《人类XBP1基因5′上游DNA序列的转录激活功能分析.pdf》由会员分享,可在线阅读,更多相关《人类XBP1基因5′上游DNA序列的转录激活功能分析.pdf(6页珍藏版)》请在三一文库上搜索。
1、 ORIGINAL ARTICLE An analysis on transcriptional regulation activity of humanXBP1gene 5 upstream DNA sequences GUO Feng-jin1, CHENG Hai-en2, YI Fa-ping2, PENG Hui-ming1, SONG Fang-zhou2 1 (Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing,400016 P . R . China . Email:
2、guofengjin919sohu . com) ; 2 (Department of Molecular Biology,Chongqing Medical University,Chongqing,400016 P . R . China) Corresponding author:SONG Fang-zhou,Email: xwx999263. net 【Abstract】 ObjectiveTo analyze the transcription activation and possible regulation mechanism of human X-box binding pr
3、otein 1(XBP1)gene 5upstream DNA sequence in different cell lines. MethodsSix kinds ofXBP1promot- er deletion mutants were cloned into pGEM-Teasy vector,which includedXBP1gene 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. Eve
4、ry deletion mutant sequence was cut from Teasy-XBP1p by Kpnand Xho,and subcloned into pCAT3-Basic to produce a set of con- structs termed as p1-XBP1p,p2-XBP1p,p3-XBP1p,p4-XBP1p,p5-XBP1p,p6-XBP1p,respectively. The transcrip- tion activity of each construct was detected after transiently transfecting
5、K562,HepG2, NIH-3T3 and L02cell 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 theXBP1 gene promoter,was detect
6、ed by ELISA after 48 hours of transfection. ResultsThe reporter vectors of six kinds ofXBP1 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
7、 and HepG2. And the ac- tivity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3. Conclusion TheXBP1gene promoter can transactivate its downstream gene to transcription. The core sequence ofXBP1promoter was implied between - 227 bp and 66 bp. This sequ
8、ence was connected with the transcriptional activity ofXBP1promot- er closely. Its transcription activity varies with different cell lines.XBP1promoter might drive gene expression with cell- type specificity. 【Key words】 X-box binding protein 1; promoter; chloramphenicol acetyltransferase reporter g
9、ene; tran- scriptional regulation INTRODUCTION Human X-box binding protein 1(XBP1)is ubiquitously expressed in adult tissues,and disruption of theXBP1gene revealed that it is essential for hepatocyte differentiation, car- diomyocyte survival,and plasma cell differentiation. XBP1 is the only transcri
10、ption factor identified to date,which is required for B-cell differentiation into plasma cells. XBP1 is highly expressed in hepatocellular carcinomas.Besides, hepatitis C virus may suppress the IRE1-XBP1 pathway to stimulate the synthesis of its viral proteins. XBP1 partici- pates in the procession
11、of unfolded or misfolded proteins in ER as the strong transcription suppressor during virus in- break 1-3.Therefore,XBP1 is connected with many diseases closely as an important transcription factor of CREB/ ATF family. XBP1 is a basic region-leucine zipper(bZIP)pro- tein and can bind to cAMP respons
12、e elements found in a large number of cellular promoters. TheXBP1sequence suggests that XBP1 is a sequence-specific DNA-binding pro- tein. TheXBP1gene 5 upstream DNA sequence includes typical TATA box,CAAT box and much higher GC abun- dance 4, 5,and also there are many more conservative tran- script
13、ion factors binding sites,such as sp1, CREB, NF-kB, AP2,CTF and some binding sites for stress regulated tran- scription factors,in this sequence region. This structure de- termines the function ofXBP1.In this report,we designed six kinds ofXBP1promoter deletion to study the characteri- zation ofXBP1
14、promoter on chloramphenicol acetyltrans- ferase reporter gene(CAT)transient express system. We thought that the sequence ofXBP1promoter from - 227 bp to 66 bp isXBP1core sequence including TATA box, CAAT box and SP1 structure. MATERIALS AND METHODS Materials K562, HepG2, NIH-3T3 and L02cell lines ca
15、me from the Cell Center of China Medical Science. DNA marker and all restriction enzymes were purchased from TaKaRa Shuzo Co. Ltd. The Wizard purefection plasmid DNA purification sys- tem,T-A clone kit and pCAT3-Basic, pCAT3-Promoter re- porter vector were got from Promega. Fugene 6 Transfection Rea
16、gent and CAT-ELISA kit were purchased from Roche. Primers were synthesized by Sangon Co. . The quick Gel Ex- traction Kit was got from BODA. And other reagents were purchased from Sigma.DNA sequence was finished by Shanghai Jikang Company. Reporter vector construction TheXBP1promoter sequence was de
17、termined based on 1中华医学遗传学杂志 2006 年 2 月第 23 卷第 1 期Chin J Med Genet,February 2006,Vol. 23,No. 1 the sequence of XBP1 and the promoter prediction server (WWW Promoter Scan,http: / / bimas. dcrt. nih. gov/ mol- bio /proscan/ or Promoter 2. 0 Prediction Server,http: / / www. fruitfly. org/ seq-tools/ pr
18、omoter. html) .TheXBP1 promoter which included sequence from - 1039 bp to 66 bp was obtained by PCR amplification of total DNA isolated from the L02cell line. Its primer was designed as follows, sense primer:5 GGTACC AATTAGCCGGGTGTGGTGC 3, antisense primer:5 CTCGAG CCCCGACAGAAGCAGAAC 3. Restriction
19、sites were Kpnand Xho(italic and lineation) . For that purpose, 1g of L02DNA was amplified in a buffer containing 2. 4 mmol/ L MgCl2, 0. 2 mmol/ L dNTP mixture,primer mix (0. 4 mol/ L each)for 35 cy- cles,pre-denaturation at 94 for 4 min,denaturation at 95 for 1 min,annealing at 63 for 1 min,and ext
20、ension at 72 for 1 min(Perkin-Elmer9600 PCR apparatus) . The 1105 bp PCR product was directly cloned in the pGEM- Teasy vector(T-A clone kit,Promega) . After sequencing, theXBP1promoter was cut down from the pGEM-Teasy vec- tor by Kpnand Xhodigestion,and the fragment was subcloned in same site locat
21、ed in upstream sequence of the chloramphenicol acetyltransferase gene (CAT)sites of the pCAT3-Basic plasmid (Promega)to obtain the pCAT3-XBP1 plasmid. Deletion mutants of theXBP1promoter fragment were generated by PCR using the pCAT3-XBP1 plasmid under the conditions described above. All PCR product
22、s were cloned in the pGEM-Teasy vector,and after sequencing,they were subcloned in the Kpnand Xhosites of the pCAT3-Ba- sic plasmid. A schematic representation of all these plasmids is shown in Fig.1. Primers used in the amplification of the XBP1promoter and the deletion mutants are shown in Table 1
23、. Six reporter vectors were named as p1-XBP1p, p2- XBP1p,p3-XBP1p,p4-XBP1p,p5-XBP1p,p6-XBP1p re- spectively. Sequence was determined by Shanghai Jikang Company. The plasmids were purified by Wizard purefection plasmid DNA purification system to transfect cell. Table 1Sequences of the primers used in
24、 the amplification of the XBP1promoter and the deletion mutants of theXBP1pro- moter NameSequence (5- 3) Position (bp) 1 SenseGGTACC AATTAGCCGGGTGTGGTGC- 1039 to 66 AntisenseCTCGAG CCCCGACAGAAGCAGAAC 2 SenseGGUACC ATAGAGGCCGAAGCGGG- 859 to 66 AntisenseCTCGAG CCCCGACAGAAGCAGAAC 3 SenseGGTACC AGGGCACG
25、ATCTCATTTC- 623 to 66 AntisenseCTCGAG CCCCGACAGAAGCAGAAC 4 SenseGGTACC TCGTCAGTCTGGAAAGC- 351 to 66 AntisenseCTCGAG CCCCGACAGAAGCAG 5 SenseGGTACC ATGACCCCAAGTATACCTTGG- 227 to 66 AntisenseCTCGAG CCCCGACAGAAGCAGAAC 6 SenseGGTACC ATGACCCCAAGTATACCTTGG- 227 to - 45 AntisenseCTCGAG ATAGCTCCAGACTACGCAC C
26、ell transient-transfection and determination of CAT K562, HepG2, NIH-3T3 and L02cells were seeded at 1 106/well in a 6-well plate (Nunc, USA)in 2 mL DMEM, respectively. DNA transfection was performed with Dosper liposomal transfecting reagent according to manufacturer s instruction. Briefly,the day
27、before transfection,every cell was incubated until 60%-80% confluence. Then the medi- um was replaced with fresh culture medium without FBS 5-8 hours during adding transfection reagent. Six different re- porter vectors, p1-XBP1p, p2-XBP1p, p3-XBP1p, p4- XBP1p, p5-XBP1p,p6-XBP1p, transfected into K56
28、2, HepG2, L02and NIH-3T3 respectively. And the transfection dose of every vector was from 1g to 4g. pCAT3-Basic was the negative control,and pCAT3-promoter was the posi- tive control. Two days after transfection,the cell was ready for reporter gene (CAT) expression measurement. The transiently trans
29、fected cells were harvested at the 48th hour after the transfection. Cell was washed with precooled phosphate buffered solution (PBS, 0. 1mol/ L, pH7. 4) and lysed with lysis buffer (Roche, USA) . Aliquots of cell ex- tracts were made for protein determination as described pre- viously. CAT was dete
30、cted by ELISA (Roche, USA) with en- zyme activity analysis method under 415 nm wavelength. Fig. 1Schematic representation ofXBP1promoter constructs used in this study. The name of the primers used in the amplification of theXBP1promoter and the dele- tion mutants are shown above the arrows. The impo
31、rtant functional binding sites are indicated in each construct 2中华医学遗传学杂志 2006 年 2 月第 23 卷第 1 期Chin J Med Genet,February 2006,Vol. 23,No. 1 Fig.2Biological information analysis of humanXBP1gene RESULTS Biological information analysis of humanXBP1gene The transcription start site (TSS)of humanXBP1gen
32、e lies in the upstream 174 bp of the start codon ATG. There are four characteristics inXBP1gene promoter sequence. At first,there are many more conservative transcription factors binding sites,such as sp1, CREB, NF-kB, AP2 and CTF etc. Secondly,the abundance of GC is much higher in XBP1promoter. Thi
33、rdly,there are typical TATA box and CAAT box in this promoter sequence. The last is that this sequence includes a cluster of binding sites for stress regu- lated transcription factors. Fig.2 shows the structure of hu- manXBP1gene 5 upstream DNA sequence. Identification of reporter vectors XBP1promot
34、er and its five deletions DNA were ampli- fied by PCR and the electrophoresis result showed six differ- ent fragments in 0. 9% agarose gel (Fig. 3) . Every product was cloned pGEM-Teasy vector, the cut products were pGEM-Teasy vector (3015 bp) ,XBP1promoter (1105 bp) and its five deletions (925 bp,
35、701 bp, 417 bp, 305 bp, 172 bp) . Each deletion was subcloned in pCAT3-Basic vector, the cut result was showed in Fig. 4 (pCAT3-Basic vector 4027 bp and 1105 bpXBP1p,925 bp, 701 bp, 417 bp, 305 bp and 172 bpXBP1pdeletions DNA fragments) . The six pCAT3-XBP1 promoter reporter vectors were named as p1
36、- XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p and p6-XBP1p respectively. DNA sequence was determined by Shanghai Jikang Company,whose result was the same as GenBank . Fig . 5 showed the partial sequence ofXBP 1 Fig.3The fragment ofXBP1promoter and its deletions amplified by PCR with L02DNA templat
37、e.Lane 1: DNA marker;lane 2: PCR products ofXBP1pro- moter (1105 bp) ;lane 3: PCR products ofXBP1promoter deletion 1 (925 bp) ; lane 4: PCR products ofXBP1promoter deletion 2 (701 bp) ;lane 5: PCR prod- ucts ofXBP1promoter deletion 3 (417 bp) ;lane 6: PCR products ofXBP1pro- moter deletion 4 (305 bp
38、) ;lane 7: PCR products ofXBP1promoter deletion 5 (172 bp) Fig.4pCAT3-Basic-XBP1promoter cut by Kpn/ Xho.Lane 1: DNA marker;lane 2: recombinant plasmid p1(1105 bp) ;lane 3: recombinant plasmid p2 (925 bp) ;lane 4: recombinant plasmid p3 (701 bp) ;lane 5: recombinant plasmid p4 (417 bp) ;lane 6: reco
39、mbinant plasmid p5(305 bp) ;lane 7: recom- binant plasmid p6 (172 bp) promoter by Shanghai Jikang Company. Fig. 6 showed the structure of pCAT3-Basic-XBP1p. 3中华医学遗传学杂志 2006 年 2 月第 23 卷第 1 期Chin J Med Genet,February 2006,Vol. 23,No. 1 Fig.5The partial sequencing of theXBP1promoter Fig.6Map of plasmid
40、 pCAT3-Basic-XBP1promoter Transcription activations ofXBP1promoter and its five deletions in different cell lines Six different reporter vectors p1-XBP1p,p2-XBP1p, p3- XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p were transfected in liver cell L02,K562, HepG2 and NIH-3T3 respectively. The absorbencies were d
41、etected by CAT ELISA analysis method. The negative control was pCAT3-Basic,The posi- tive control was pCAT3-promoter. As shown in Table 2,the activities of p4-XBP1p and p5-XBP1p were higher than those of the other deletion mutants. And the activity of p5- XBP1p was the highest in HepG2,which was 27.
42、4 multiple of pCAT3-Basic. The transcription activation ofXBP1pro- moter and its five deletions in K562 and HepG2 cells were higher than that of normal liver cell L02on average. Be- sides,there is no activity detected in any transfected NIH- 3T3. All results were shown in Table 2. Fig.7 showed the e
43、ffects of deletion mutations on the transcription activity of theXBP1promoter in different cell lines. DISCUSSION Human X-box binding protein 1(XBP1)has many bio- logical functions. Previous reports have shown that XBP1 is essential for liver growth. Mice lacking XBP1 displayed hy- poplastic fetal l
44、ivers,andFP were identified as the specif- ic target genes of XBP1 in the liver 6, 7. As an important transcription factor,XBP1 participates in the unfolded pro- tein response(UPR) . The UPR is essential for survival of all eukaryotic cells under conditions of ER stress and is also essential for dif
45、ferentiation and/ or survival of eukaryotic cells that secrete high levels of proteins. The UPR is also implicated in the pathogenesis of a number of diseases. And during UPR,XBP1is a member of the ER stress-response genes. XBP1 can produce lecithin in cell, which is the im- portant material of endo
46、plasmic reticulum (ER) . XBP1 can rebuild the ER and improve the ability of refolding and transporting protein in cell . Anyway ,XBP 1 is directly Table 2Transient-transfection assays ofXBP1promoter and its deletions in different cell lines Cell linespCAT3-BasicpCAT3-promoterp1-XBP1pp2-XBP1pp3-XBP1p
47、p4-XBP1pp5-XBP1pp6-XBP1p K5620.0371.0790.7710.3010.7321.1301.3280.068 HepG20.0531.2160.6950.2780.8011.3441.4500.071 L020.0290.9130.5480.2260.6740.6210.7320.060 NIH-3T30.0410.9720.0910.0780.0790.1100.0930.079 Fig.7Effects of deletion mutations on the transcription activities of theXBP1promoter in dif
48、ferent cell linesa: Schematic presentation of theXBP1promoter region and its five kinds truncated forms. Putative cis-acting regulatory motifs in the reporter constructs are denoted,and the numbers identify the position relative to the tran- scription initiation site;b: chloramphenicol acetyltransfe
49、rase (CAT)activities in cells transfected with each truncated reporter construct. The transfected cells were deter- mined by CAT after transfection between 24 h and 48 h 4中华医学遗传学杂志 2006 年 2 月第 23 卷第 1 期Chin J Med Genet,February 2006,Vol. 23,No. 1 involved in many metabolism procession 8-10. To study the transcriptional regulation activity ofXBP1 gene promoter sequence,we designed six kinds ofXBP1 promoter deletion mutants. And we constructed six kinds of CAT-reporter gene vectors for this goal. After transfecting six different reporter vect
链接地址:https://www.31doc.com/p-3698968.html