Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2011) 35, 967–971 (Printed in Great Britain)
Identification of the VIL2 enhancer in human embryonic kidney cells
Shu‑Ying Gao, Yan‑Peng Dai, Xia Long, Fang‑Ting Zhang and Jie Yu1
Central Laboratory, Peking University Shenzhen Hospital, Shenzhen PKUHKUST Medical Center, Shenzhen 518036, Peoples Republic of China


We previously demonstrated that the VIL2 −87/+134 region exhibited promoter activity in some human cells, and a region further upstream of this promoter might contain an enhancer. However, the properties and location of this VIL2 enhancer remain unclear. In this study, we cloned the VIL2 −1541/−706 segment and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected HEK-293 cells (human embryonic kidney cells). The VIL2 −1541/−706 was found to exhibit promoter activity. Furthermore, when this segment was located upstream of the VIL2 or SV40 (simian virus 40) promoters in the forward orientation, the expression levels of luciferase were dramatically enhanced. However, this transcriptional enhancement disappeared when this segment was located upstream of the promoter in the reverse orientation or downstream of the reporter gene in the forward or reverse orientation. In deletion experiments, we found several potential regulatory regions within the VIL2 −1541/−706. When these regions were separately located upstream of the VIL2 or SV40 promoters, only the −1297/−1186 considerably enhanced the activity of these promoters. Although the other regulatory regions exhibited significant transcriptional regulation in deletion experiments, they weakly enhanced VIL2 promoter activity and/or did not regulate SV40 promoter activity. These results suggest that the DNA sequence upstream of the VIL2 promoter functions as an enhancer in a position- and orientation-dependent manner, and the VIL2 −1297/−1186, which acts as a key enhancer, probably regulates VIL2 transcription in combination with other potential regulatory regions located upstream of the VIL2 promoter.


Key words: enhancer, ezrin, promoter, transcriptional regulation, VIL2

Abbreviations: EC109 cells, oesophageal carcinoma 109 cells, HEK-293 cells, human embryonic kidney cells, SV40, simian virus 40

1To whom correspondence should be addressed (email yujie007@hotmail.com).


1. Introduction

Ezrin, encoded by VIL2, is a membrane–cytoskeleton linker protein involved in a wide variety of cellular processes such as adhesion (Hiscox et al., 1999), survival (Gautreau et al., 1999), motility (Crepaldi et al., 1997), signal transduction (Hatzoglou et al., 2007) and tumour generation (Kang et al., 2010). Recent results have provided further evidence for the novel roles of ezrin in the control of cyclin A gene transcription and endothelial cell proliferation (Kishore et al., 2005), in bacterial uptake by trophoblast giant cells (Watanabe et al., 2009), as a mediator of c-Myc-induced tumorigenesis in prostate cancer cells (Chuan et al., 2010), and as a potentially new prognostic marker and/or therapeutic target for certain carcinomas (Kim et al., 2009; Kang et al., 2010).

Our recent study on ezrin regulation demonstrated that the co-operativity of Sp1 (specificity protein 1) and AP-1 (activator protein 1; c-Jun–c-Fos heterodimer) regulated VIL2 (human ezrin gene) promoter activity and ezrin expression in EC109 cells (oesophageal carcinoma 109 cells; Gao et al., 2009). We also found that the VIL2 promoter is located within −87/+134, and the region −1324/−890 positively regulated transcription. The DNA sequence upstream of the VIL2 promoter might contain an enhancer, but the properties and location of the VIL2 enhancer remain unclear.

In the present study, we investigated the transcriptional regulatory properties of the DNA sequence upstream of the VIL2 promoter, sublocalized transcriptional regulatory regions of VIL2 within the −1541/−706, and identified the VIL2 enhancer via luciferase assays in transiently transfected HEK-293 cells (human embryonic kidney cells). We found that the DNA sequence upstream of the VIL2 promoter functioned as an enhancer in a position-, orientation- and relative promoter-dependent manner, and VIL2 −1297/−1186, acting as a key enhancer, probably regulated VIL2 transcription in combination with other potential regulatory regions upstream of the VIL2 promoter.

2. Materials and methods

2.1. Materials

pGL3-Basic, pGL3-Promoter, pRL-TK and the Dual-luciferase® reporter assay system were purchased from Promega (Madison, WI, U.S.A.). Lipofectamine™ 2000 transfection reagent was purchased from Invitrogen (Carlsbad, CA, U.S.A.). Endonucleases, T4 DNA polymerase and ligase were purchased from Takara. All other reagents were of analytical grade.

2.2. Plasmids construction

All VIL2 segments (GenBank® Nucleotide Sequence Database accession number AL589931) originated from genomic DNA of EC109 cells and were generated by PCR. Plasmids were constructed using standard methods (Sambrook et al., 2000). The backbone of these plasmids is pGL3 luciferase reporter vector. Schematic representations of some plasmids are shown in Figures 13. In the name of plasmids, the letters ‘P(hE)’ and ‘P(SV40)’ indicate that the plasmids carry the VIL2 −87/+134 and the SV40 (simian virus 40) promoter respectively and the letters ‘U’, ‘D’ and ‘R’ are abbreviations for ‘Upstream’, ‘Downstream’ and ‘Reverse’, indicating that the VIL2 segments are located upstream of the promoter, downstream of the reporter gene and in reverse orientation relative to the genomic orientation respectively.

2.3. Cell culture and transfection

HEK-293 cells were maintained in DMEM (Dulbecco's modified Eagle's medium; Invitrogen) supplemented with 10% (v/v) foetal bovine serum (Invitrogen) at 37°C in a 5% CO2 environment. For transfection, cells were seeded on 96-well plates at 1.5×105 cells/ml, grown to 50–80% confluency and transfected with pGL3 luciferase reporter plasmid and pRL-TK using Lipofectamine™ 2000 reagent according to the manufacturer's protocol. After transfection, cells were incubated for another 24–48 h before being harvested for the luciferase assay. Transfections were performed in at least two independent experiments and in triplicate for each experiment.

2.4. Luciferase assay

Transfected cells were harvested in passive lysis buffer (Promega), and the cell lysates were analysed for luciferase activity with the dual-luciferase reporter assay system according to the manufacturer's recommendations. Luciferase activity was normalized to Renilla luciferase activity.

2.5. Statistical analysis

Data analysis was conducted using SPSS 13.0 (SPSS, Chicago, IL, U.S.A.). A two-tailed independent-sample Student's t test was used to determine the significance of the differences between groups. Differences were considered statistically significant at P<0.05. Data are plotted as means±S.D.

3. Results

3.1. The DNA sequence upstream of the VIL2 promoter exhibits enhancer and promoter activities

To investigate the transcriptional regulatory properties of the DNA sequence upstream of the VIL2 promoter, we cloned the VIL2 −1541/−706 segment, which contained the positive transcriptional regulatory region −1324/−890, and analysed this region via luciferase assays in HEK-293 cells. When the −1541/−706 segment was located upstream of the VIL2 or SV40 promoter, it increased the luciferase activity by 2–3-fold. In addition, the VIL2 −1541/−706 induced luciferase activity similar to those induced by the SV40 promoter and the VIL2 −87/+134 (Figure 1). These results suggested that the VIL2 −1541/−706 could act both as an enhancer to enhance the transcription of genes controlled by the VIL2 or SV40 promoter and as a promoter to activate the transcription of genes without another promoter.

3.2. Transcriptional enhancement of the VIL2 −1541/−706 characterized a position and orientation dependence

To explore whether the transcriptional enhancement of the VIL2 −1541/−706 on VIL2 promoter depends on position and orientation, a series of pGL3 luciferase reporter vectors carrying the −1541/−706 and VIL2 promoter were constructed and analysed. Transient transfection of HEK-293 cells showed that when the −1541/−706 was located upstream of the VIL2 promoter in the forward orientation, it enhanced the transcription of luciferase significantly; when this segment was located upstream of the VIL2 promoter in the reverse orientation or downstream of the reporter gene in the forward or reverse orientation, it did not exhibit transcriptional regulation (Figure 2A). These results suggested that the −1541/−706 could enhance the expression of a reporter gene controlled by the VIL2 promoter in HEK-293 cells in a position- and orientation-dependent manner.

To further investigate whether the transcriptional regulatory characteristic of the VIL2 −1541/−706 on the SV40 promoter is similar to that of on the VIL2 promoter, a series of pGL3 luciferase reporter vectors carrying the VIL2 −1541/−706 and SV40 promoter were constructed and analysed. The results showed that the VIL2 −1541/−706 could also enhance the expression of a reporter gene controlled by the SV40 promoter in HEK-293 cells in a position- and orientation-dependent manner (Figure 2B).

3.3. Sublocalization of potential transcriptional regulatory regions within the VIL2 −1541/−706

To sublocalize potential transcriptional regulatory regions within the VIL2 −1541/−706 and avoid the influence of the activity of other promoters, a series of 5′-deletion mutants without the VIL2 or SV40 promoter was constructed from pGL3-hE(−1541/−706) and analysed using luciferase assays. In HEK-293 cells, when compared with the −1541/−706, further deletions, i.e. −1445/−706 and −1297/−706, did not markedly change the reporter activity (Figure 3A). Sequence 5′-deletion from −1297 to −1025 nearly abolished the activity, that from −1025 to −946 increased the activity slightly and that from −946 to −768 reduced the activity slightly. Obviously, the −1297/−1025 was a strongly positive transcriptional regulatory region, whereas the −1025/−946 and −946/−768 were weakly negative and positive transcriptional regulatory regions in HEK-293 cells respectively.

To further confirm the results of the 5′-deletion experiments, a series of 3′-deletion mutants was constructed and analysed. The results showed that several potential transcriptional regulatory regions existed within the VIL2 −1541/−706 (Figure 3B). The −1293/−1186 and −1186/−1102 positively regulated transcription, and the −1029/−921 negatively regulated transcription of human VIL2 in HEK-293 cells, which was consistent with the results of the 5′-deletion experiments.

3.4. Enhancement of VIL2 potential transcriptional regulatory regions on the VIL2 and SV40 promoters

Considering enzyme sites within pGL3-P(hE)-hE(−1541/−706)U and the results of the deletion experiments, plasmids, carrying VIL2 potential transcriptional regulatory region −1297/−1186, −1186/−1102, −1097/−1029, −1025/−921 or −919/−773 located upstream of the VIL2 promoter in pGL3-hE(−87/+134), were constructed and analysed. Among these detected VIL2 potential transcriptional regulatory regions, the −1097/−1029 did not affect luciferase activity, whereas other regions increased the activity. The VIL2 −1297/−1186, enhancing transcription more strongly than other detected regions, appeared to be a key enhancer. In contrast, the negative regulatory region −1025/−921 in deletion experiments exhibited transcriptional enhancement of the VIL2 promoter (Figure 4A). The functions elucidated from separate region detection were not absolutely in agreement with those elucidated from deletion experiments, suggesting that the transcriptional regulation of one element or region was probably affected by its adjacent or distant DNA sequence.

To further investigate whether these VIL2 potential transcriptional regulatory regions regulating the expression of the reporter gene controlled by the SV40 promoter are similar to those regulated by the VIL2 promoter, a series of reporter gene expression vectors carrying the SV40 promoter and VIL2 potential transcriptional regulatory region was constructed and analysed. The results showed that the VIL2 −1297/−1186 increased the expression of the luciferase gene controlled by the SV40 promoter, whereas other detected VIL2 regions did not alter the reporter activity (Figure 4B). These results testified again that the VIL2 −1297/−1186 acted as a key enhancer in transcriptional regulation, and other functional regions might have secondary roles and participate in regulation in a co-operative or synergistic manner.

4. Discussion and conclusions

The human VIL2 sequence contained a promoter within −87/+134 and a positive regulatory region within −1324/−890 (Gao et al., 2009). The VIL2 −1324/−890 was difficult to be accurately amplified via PCR for its GC content reached 80%. Primer Premier 5.0 program revealed that the VIL2 −1541/−706 was the shortest obtainable segment containing −1324/−890. Here, we cloned the VIL2 −1541/−706 segment, investigated the transcriptional regulatory properties of the DNA sequence upstream of the VIL2 promoter and identified the VIL2 enhancer in HEK-293 cells.

Analysis of the VIL2 −1541/−706 via luciferase assays in HEK-293 cells suggested that the −1541/−706 had not only promoter activity but also enhancer activity. Enhancers are DNA sequences that serve as binding sites for regulatory proteins and stimulate transcriptional activity depending on the promoter. The properties of enhancers have been widely known. Indeed, most enhancers can activate transcription in a position- and orientation-independent manner, including the enhancers of the human M creatine kinase (Trask et al., 1992) and dystrophin genes (Klamut et al., 1996). Some enhancers activate transcription in a position- and orientation-dependent manner, such as intron-1 enhancer of the human purine nucleoside phosphorylase-encoding gene (Jonsson et al., 1994). Our present results showed that the VIL2 −1541/−706 enhanced transcription in a position- and orientation-dependent manner; it played an enhancer role only when it was located upstream of the promoter in the forward orientation.

From 5′- and 3′-deletion experiments, we sublocalized and identified some potential transcriptional regulatory regions within the VIL2 −1541/−706. Further investigation revealed that among these potential regulatory regions, only the −1297/−1186 exhibited considerable enhancer activity on both the VIL2 promoter and the SV40 promoter. Other regions, including −1186/−1102, −1025/−921 and −919/−773, although exhibiting significantly positive or negative transcriptional regulation in deletion experiments, showed weaker enhancement of VIL2 promoter activity or lack of regulation of the SV40 promoter. It has been reported that the SV40 enhancer was composed of at least two DNA domains that exhibited very little enhancing activity individually, whereas their association resulted in a 400-fold enhancement of transcription (Zenke et al., 1986). Another work on the SV40 enhancer indicated that the enhancer was composed of multiple functional elements that could compensate for one another (Herr et al., 1986). Therefore we speculated that the VIL2 enhancer might be similar to the SV40 enhancer in requiring several domains or elements participating together in transcriptional enhancement.

In conclusion, our results suggest that the DNA sequence upstream of the VIL2 promoter functions as an enhancer in a position- and orientation-dependent manner; further, the VIL2 −1297/−1186 acts as a key enhancer and probably regulates VIL2 transcription in combination with other potential regulatory regions upstream of the VIL2 promoter.

Author contribution

Shu-Ying Gao designed the research, performed the experiments, analysed the data and wrote the manuscript. Yan-Peng Dai, Xia Long and Fang-Ting Zhang assisted in conducting the experiments. Jie Yu revised the manuscript.

Funding

This work was supported by grants from the China Postdoctoral Science Foundation [grant numbers 20090450100 and 201003361]; the Natural Science Foundation of Guangdong Province [grant numbers 9152800001000017 and 9151030002000008]; and Basic Research Projects of Shenzhen Bureau of Science, Technology and Information [grant numbers JC200903180676A, 200901012 and 200801001].

REFERENCES

Chuan, YC, Iglesias-Gato, D, Fernandez-Perez, L, Cedazo-Minguez, A, Pang, ST and Norstedt, G (2010) Ezrin mediates c-Myc actions in prostate cancer cell invasion. Oncogene 29, 1531-1542
Crossref   Medline   1st Citation  

Crepaldi, T, Gautreau, A, Comoglio, PM, Louvard, D and Arpin, M (1997) Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J Cell Biol 138, 423-434
Crossref   Medline   1st Citation  

Gao, SY, Li, EM, Cui, L, Lu, XF, Meng, LY and Yuan, HM (2009) Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells. J Biol Chem 284, 7995-8004
Crossref   Medline   1st Citation   2nd  

Gautreau, A, Poullet, P, Louvard, D and Arpin, M (1999) Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci USA 96, 7300-7305
Crossref   Medline   1st Citation  

Hatzoglou, A, Ader, I, Splingard, A, Flanders, J, Saade, E and Leroy, I (2007) Gem associates with ezrin and acts via the Rho-GAP protein Gmip to down-regulate the Rho pathway. Mol Biol Cell 18, 1242-1252
Crossref   Medline   1st Citation  

Herr, W and Clarke, J (1986) The SV40 enhancer is composed of multiple functional elements that can compensate for one another. Cell 45, 461-470
Crossref   Medline   1st Citation  

Hiscox, S and Jiang, WG (1999) Ezrin regulates cell–cell and cell–matrix adhesion, a possible role with E-cadherin/beta-catenin. J Cell Sci 112, 3081-3090
Medline   1st Citation  

Jonsson, JJ, Converse, A and McIvor, RS (1994) An enhancer in the first intron of the human purine nucleoside phosphorylase-encoding gene. Gene 140, 187-193
Crossref   Medline   1st Citation  

Kang, YK, Hong, SW, Lee, H and Kim, WH (2010) Prognostic implications of ezrin expression in human hepatocellular carcinoma. Mol Carcinog 49, 798-804
Medline   1st Citation   2nd  

Kim, C, Shin, E, Hong, S, Chon, HJ, Kim, HR and Ahn, JR (2009) Clinical value of ezrin expression in primary osteosarcoma. Cancer Res Treat 41, 138-144
Crossref   Medline   1st Citation  

Kishore, R, Qin, G, Luedemann, C, Bord, E, Hanley, A and Silver, M (2005) The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-alpha-induced transcriptional repression of cyclin A. J Clin Invest 115, 1785-1796
Crossref   Medline   1st Citation  

Klamut, HJ, Bosnoyan-Collins, LO, Worton, RG, Ray, PN and Davis, HL (1996) Identification of a transcriptional enhancer within muscle intron 1 of the human dystrophin gene. Hum Mol Genet 5, 1599-1606
Crossref   Medline   1st Citation  

Sambrook, J and Russell, D (2000) Molecular Cloning: A Laboratory Manual, 3rd edn., Cold Spring Harbor Laboratory Press, Plainview, NY
1st Citation  

Trask, RV, Koster, JC, Ritchie, ME and Billadello, JJ (1992) The human M creatine kinase gene enhancer contains multiple functional interacting domains. Nucleic Acids Res 20, 2313-2320
Crossref   Medline   1st Citation  

Watanabe, K, Tachibana, M, Kim, S and Watarai, M (2009) Participation of ezrin in bacterial uptake by trophoblast giant cells. Reprod Biol Endocrinol 7, 95
Crossref   Medline   1st Citation  

Zenke, M, Grundstrom, T and Matthes, H (1986) Multiple sequence motifs are involved in SV40 enhancer function. EMBO J 5, 387-397
Medline   1st Citation  


Received 26 November 2010/19 April 2011; accepted 18 May 2011

Published as Cell Biology International Immediate Publication 18 May 2011, doi:10.1042/CBI20100854


© The Author(s) Journal compilation © 2011 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)