2017 Volume 40 Issue 5 Pages 733-737
Increased expression of β4-galactosyltransferase (β4GalT) 4 has been shown to be associated with metastatic ability and poor prognosis of colon cancer cells. To solve the up-regulation of β4GalT4 in colon cancer cells at transcriptional level, we examined the transcriptional mechanism of the β4GalT4 gene in SW480 human colon cancer cell line. Luciferase assay using the deletion constructs revealed that the promoter activity of the β4GalT4 gene is associated with the region between nucleotides −122 and −55 relative to the transcriptional start site, which contained one Specificity protein 1 (Sp1)-binding site. The mutation into the Sp1-binding site resulted in dramatic decreased promoter activity. Meanwhile, ectopic Sp1 expression stimulated the promoter activity significantly. The present study suggests that the expression of the β4GalT4 gene is controlled by Sp1, and Sp1 plays a key role in the activation of the β4GalT4 gene in colon cancer cells.
It is well documented that the structures of glycans attached to proteins and lipids on cell surface change dramatically upon malignant transformation of cells.1–3) In general, altered glycosylation modulates the function of cell adhesion molecules and receptors,4,5) and the cell membrane properties.6,7) The background of the altered glycosylation has been shown to be attributed to the altered expression of glycosyltransferases at the transcriptional level.
The Galβ1→4GlcNAc/Glc/Xyl groups, in which Galβ1→4 structures are synthesized by seven members in the β4-galactosyltransferase (β4GalT) family, are common and make the glycan backbone structures in glycoconjugates.8) Among the β4GalT family β4GalT4 has been shown to be involved in the biosynthesis of neolacto-series glycolipids,9) and poly-N-acetyllactosamine on O-glycans.10) Moreover, β4GalT4 can transfer galactose from uridine 5′-diphosphate-Gal to the GlcNAc-6-O-sulfate residues, which are included in the keratan sulfate chains and 6-sulfosialyl-Lewis X group.11)
The β4GalT family shows different tissue distribution. The β4GalT4 gene is expressed in various tissues such as placenta, pancreas, kidney, and colon.9,11,12) Increased expression of β4GalT4 has been shown to be associated with metastatic ability and poor prognosis of human colon cancer.13) Thus, β4GalT4 is key enzyme for expressing malignant properties of colon cancer.
So far the transcriptional mechanisms of the human β4GalT1, β4GalT2, and β4GalT5 genes in cancer cells have been reported.14–16) However, the transcriptional mechanism of the human β4GalT4 gene remains to be elucidated. Herein, we defined the core promoter region of the β4GalT4 gene and the transcription factor that regulates the promoter activity of the β4GalT4 gene in human colon cancer cells.
SW480 human colon cancer cell line was cultivated in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 50 units/mL penicillin and 50 µg/mL streptomycin.
Determination of Transcriptional Start SiteTo determine the transcriptional start site, the cDNA was synthesized from SW480 cells with a GeneRacer kit (Invitrogen, Carlsbad, CA, U.S.A.) as described previously.16) Two reverse gene-specific primers, which correspond to the coding region of the β4GalT4 cDNA, were used for the PCR. The first PCR was performed using the cDNA as templates, the gene-specific primer TS27-4 (5′-CTC TTG AAT GGC ACC CAC GAA GTA G-3′; complementary to nucleotides +96/+120 relative to the translational start site), and the GeneRacer 5′-Primer included in the kit. The nested PCR was performed using the first PCR products as templates, the gene-specific primer TS27-3 (5′-CCC AAC CAC TGT CAG GCA CAA AGT C-3′; complementary to nucleotides +57/+81 relative to the translational start site), and the GeneRacer 5′-Nested Primer included in the kit. The nucleotide sequence of the nested PCR products was determined.
Isolation of 5′-Region of Human β4GalT4 GeneThe 5′-flanking region of the transcriptional start site of the human β4GalT4 gene was amplified by PCR using the human whole blood genomic DNA as a template according the method as described previously.17) The following 5′- and 3′-primer pairs were synthesized based on the nucleotide sequence of the human chromosome 3q BAC RP11-484M3 (accession no. AC083800) and used: TS27-50 (5′-CAG AGA CAC TCC TCA GTT CCT AGA C-3′) and TS27-42 (5′-CGG AGC CAG CGT ACT CAC CCC GGA G-3′) for the amplification of the region −1767 to +47 relative to the transcriptional start site, which was mostly used in mRNA and is tentatively designed as +1.
Reporter Plasmid ConstructionTo identify the core promoter region of the β4GalT4 gene, the deletion constructs of the 5′-regions were made using the firefly luciferase reporter vector, pGL3-Basic (Promega, Madison, WI, U.S.A.), with the numbers indicating the nucleotide positions relative to the transcriptional start site. After the KpnI–BglII fragment was amplified by PCR with the 5′- and 3′-primer pairs, in which newly created KpnI and BglII sites are underlined, respectively, as described below, each fragment was inserted between KpnI and BglII sites of pGL3-Basic vector. 1) pGL3-1.8 (−1767/+47), TS27-67 (5′-GGGGTACCCAG AGA CAC TCC TCA GTT CCT A-3′) and TS27-68 (5′-CGAGATCTCGG AGC CAG CGT ACT CAC CCC G-3′) were used. 2) pGL3-1.5 (−1453/+47), TS27-78 (5′-GGGGTACCCTG GGA GGT GGA GGT TGT AGC G-3′) and TS27-68 were used. 3) pGL3-1.0 (−953/+47), TS27-79 (5′-GGGGTACCGAT TGT ATA ACG CCA CTG GAT G-3′) and TS27-68 were used. 4) pGL3-0.5 (−453/+47), TS27-80 (5′-GGGGTACCTCT CAG ACC TGC AAC ATG GGA A-3′) and TS27-68 were used. 5) pGL3-0.3 (−253/+47), TS28-1 (5′-GGGGTACCACC CAG TCT ACT ATT GGC ACA G-3′) and TS27-68 were used. 6) pGL3-0.23 (−189/+47), TS28-21 (5′-GGGGTACCTCC CTC CCG GTG CTC CTT CCA G-3′) and TS27-68 were used. 7) pGL3-0.17 (−122/+47), TS28-22 (5′-GGG GTA CCC AGA GCC GGG CGG ACC AGC CTC-3′) and TS27-68 were used. 8) pGL3-0.1 (−54/+47), pGL(−453/+47) was digested with KpnI and SmaI. The end of the KpnI site was blunted by the treatment with T4 DNA polymerase, and the plasmid was then self-ligated. 9) pGL3-Sp1m, the mutation was introduced into the Specificity protein 1 (Sp1)-binding site of pGL3-0.17 using a KOD –Plus– Mutagenesis Kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. The mutation is underlined in nucleotides −88 to −76 relative to the transcriptional start site (GGATTCGG AG instead of the wild type, GGA GGC GGA G). The correct sequences of all plasmids were confirmed by nucleotide sequencing.
Quantitative Real-Time RT-PCR AnalysisThe expression levels of the β4GalT4 mRNA 5′-untranslated region (UTR) 1 and 5′-UTR 2 were examined by quantitative real-time RT-PCR analysis as described previously.18) In brief, we designed the primer pairs for amplification of the β4GalT4 mRNA 5′-UTR 1, TS27-36 (5′-AGC TCG CCG CGG CCG CCT C-3′) and TS27-38 (5′-ATG AAG CCA CAA AGT GCC AC-3′), while for amplification of the β4GalT4 mRNA 5′-UTR 2, TS27-37 (5′-ACC TTT GAC CTT TCT CCA AA-3′) and TS27-38. The amplification efficiency of each primer pairs was calculated from the slope of the standard curve, and was 123.1 and 122.8% for the amplicons of the β4GalT4 mRNA 5′-UTR 1 and 5′-UTR 2, respectively.
Luciferase AssayThe promoter activities of the reporter plasmids were measured by luciferase assay as described previously.16,17)
Statistical AnalysisA significant difference in the experiments was determined with Student’s t-test for comparing the means of two independent, and with one-way ANOVA followed by Tukey’s post hoc test for comparing the means of more than two independent groups.
Determination of the transcriptional start sites showed five transcriptional start sites in SW480 cells (Fig. 1). Whether or not multiple transcriptional start sites are observed for other cancer cells was analyzed using the total RNA preparations prepared from A549 human lung carcinoma cell line and SH-SY5Y human neuroblastoma cell line. The results showed that six and four transcriptional start sites are identified in A549 cells and SH-SY5Y cells, respectively (data not shown). Since one of the transcriptional start sites, which was located 11 kb upstream from the initiation codon of the β4GalT4 gene (Fig. 2), was used mostly and common in three cancer cell lines, this transcriptional start site was numbered as +1 (Fig. 1). Besides these transcriptional start sites in exon X, one transcriptional start site was identified in exon Y (Fig. 2). Taken together, these results indicate that β4GalT4 has two transcripts in SW480 cells. The transcriptional start site in exon Y was also observed for A549 cells and SH-SY5Y cells (data not shown). It is considered that two transcripts containing 5′-UTR 1 and 5′-UTR 2 exist as the β4GalT4 mRNA by alternative splicing of exons X and Y in cancer cells. The expression level of the β4GalT4 mRNA containing 5′-UTR 1 showed 26-times higher than that of the β4GalT4 mRNA containing 5′-UTR 2 in SW480 cells (Fig. 3), indicating that the β4GalT4 mRNA 5′-UTR 1 containing exon X is expressed predominantly in SW480 cells. Therefore, the transcriptional mechanism of the β4GalT4 gene was examined by focusing on the 5′-upstream region of exon X.
The arrows indicate the transcriptional start sites. The number of arrow shows the number of plasmid clone containing the indicated nucleotide as the transcriptional start site by nucleotide sequencing of eight plasmid clones isolated. The numbers on the left side were determined based on the transcriptional start site that was used mostly in SW480 cells. Capital letters show coding sequences. The box indicates the initiation codon. Five (−76, −29, −26, +1, and +3) and three (−72, −29, and +1) transcriptional start sites in exon X were identified in A549 and SH-SY5Y cells, respectively.
The filled and opened boxes indicate the UTR exons and coding regions, respectively.
Data show mean±S.D. (n=3). ** p<0.01.
To examine the promoter activity of the β4GalT4 gene, the 1.8 kb region containing exon X was subcloned into the firefly luciferase gene to produce the reporter plasmid, pGL3-1.8. When pGL3-1.8 was transiently transfected into SW480 cells, significant promoter activity was observed when compared with that of the pGL3-Basic vector as a promoterless (Fig. 4, right panel). In order to identify the core promoter region, seven additional reporter plasmids containing the 1.5, 1.0, 0.5, 0.3, 0.24, 0.17, and 0.1 kb DNA fragments were constructed (Fig. 4, left panel), and the promoter activities were measured. The results showed that the highest promoter activity is associated with pGL3-1.0 (Fig. 4, right panel). Furthermore, pGL3-0.17 retained relatively high promoter activity, while the promoter activity of pGL3-0.1 was dramatically reduced to 16% of pGL3-1.8 (Fig. 4, right panel). These results indicate that the region between nucleotides −122 and −55 is involved in the promoter activation in SW480 cells, and contains the important positive regulatory elements.
SW480 cells were transfected with the reporter plasmids shown in left panel. The luciferase activity of pGL3-1.8 was set at 100%. Data show mean±S.D. (n=3). ** p<0.01.
In order to predict the putative binding sites of transcription factors, the region between nucleotides −122 and −55 was analyzed by TFBIND program.19) The analysis revealed that one Sp1-binding site at nucleotide positions −88/−76 is included in the region. When the Sp1-binding site was mutated, the promoter activity decreased to 18% of pGL3-0.17 that is similar to the activity of pGL3-0.1 (Fig. 5). This mutation in the Sp1-binding site could reduce the Sp1-binding to the promoter region.16) These results indicate that the Sp1-binding site is important for the promoter activity of the β4GalT4 gene. Sp1, a well-characterized transcription factor that regulates many cellular genes, has been shown to increase in various cancer cells.20) Elevated Sp1 expression has been observed in colon cancer as compared with normal counterpart.21) Therefore, the effect of Sp1 expression on the promoter activity was examined by introduction of Sp1-expression vector into SW480 cells as described previously.16) Ectopic Sp1 expression enhanced the promoter activity by 2.4–4.2-fold (Fig. 6). These results suggest that elevated Sp1 expression is involved in the promoter activation of the β4GalT4 gene in colon cancer cells.
SW480 cells were transfected with the reporter plasmids shown in left panel. The luciferase activity of pGL3-0.17 was set at 100%. Data show mean±S.D. (n=3). ** p<0.01.
SW480 cells were transfected with pGL3-0.17 and either pcDNA3.1 or Sp1-expression vector. The luciferase activity of pGL3-0.17 in SW480 cells co-transfected with pcDNA3.1 was set at 1.0. Data show mean±S.D. (n=3). ** p<0.01.
The present study describes the transcriptional start sites, 5′-UTR structures, and transcriptional mechanism of the β4GalT4 gene in SW480 cells. We found multiple transcriptional start sites and two transcripts with different 5′-UTRs of the β4GalT4 mRNA. In general, 5′-UTRs regulate the tissue-specific expression and the translational efficiency of genes.22) In the case of human α2,6-sialyltransferase I, α2,3-sialyltransferase IV, and α1,3-fucosyltransferase 4, the gene expression patterns of these transferases showed tissue-specific manners.23–25) Whether two transcripts of the β4GalT4 gene are expressed in tissue-specific manner, and different 5′-UTR structures regulate the translational efficiency of the β4GalT4 gene remain to be clarified.
Although we demonstrated that Sp1 is involved in the promoter activity of the β4GalT4 gene, the possibility that other transcription factors are also involved in the regulation of the β4GalT4 gene in colon cancer cells can not be excluded. Interestingly, the promoter activity of the β4GalT4 gene increased significantly by deleting the region between nucleotides −1453 and −954, suggesting that the region contains negative regulatory elements. TFBIND program showed that ten binding sites for MZF1 are included in this region. Since MZF1 has been shown to regulate gene expression negatively,26) the deletion of the MZF1-binding sites may contribute to the increased promoter activity. The glycans with the carbohydrate antigens, sialyl Lewis X and sialyl Lewis A, which promote extravasation of cancer cells, are expressed in colon cancer cells with high metastatic ability.27) The gene expression levels of α2,3-sialyltransferase I/III/IV and α1,3-fucosyltransferase 3, which are involved in the biosynthesis of sialyl Lewis X and sialyl Lewis A, have been shown to be up-regulated by c-Myc upon epithelial–mesenchymal transition of colon cancer cells.28) The expression of c-Myc was reported to overexpress in colon cancer cells.29) In addition, during the process of this study, we found that three binding sites for Runt-related transcription factors (Runx) are present in the region between nucleotides −1767 and +47 by TFBIND program. The gene expression of Runx has been shown to increase in colon cancer cells.30) Our preliminary experiments demonstrated that the promoter activity of the β4GalT4 gene increases significantly by ectopic expression of Runx1, one of the Runx proteins. Therefore, it is of interest to examine whether c-Myc and/or Runx regulate the gene expression of the β4GalT4 gene cooperatively with Sp1.
In conclusion, this study is the first report describing the transcriptional mechanism of the β4GalT4 gene in colon cancer cells, and the increased promoter activity of the β4GalT4 gene by Sp1. Based on the mechanism, a novel screening system for anti-colon cancer drugs can be developed by focusing on the cancer-related glycan glycosylation.
We are grateful to Dr. Robert Tjian in the University of California, Berkeley, for kindly supplying Sp1-expression vector, CMV-Sp1, and to Mr. Yasuyuki Tsukahara in our laboratory for providing his technical assistance. This work was supported by Grant-in-Aid for the Scientific Research (15K07924) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to TS.
The authors declare no conflict of interest.
