The Regulation Of Human CD226 Gene Expression

Objective: To investigate the mechanism of regulation of human CD226 gene expression. To identify the promoter of CD226 gene and determine how cytokines, stimuli and transcription factors to regulate the expression of CD226 gene.Methods: Freshly extracted mRNA from 50ng/ml PMA treated Jurkat cells were reversely transcripted into cDNA, and the transcription start site was identified by 5'-RACE after nest PCR. The sequence of upstream regulation region of human CD226 gene obtained from GenBank was scanned for predicting transcription factors binding sites by tfsitescan program in www.ifti.org, then higher possibility transcrition factors were selected manually for further functional research. A 2kb DNA fragment in the upstream regulation region of CD226 gene was cloned by PCR, by using the genomic DNA from human normal PBMC and the primers designed according to the data from GenBank. Based on the obtained DNA sequence, a series of truncated fragments were constructed and ligated into the pGL3 luciferase reporter vector. These vectors were transfected into Jurkat cells and the luciferase activity was analyzed at 48h after transfection to determine the promoter sites. These reporter vectors containing CD226 promoters were transfected into Jurkat cells, which were treated with 50ng/ml PMA and 0.5 μ g/ml A23187 for 4h and 1h respectively. To investigate how the promoters were regulated by PMA and A23187, the luciferase activity was analyzed 48h after transfection. Putative negative regulation element (NRE) was induced tothe downstream of CMV promoter by PCR, and ligated into pGL3 reporter vector after sequencing. The inhibition effect of NRE on CMV promoter was indicted by the luciferase activity of CMV-NRE pGL3 transfected HHCC cells. These reporter vectors containing CD226 promoters were transfected into HHCC cells and cultured for 24h before the cells were cultured for another 24h in the presence of 500u/ml TNF-α and 100u/ml IL-2. The regulation of TNF- a and IL-2 on CD226 promoters was indicted by the luciferase activity. The reporter vectors of CD226 promoters were co-transfected with c-jun,c-fos,etsl,AP-l or AP-1 plus etsl respectively and the luciferase activities were tested at 48 hours after transfection to investigate the role of these transcription factors on CD226 promoters. Nuclear extraction of HHCC cells was incubated with ^γ-32P labeled CD226 promoters, and meantime a negative control, a positive control, non-specific competition and specific competition groups were set up. Non-denaturing SDS- PAGE was carried out to separate protein, and the dried gel was exposed at -70℃ overnight. Binding of transcription factors with CD226 promoters was pointed out by the protein retardation.Results: The transcription start site was determined at 229bp upstream ofATG in mRNA after sequencing three clones selected randomly.Bioinformatics analysis showed that there were two TATA-box, one GC-boxand some putative transcription factors binding sites such as AP-1,etsl,GATA-l,Spl and AP2 in the upstream regulation region. Among theseven truncated fragments, T1, which containing 2kb regulation region, wasshown to have relative low basic transcription activity, while the other twofragments had higher promoter activity, which located at -811bp^-287bp( T4/P1 ) and +32bp +214bp(T7/P2)respectively. Between the twopromoters there may be a negative regulation element (NRE) as it couldinhibit PI and P2 activities. When the NRE was cloned into the downstreamof CMV promoter the NRE could inhibit CMV promoter markedly. In Jurkatcells PMA could up-regulate PI activity while inhibit P2 activity, whereasA23187 could enhance activity both PI and P2, especially P2. The activitiesof Tl and P2,but not PI, could be upregulated by TNF- α and activities of allT1,P1 and P2 could be enhanced by IL-2 which was quite similar with thepattern of CD226 protein regulation by the two cytokines. In HHCC cellstranscription factors c-jun, c-fos or etsl could not regulate PI and P2 whileAPI could up-regulate both of them, especially PI. Etsl could enhance AP-1induced PI activity and promote AP-1 induced P2. A composite site of AP-1and etsl in PI may be responsible for AP-1 induction of PI and etsl inhibitionof AP-1 induction. AP-1 could not bind with PI in vitro as indicted by EMS A,...