Transcriptional Mechanism of the Β4-Galactosyltransferase 4 Gene In

Vol. 40, No. 5 Biol. Pharm. Bull. 40, 733–737 (2017) 733 Note

Transcriptional Mechanism of the β4-Galactosyltransferase 4 Gene in SW480 Human Colon Cancer Cell Line Atena Sugiyama, Naomichi Fukushima, and Takeshi Sato* Laboratory of Glycobiology, Department of Bioengineering, Nagaoka University of Technology; Nagaoka, Niigata 940–2188, Japan. Received January 19, 2017; accepted February 15, 2017; advance publication released online February 23, 2017

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 stimu- lated 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. Key words β4-galactosyltransferase 4; transcriptional mechanism; colon cancer; transcription factor; Speci- ficity protein 1 (Sp1)

It is well documented that the structures of glycans attached 10% fetal calf serum, 50 units/mL penicillin and 50 µg/mL to proteins and lipids on cell surface change dramatically streptomycin. upon malignant transformation of cells.1–3) In general, altered Determination of Transcriptional Start Site To deter- glycosylation modulates the function of cell adhesion mol- mine the transcriptional start site, the cDNA was synthesized ecules and receptors,4,5) and the cell membrane properties.6,7) from SW480 cells with a GeneRacer kit (Invitrogen, Carlsbad, The background of the altered glycosylation has been shown CA, U.S.A.) as described previously.16) Two reverse gene- to be attributed to the altered expression of glycosyltransfer- specific primers, which correspond to the coding region of the ases at the transcriptional level. β4GalT4 cDNA, were used for the PCR. The first PCR was The Galβ1→4GlcNAc/Glc/Xyl groups, in which Galβ1→4 performed using the cDNA as templates, the gene-specific structures are synthesized by seven members in the β4- primer TS27-4 (5′-CTC TTG AAT GGC ACC CAC GAA GTA galactosyltransferase (β4GalT) family, are common and make G-3′; complementary to nucleotides +96/+120 relative to the the glycan backbone structures in glycoconjugates.8) Among translational start site), and the GeneRacer 5′-Primer included the β4GalT family β4GalT4 has been shown to be involved in in the kit. The nested PCR was performed using the first PCR the biosynthesis of neolacto-series glycolipids,9) and poly-N- products as templates, the gene-specific primer TS27-3 (5′-CCC acetyllactosamine on O-glycans.10) Moreover, β4GalT4 can AAC CAC TGT CAG GCA CAA AGT C-3′; complementary to transfer galactose from uridine 5′-diphosphate-Gal to the nucleotides +57/+81 relative to the translational start site), and GlcNAc-6-O-sulfate residues, which are included in the kera- the GeneRacer 5′-Nested Primer included in the kit. The nu- tan sulfate chains and 6-sulfosialyl-Lewis X group.11) cleotide sequence of the nested PCR products was determined. The β4GalT family shows different tissue distribution. The Isolation of 5′-Region of Human β4GalT4 Gene The β4GalT4 gene is expressed in various tissues such as pla- 5′-flanking region of the transcriptional start site of the human centa, pancreas, kidney, and colon.9,11,12) Increased expression β4GalT4 gene was amplified by PCR using the human whole of β4GalT4 has been shown to be associated with metastatic blood genomic DNA as a template according the method ability and poor prognosis of human colon cancer.13) Thus, as described previously.17) The following 5′- and 3′-primer β4GalT4 is key enzyme for expressing malignant properties of pairs were synthesized based on the nucleotide sequence of colon cancer. the human chromosome 3q BAC RP11-484M3 (accession no. So far the transcriptional mechanisms of the human AC083800) and used: TS27-50 (5′-CAG AGA CAC TCC TCA β4GalT1, β4GalT2, and β4GalT5 genes in cancer cells have GTT CCT AGA C-3′) and TS27-42 (5′-CGG AGC CAG CGT ACT been reported.14–16) However, the transcriptional mechanism of CAC CCC GGA G-3′) for the amplification of the region −1767 the human β4GalT4 gene remains to be elucidated. Herein, we to +47 relative to the transcriptional start site, which was defined the core promoter region of the β4GalT4 gene and the mostly used in mRNA and is tentatively designed as +1. transcription factor that regulates the promoter activity of the Reporter Plasmid Construction To identify the core β4GalT4 gene in human colon cancer cells. promoter region of the β4GalT4 gene, the deletion constructs of the 5′-regions were made using the firefly lu- MATERIALS AND METHODS ciferase reporter vector, pGL3-Basic (Promega, Madison, WI, U.S.A.), with the numbers indicating the nucleotide Cell Culture SW480 human colon cancer cell line was positions relative to the transcriptional start site. After cultivated in Dulbecco’s modified Eagle’s medium containing the KpnI–BglII fragment was amplified by PCR with the

5′- and 3′-primer pairs, in which newly created KpnI and designed the primer pairs for amplification of the β4GalT4 BglII sites are underlined, respectively, as described below, mRNA 5′-UTR 1, TS27-36 (5′-AGC TCG CCG CGG CCG CCT each fragment was inserted between KpnI and BglII sites C-3′) and TS27-38 (5′-ATG AAG CCA CAA AGT GCC AC- of pGL3-Basic vector. 1) pGL3-1.8 (−1767/+47), TS27-67 3′), while for amplification of the β4GalT4 mRNA 5′-UTR (5′-GGGGTACCCAG AGA CAC TCC TCA GTT CCT A-3′) 2, TS27-37 (5′-ACC TTT GAC CTT TCT CCA AA-3′) and and TS27-68 (5′-CGAGATCTCGG AGC CAG CGT ACT CAC TS27-38. The amplification efficiency of each primer pairs CCC G-3′) were used. 2) pGL3-1.5 (−1453/+47), TS27-78 was calculated from the slope of the standard curve, and was (5′-GGGGTACCCTG GGA GGT GGA GGT TGT AGC G-3′) 123.1 and 122.8% for the amplicons of the β4GalT4 mRNA and TS27-68 were used. 3) pGL3-1.0 (−953/+47), TS27-79 5′-UTR 1 and 5′-UTR 2, respectively. (5′-GGGGTACCGAT TGT ATA ACG CCA CTG GAT G-3′) Luciferase Assay The promoter activities of the reporter and TS27-68 were used. 4) pGL3-0.5 (−453/+47), TS27-80 plasmids were measured by luciferase assay as described pre- (5′-GGGGTACCTCT CAG ACC TGC AAC ATG GGA A-3′) viously.16,17) and TS27-68 were used. 5) pGL3-0.3 (−253/+47), TS28-1 Statistical Analysis A significant difference in the ex- (5′-GGGGTACCACC CAG TCT ACT ATT GGC ACA G-3′) and periments was determined with Student’s t-test for comparing TS27-68 were used. 6) pGL3-0.23 (−189/+47), TS28-21 the means of two independent, and with one-way ANOVA (5′-GGGGTACCTCC CTC CCG GTG CTC CTT CCA G-3′) and followed by Tukey’s post hoc test for comparing the means of TS27-68 were used. 7) pGL3-0.17 (−122/+47), TS28-22 more than two independent groups. (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) RESULTS AND DISCUSSION was digested with KpnI and SmaI. The end of the KpnI site was blunted by the treatment with T4 DNA polymerase, Expression of β4GalT4 mRNA with Different 5′-UTRs and the plasmid was then self-ligated. 9) pGL3-Sp1m, the in SW480 Cells Determination of the transcriptional start mutation was introduced into the Specificity protein 1 (Sp1)- sites showed five transcriptional start sites in SW480 cells binding site of pGL3-0.17 using a KOD –Plus– Mutagenesis (Fig. 1). Whether or not multiple transcriptional start sites are Kit (TOYOBO, Osaka, Japan) according to the manufacturer’s observed for other cancer cells was analyzed using the total instructions. The mutation is underlined in nucleotides −88 to RNA preparations prepared from A549 human lung carci- −76 relative to the transcriptional start site (GGATTCGG AG noma cell line and SH-SY5Y human neuroblastoma cell line. instead of the wild type, GGA GGC GGA G). The correct The results showed that six and four transcriptional start sites sequences of all plasmids were confirmed by nucleotide se- are identified in A549 cells and SH-SY5Y cells, respectively quencing. (data not shown). Since one of the transcriptional start sites, Quantitative Real-Time RT-PCR Analysis The expres- which was located 11 kb upstream from the initiation codon sion levels of the β4GalT4 mRNA 5′-untranslated region of the β4GalT4 gene (Fig. 2), was used mostly and common (UTR) 1 and 5′-UTR 2 were examined by quantitative real- in three cancer cell lines, this transcriptional start site was time RT-PCR analysis as described previously.18) In brief, we numbered as +1 (Fig. 1). Besides these transcriptional start

Fig. 1. The Human β4GalT4 Gene Promoter Sequence and Its Transcriptional Start Sites 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. Vol. 40, No. 5 (2017) Biol. Pharm. Bull. 735 sites in exon X, one transcriptional start site was identified reporter plasmids containing the 1.5, 1.0, 0.5, 0.3, 0.24, 0.17, in exon Y (Fig. 2). Taken together, these results indicate that and 0.1 kb DNA fragments were constructed (Fig. 4, left β4GalT4 has two transcripts in SW480 cells. The transcrip- panel), and the promoter activities were measured. The re- tional start site in exon Y was also observed for A549 cells sults showed that the highest promoter activity is associated and SH-SY5Y cells (data not shown). It is considered that with pGL3-1.0 (Fig. 4, right panel). Furthermore, pGL3-0.17 two transcripts containing 5′-UTR 1 and 5′-UTR 2 exist as retained relatively high promoter activity, while the promoter the β4GalT4 mRNA by alternative splicing of exons X and Y activity of pGL3-0.1 was dramatically reduced to 16% of in cancer cells. The expression level of the β4GalT4 mRNA pGL3-1.8 (Fig. 4, right panel). These results indicate that the containing 5′-UTR 1 showed 26-times higher than that of the region between nucleotides −122 and −55 is involved in the β4GalT4 mRNA containing 5′-UTR 2 in SW480 cells (Fig. 3), promoter activation in SW480 cells, and contains the impor- indicating that the β4GalT4 mRNA 5′-UTR 1 containing exon tant positive regulatory elements. X is expressed predominantly in SW480 cells. Therefore, the Regulation of β4GalT4 Gene Promoter by Sp1 In order transcriptional mechanism of the β4GalT4 gene was examined to predict the putative binding sites of transcription factors, by focusing on the 5′-upstream region of exon X. the region between nucleotides −122 and −55 was analyzed Identification of Core Promoter Region To examine the by TFBIND program.19) The analysis revealed that one Sp1- promoter activity of the β4GalT4 gene, the 1.8 kb region con- binding site at nucleotide positions −88/−76 is included in the taining exon X was subcloned into the firefly luciferase gene region. When the Sp1-binding site was mutated, the promoter to produce the reporter plasmid, pGL3-1.8. When pGL3-1.8 activity decreased to 18% of pGL3-0.17 that is similar to the was transiently transfected into SW480 cells, significant pro- activity of pGL3-0.1 (Fig. 5). This mutation in the Sp1-binding moter activity was observed when compared with that of the site could reduce the Sp1-binding to the promoter region.16) pGL3-Basic vector as a promoterless (Fig. 4, right panel). In order to identify the core promoter region, seven additional

Fig. 2. Schematic Illustration of the Genomic DNA Structure and Two Transcripts with Different 5′-UTRs of the Human β4GalT4 Gene Fig. 3. Comparison of the Expression Levels between the β4GalT4 mRNA 5′-UTR 1 and 5′-UTR 2 in SW480 Cells The ﬁlled and opened boxes indicate the UTR exons and coding regions, respectively. Data show mean±S.D. (n=3). ** p<0.01.

Fig. 4. Identiﬁcation of the Core Promoter Region of the Human β4GalT4 Gene 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. 736 Biol. Pharm. Bull. Vol. 40, No. 5 (2017)

Fig. 5. Involvement of the Sp1-Binding Site in the Promoter Activity of the β4GalT4 Gene 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.

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. Inter- estingly, 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 extravasa- tion of cancer cells, are expressed in colon cancer cells with Fig. 6. Stimulation of the β4GalT4 Gene Promoter by Sp1 high metastatic ability.27) The gene expression levels of α2,3- SW480 cells were transfected with pGL3-0.17 and either pcDNA3.1 or Sp1-ex- sialyltransferase I/III/IV and α1,3-fucosyltransferase 3, which pression vector. The luciferase activity of pGL3-0.17 in SW480 cells co-transfected are involved in the biosynthesis of sialyl Lewis X and sialyl with pcDNA3.1 was set at 1.0. Data show mean±S.D. (n=3). ** p<0.01. Lewis A, have been shown to be up-regulated by c-Myc upon epithelial–mesenchymal transition of colon cancer cells.28) These results indicate that the Sp1-binding site is important The expression of c-Myc was reported to overexpress in colon for the promoter activity of the β4GalT4 gene. Sp1, a well- cancer cells.29) In addition, during the process of this study, characterized transcription factor that regulates many cellular we found that three binding sites for Runt-related transcription genes, has been shown to increase in various cancer cells.20) factors (Runx) are present in the region between nucleotides Elevated Sp1 expression has been observed in colon cancer −1767 and +47 by TFBIND program. The gene expression as compared with normal counterpart.21) Therefore, the effect of Runx has been shown to increase in colon cancer cells.30) of Sp1 expression on the promoter activity was examined by Our preliminary experiments demonstrated that the promoter introduction of Sp1-expression vector into SW480 cells as activity of the β4GalT4 gene increases significantly by ectopic described previously.16) Ectopic Sp1 expression enhanced the expression of Runx1, one of the Runx proteins. Therefore, it promoter activity by 2.4–4.2-fold (Fig. 6). These results sug- is of interest to examine whether c-Myc and/or Runx regulate gest that elevated Sp1 expression is involved in the promoter the gene expression of the β4GalT4 gene cooperatively with activation of the β4GalT4 gene in colon cancer cells. Sp1. The present study describes the transcriptional start sites, In conclusion, this study is the first report describing the 5′-UTR structures, and transcriptional mechanism of the transcriptional mechanism of the β4GalT4 gene in colon can- β4GalT4 gene in SW480 cells. We found multiple transcrip- cer cells, and the increased promoter activity of the β4GalT4 tional start sites and two transcripts with different 5′-UTRs gene by Sp1. Based on the mechanism, a novel screening sys- of the β4GalT4 mRNA. In general, 5′-UTRs regulate the tem for anti-colon cancer drugs can be developed by focusing tissue-specific expression and the translational efficiency of on the cancer-related glycan glycosylation. Vol. 40, No. 5 (2017) Biol. Pharm. Bull. 737

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