Study of transcription activity of X-box binding protein 1 gene in human different cell lines.
Human X-box binding protein 1 (XBP1), an important transcription factor, participates in many signal transduction processes. To further investigate the biological function of XBP1, sequences of XBP1 promoter and its two deletion mutants were first determined using bioinformatic analysis. The report vectors containing XBP1 promoter and its deletion mutants were then constructed, namely, p1-XBP1p, p2-XBP1p, and p3-XBP1p. Each reporter vector was separately transfected into HepG(2), L0(2), K562, SMMC-7721, HSF, and Lipocyte Ito Cell line using FuGENE 6 transfection reagents. The activity of chloramphenicol acetyltransferase (CAT) in each group of transfected cells was detected by ELISA assay, which in turn reflects the transcription activity of the XBP1 gene promoter. The activity involving p3-XBP1p was the highest in HepG(2), which was 12.4-fold of that of pCAT3-Basic. The activities of p3-XBP1p in K562 and SMMC-7721 were the second and the third highest, which were 10.9-fold and 10.0-fold of that of the pCAT3-Basic, respectively. The CAT activity in L0(2) was lower than that in the above-mentioned abnormal cell, and no reporter activity was detected in HSF and Ito Cell. The XBP1 transcription and expression in K562, HepG(2) and SMMC-7721 were found to be higher than that in L0(2), HSF and Ito cells, based on the results of real-time RT-PCR and Western blot. The XBP1 transcription and expression in L0(2), HSF was lower, whereas that in Ito cells was totally lacking. The result was similar to that of CAT-ELISA. Therefore, the XBP1 gene promoter can drive its downstream gene expression and its activity is cell line-dependent. The core sequence of XBP1 promoter was found between -227bp and 66bp sequence. This sequence was closely associated with the transcriptional activity of XBP1 promoter.
Guo
Fengjin
F
Department of Cell Biology and Genetics, Institute of Basic Medicine Sciences, Chongqing Medical University, Chongqing 400016, China. guofengjin919@vip.sohu.com
Song
Fangzhou
F
Zhang
Jing
J
Li
Jing
J
Tang
Yong
Y
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; D015500:Chloramphenicol O-Acetyltransferase