Analysis Of Related Elements In The5’ Upstream Region Of Rat GCLC Gene

Oxidative stress is closely related to the development of a variety of majordiseases, serious threat to human health. GSH is one of the most importantantioxidant and free radical scavenger, plays a vital role in maintaining the normalredox state. GCL is the rate limiting enzyme in GSH synthesis, including catalyticsubunit (GCLC) and regulatory subunit (GCLM), where GCLC is the catalyticactivity of GCL of all, is the primary rate limiting step in the synthesis of GSH.Previous studies in5’ upstream region of rats GCLC gene1759bp found twoadjacent E-box elements (-804~-779,-729~-724), have inhibitory effect on GCLCtranscription. The physiological activity of the two E-box component was found dueto the presence of other E-box components will affect the result of the experiment.So we build5’ upstream region of rats GCLC gene876bp of luciferase reportcarriers, in this fragment only have three classical E-box elements, E-box1(-590~-585) is not discussed before. So we construct a serious of GCLC upstream (1759bp,876bp) luciferase reporter vectors, delete different E-box1(-590~-585), E-box2(-729~-724), E-box3(-804~-779), to observe the possible role of E-box elementsand physiological function. At the same time, we find the phenomenon of GCLCprotein express in the nucleus, so we explore mechanisms of how its import nucleusin many aspects. Results show that the upstream of GCLC gene, TATA-box (-306~-301) is reported of the classic GCLC promoter, there may be other initiators toregulate the GCLC gene, and then GCLC protein will express in the nucleus.ObjectiveTo analysis the role of the three E-box elements on GCLC gene5’ upstreamregion about876bp on GCLC gene expression regulation and study about thepossible physiological function of E-box element. And explore the TATA-box (-306 ~-301) in the rat GCLC gene5’ upstream region, analysis the existence oftranscription initiation of GCLC gene promoter elements of non TATA-box, discussthe mechanism of GCLC protein place into the nucleus.Methods1. Construction of luciferase reporter vectors.(1) Construct the luciferase reporter vectors, one is containing upstream regulatory sequences5892bp of rat GCLC gene, GCLC5892-luc and another is GCLC4347-luc which is missing a period of about1500bp, rich in E-box elements.(2) Construction of deletion of E-box1(-590~-585) luciferase reporter vectorof rat GCLC gene upstream regulatory sequence in different length (1759bp,876bp), study the function of E-box1in transcription regulation.(3) Establishment of luciferase reporter vectors, containing different deletions E-box element in rat GCLC gene1759bp and876bp upstream regulator sequences: used GCLC1759-luc, GCLC1759-del-E-box2-luc, GCLC1759-del-E-box3-luc,GCLC1759-del-E-box2/3-luc as template, designed some primers, constructed GCLC1759-del-E-box1-luc, GCLC1759-del-E-box1/2-luc, GCLC1759-del-E-box1/3-luc,GCLC1759-del-E-box1/2/3-luc (GCLC1759-luc series); GCLC876-luc, GCLC876-del-E-box1-luc, GCLC876-del-E-box2-luc, GCLC876-del-E-box3-luc, GCLC876-del-E-box1/2-luc, GCLC876-del-E-box1/3-luc, GCLC876-del-E-box2/3-luc, GCLC876-del-E-box1/2/3-luc (GCLC876-luc series).(4) Construction of mutant TATA (-306~-301) luciferase reporter vectors, GCLC1759-mu-TATA-luc and GCLC876-mu-TATA-luc.(5) Construction of a suspected mutant promoter INR1(-107~-101), INR2(-616~-610), INR3(-862~-856) luciferase reporter vectors, GCLC876-mu-INR1-luc, GCLC876-mu-INR2-luc, GCLC876-mu-TATA-mu-INR1-luc, GCLC876-mu-TATA-mu-INR2-luc, GCLC1759-mu-INR3-luc, GCLC1759-mu-TATA-mu-INR3-luc.2. The reporter vectors and PRL-SV40are co-transfected L2cells, luciferase activityis detected after24hours, by comparing wild type and mutant transcriptional activity of reporter vector to determine the effect of the E-box elements, TATA-box and thesuspected INR elements on the transcription activity for GCLC gene.3. Detection of different transcription products may exist by PCR method.4. Western Blot is used to detect the protein expression of GCLC gene place into thenucleus.5. Electrophoretic mobility shift assays (EMSA) is performed to confirm if the TATAand INR elements can specific combined with nucleus protein.Results1. All of the recombination reporter vectors are successfully constructed aftersequencing and blasting.2. Firefly luciferase activity assay results:(1) GCLC5892-luc, GCLC4347-luc transfected L2cells, found that deletion of afragment of approximately1.5kb the GCLC gene transcription, rich in E-boxelements, can make the luciferase activity significantly increased (P<0.05).(2) The delete E-box elements luciferase reporter vectors respectively transfected L2cells, in different length of rat upstream regulatory sequences (1759bp,876bp), found that missing either a single, dual or three E-box elementsvectors compared with wild-type, the luciferase activity are all significantly in-creased (P<0.05), these described that three E-box elements are all inhibit thetranscription of GCLC gene.(3) GCLC1759-mu-TATA-luc, GCLC876-mu-TATA-luc vector is compared withthe wild-type, luciferase activity decreased significantly (P<0.05), TATA-boxmutation lead GCLC gene expression fall; and compared to the control vectorpGL3-enhancer found that after mutation TATA-box the activity also persists,which means that there may be another start site guide a different expressionresult.(4) Analyze the suspected INRs, GCLC876-mu-INR1-luc, GCLC876-mu-INR2-luc, GCLC876-mu-TATA-mu-INR1-luc, GCLC876-mu-TATA-mu-INR2-luc, GCLC1759-mu-INR3-luc, GCLC1759-mu-TATA-mu-INR3-luc vectors are compared with wild-type, expression of luciferase activity varied. Then select the elementswhich showed luciferase activity decline to carry further experiments.3. DNA expression analysis presumed the possible INRs location and spliceversions.4. Western Blot detected that the protein expression of GCLC gene place inthe nucleus in many sizes and shapes.5. EMSA showed that the TATA-box can specific combined with nucleus protein, while the suspected INR elements cannot.Conclusion1. Many E-boxs in rat GCLC gene’s promoter sequence play a negative regulationrole in GCLC gene expression and we built the blank group successfully, which isGCLC876-del-E-box1/2/3-luc.2. There exist other rat GCLC gene promoters, not the TATA-box (-306~-301),may lead to different splicing version, which lead to the expression of GCLC geneprotein place into the nucleus.