An analysis on transcriptional regulation activity of human XBP1 gene 5' upstream DNA sequences.
OBJECTIVE
To analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.


METHODS
Six kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.


RESULTS
The reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.


CONCLUSIONS
The XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.
Guo
Feng-jin
FJ
Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, P.R.China.
Cheng
Hai-en
HE
Yi
Fa-ping
FP
Peng
Hui-ming
HM
Song
Fang-zhou
FZ
D016475:3T3 Cells; D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics*; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C000605544:Xbp1 protein, mouse; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase