Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury
ALI急性肺损伤机制——>氧化应激——(positional cloning in mice)——>Nrf2（NRF2）结合ARE，上调保护性解毒酶——>NRF2是ALI机制的候选易感性基因
NRF2基因——sequence SNP（-738 ~ -461region）——报告基因实验——>得到两个SNPs有功能相关性
2.1 sequence SNP
We sequenced the –738 to –461 region of the NRF2 promoter to detect the two polymorphisms we found to be functionally relevant using our reporter gene assays (see below). For adequate coverage of the polymorphic region, primers were designed at least 50 to 100 basepairs (bp) away from the SNP site. To obtain a GC clamp for robust sequencing, we amplified the region from 738 to 461. We amplified the site from 100 ng of genomic DNA using the Epicenter Failsafe system (Madison, WI, USA), purified products with the GenElute PCR cleanup kit (Sigma Chemical, St. Louis, MO, USA), and performed sequencing reactions with dGTP dye terminator kits (Applied Biosystems, Foster City, CA, USA). Purified products were analyzed on an ABI 377 Automated DNA Sequencer (Applied Biosystems).
发现3个位点：（文献调研SNP相关性和连锁）Thr72Ala (A/G), Thr92Ile (C/T), and Leu177Val (T/G)
These analyses revealed that variations at -653/-651 and -617 may potentially alter the consensus recognition sequences for MZF1 and NRF2, respectively, suggesting that these polymorphisms may affect NRF2 transcription.
Reporter gene assays
The pGL3 basic reporter vector
缺乏promoter的质粒 vs. 克隆promoter区域的质粒——> 后者强转录活性和荧光素酶表达
mutations（e.g., insertions/deletions and SNPs）——> 影响转录活性
a. 两段序列：[-727 to -131 bp, (727-Luc) ；–538 to -131bp (538-Luc, lacking the SNPs)]；
Constructs were verified by sequencing and transiently transfected into A549 cells (American Type Culture Collection, Manassas, VA, USA) as described previously (17).
These results indicate that the -538 to -131 bp region acts as a basal promoter, while the -538 to -727 region acts as an enhancer.
克隆 727bp NRF2 promoter bearing the indicated polymorphisms upstream of the Luc gene
Variants and putative cis-elements, such as AP-1-like and ARE-like sites, encompassed the SNP region (-659~-608; Fig. 4).
The MZF1, AP1-like, and ARE-like sites mediate transcriptional responses and have been shown to play key regulatory roles in various cellular responses after toxic and oxidant insults(13, 24, 25).
In transient transfection assays, luciferase activity was significantly (P<0.001) higher in – 617 C wild-type compared to promoter constructs bearing –617 A and –651 A variants (Fig. 4).
Basal activity of cells transfected with a promoter construct bearing – 651 A and – 653 G polymorphisms differed significantly (P=0.014) from that of cells transfected with – 651 A alone.
Electrophoretic mobility shift assay (EMSA) allows visualization of transcription factor binding to promoter regulatory sequences and is used to assess effects of mutations at regulatory regions of genes.
Radioactively labeled promoter DNA sequences are incubated with the nuclear proteins isolated from mammalian cells yielding DNA:protein complexes.Migration of the labeled probe on a polyacrylamide gel will be retarded or shifted due to the binding of aprotein(s). Competition with the unlabeled probe or mutations or deletions in the DNA sequence of the probe will diminish or abolish the binding. The specificity of the shifted bands may be further confirmed using antibodies specific to the protein of interest. In the present study, an aliquot (5 ug) of human lung nuclear protein (Active Motif, Carlsbad, CA, USA) was incubated on ice with binding buffer for 15 min, followed by addition of 3*104 cpm [γ32 P]ATP (Amersham Biosciences, Piscataway, NJ, USA) end-labeled wild-type or variant probes (Table 1) and incubated for 30 min at room temperature. We used short (26 bp) and long (62bp)fragment sequences for the EMSA experiments. The short fragment overlaps only the consensus sequence for NRF2, whereas the long fragment overlaps potential binding sites for AP-1, myeloid zinc finger-1 MZF1, and NRF2. The binding of transcription factors to their cognate sites is also influenced by the presence of flanking DNA sequences. Thus, to assess the influence of flanking MZF1 and AP-1 sites on the binding of NRF2 at the ARE-like element, we performed EMSA assays using a fragment containing all three sites. The protein binding of this fragment was compared with that of a shorter fragment, which contains only the ARE-like site. A higher degree of specificity is conferred with shorter oligonucleotide fragments, since there is a lower likelihood of nonspecific hybridization. To analyze specific binding of NRF2 at the –617 polymorphic site, nuclear proteins (5 ug) were preincubated with polyclonal rabbit anti-NRF2 antiserum (2 ul; ref15) or control serum (2 ul) and processed for EMSA as described above. Samples were electrophoresed and autoradiographed with an intensifying screen at –70°C. We tested for statistically significant differences in DNA-NRF2-antibody complex formation between wild-type, heterozygous, and variant genotypes using one-way ANOVA with SNK a posteriori comparisons of means (Sigma Stat).
Importantly, formation of protein-DNA complex was significantly diminished in heterozygotes (P<0.001) and variants (P<0.001) for the -617 polymorphism in the ARE-like sequence (Fig. 5).
蛋白-DNA复合体形成的显著下降表明，-617 SNP影响蛋白的有效结合，比如NRF2和ARE-like site的结合；
Supershift assays with anti-NRF2 antiserum revealed binding of NRF2 to the wild-type ARE-like sequence, suggesting that NRF2 autoregulates transcription through this promoter region.
In support of this notion, Kwak et al.(26) showed that NRF2 binds to the ARE-like element of the mouse Nrf2 promoter (-754) and up-regulates its transcription.
The –617 polymorphism was associated with increased risk of ALI (Table 3). Relative to wild type (-617 C), subjects with at least one A allele at position -617 were at significantly greater risk of ALI (OR 6.44; 95% CI 1.34, 30.8;P=0.021). No significant confounding of this association was found with adjustment for clinical variables, including age, gender, mechanism of trauma, APACHE II score, and volume of fluids and blood products administered (Table 4).
In conclusion, we identified functional polymorphisms in the promoter of NRF2 that are found in relatively high frequency among multiple ethnic populations. We also present epidemiological evidence of an association of an NRF2 polymorphism with ALI risk in a well-characterized clinical at-risk group. This study demonstrates the utility of inbred mouse models for identification of candidate genes in human disease processes when traditional family-based approaches are not feasible and illustrates a candidate gene association approach based on solid laboratory evidence of altered gene function. Future investigations will be instrumental in confirming our observations in additional populations at risk for ALI, as well as investigating the role of NRF2 in other oxidant-related disease processes such as carcinogenesis, aging, atherosclerosis, and chronic inflammatory disorders.