Analysis Novel NRF2 ARE Genes Using Luciferase Reporter System

BackgroundThe antioxidant response element(ARE)is a unique cis-acting regulatory sequence present in the upstream(promoter)region of a number of genes encoding enzymes involved in the phase ?/? metabolism of xenobiotics,cytoprotective proteins and its activity mainly regulated by NRF2.NRF2 is a master regulator of the antioxidant response pathway and orchestrates the transcriptional activation of cellular defense mechanism by combating oxidative stress.Presence of ARE is a common feature of the 5'flanking region of genes which are regulated by NRF2.There are number of different detection methods used to determine the binding sites of transcription factors in which most commonly used experimental methods are luciferase report gene,electrophoretic mobility shift assays,chromatin precipitation and DNase footprinting.In the recent years,emerging techniques such as Chromatin immunoprecipitation followed by high-throughput sequencing data and different statistical algorithms are been widely used to identify the transcription factor binding sites(TFBS)in different research platforms.Using ChIP-Seq,microarray and bioinformatics analysis tools,we carried out the identification of NRF2-TFBS in this study.There are many freely available TFBS identification software's such as TRANSFAC,JASPAR,LASAGNA,and Transcription Factor Affinity Prediction(TRAP).We specifically used TRAP web tool to identify the NRF2-TFBS because of its robustness and accuracy.TRAP protocol covers two major applications:(i)determining which transcription factors have the highest affinity in a set of sequences(ii)finding which factor is the most affected by a regulatory single-nucleotide polymorphism.A high-throughput ChIP-sequencing analysis performed in NRF2 overexpressing A549 Non Small Cell Lung Cancer(NSCLC)cells revealed a specific interaction between NRF2 and the promoter region of LAMC1 gene,the first exon of the human GCNT3 gene,and all the four exons of the human MKP1gene.The length of NRF2 binding site(peak)is 151 bp.We hypothesized that the above three genes interact and regulated by NRF2 with their respective binding sites.ObjectiveThis research focused on prediction and validation of LAMC1,GCNT3 and MKP1 gene NRF2 binding sites,which lay the foundation for follow-up study of the interaction between NRF2 and these genes.Method1.Identification and prediction of above three genes NRF2 ARE's in their TFBS(151bp sequence)and also in promoter region(within-2kb)of mice through TRAP.2.Construction of the vector for above three gene using molecular biology techniques.3.Detection of luciferase activity of above three genesResult1.Two putative NRF2 ARE identified in the 151 bp binding site of human LAMC1 gene where as mouse Lamcl promoter region contains two putative ARE sequences with in-2kb upstream(at-1070 and-1101)distance to transcription starting site(TSS).One putative NRF2 ARE identified in the 151 bp binding site of human GCNT3 gene.We didn't find any ARE in four exons of human MKP1 gene,but we found two putative AREs on the promoter region of mouse Mkpl within-2kb distance to TSS,which are located at-1136 and-1720 bp respectively.2.The 151 bp of human NRF2 binding site of LAMC1 promoter region along with 1180 bp fragment mouse promoter region of Lamcl were cloned and ARE deletion luciferase expression vectors were successfully constructed.Similarly,the 151 bp fragment of human GCNT3 gene were cloned and ARE deletion luciferase expression vectors were successfully constructed.1880bp fragment of mouse Mkpl gene were also cloned and ARE deletion luciferase expression vector were successfully constructed.3.151 bp and 1180 bp of human and mouse LAMC1 ARE sites have shown significant induction activity.Similarly 151 bp ARE of human GCNT3 has also shown significant induction activity.Whereas one of ARE present in mouse Mkpl promoter region at 1880bp has shown significant induction activity.ConclusionAltogether,both of ARE in human and mouse LAMC1 gene,one of the two ARE's in mouse Mkpl gene,at 1880bp,they ARE present within GCNT3 exonl which were identified by ChIP-Seq and insilico analysis confirmed as functional AREs.