from an adjacent strong negative regulatory region located downstream (+49 to +710 bp), suggesting that NEIL2 transcription is influenced by both these regions. We also found that oxidative stress, induced by glucose oxidase treatment, reduced the positive regulatory region expression levels, suggesting that ROS may play a significant role in regulating NEIL2 transcription. In an initial attempt to characterize the underlying mechanisms, we used in silico analysis to identify putative cis-acting binding sites for ROS-responsive transcription factors within this region and then used site-directed mutagenesis to investigate their role. A single-base change in the region encompassing nucleotides -206 to +90 abolished the effect of oxidative stress that was observed in the absence of the mutation. Our study is the first to provide an initial partial characterization of the NEIL2 promoter and opens the door for future research aimed at understanding the role of genetic and environmental factors in regulating NEIL2 expression.

Fig. 1

Schematic presentation of the NEIL2 gene and the relative locations of the promoter fragments isolated, cloned and sub-cloned into luciferase reporter vectors. The identified transcriptional start site is designated as +1.

Fig. 2

The DNA sequence, encompassing the pSTEP1 fragment, that includes the NEIL2 gene sequence flanking 5′ upstream and 3′ downstream of the identified transcriptional start site (+1). Putative consensus sequences for binding to regulatory factors are activator protein 1 (AP-1) (highlighted), CREB (italicized), NFκB (underlined) and PEA3 (bold and italicized). Mutation of a putative NFκB/Sp-1 site denoted in rectangle.

Fig. 3

(A) Luciferase constructs containing DNA fragments of the 5′-regulatory region that were cloned upstream of the luciferase-coding sequence; (B) promoter activity (determined as described in Materials and methods). *P < 0.05.

Fig. 4

(A) Luciferase constructs containing the pSTEP3 DNA fragment inserted 5′ of a SV40 promoter; (B) promoter activity relative to a pSV40 control vector is shown in relative light units (RLUs) per millilitre, normalized to total protein in the sample. *P < 0.05.

Fig. 5

(A) Luciferase sub-clone constructs containing fragments (A, B and C) of the p1200 DNA fragment inserted upstream of luciferase-coding sequence; (B) promoter activity (determined as described under Materials and methods). *P < 0.05.

Fig. 6

Luciferase reporter activity in cells transfected with the p1200C constructs and then treated with GO (100 ng/ml) for 1, 6 and 12 h. Comparisons are between GO treated at 1, 6 and 12 h versus untreated. *P < 0.05.

Fig. 7

Promoter activity for p1200C wild type and p1200C + mutation in NFκB/Sp-1 site (104 G to C) with and without 1 h GO treatments (determined as described in Materials and methods). *P < 0.05.