Genomic structure and transcriptional regulation of human Galß1,3GalNAc {alpha}2,3-sialyltransferase (hST3Gal I) gene

Akiyoshi Taniguchi1, Itaru Yoshikawa, and Kojiro  Matsumoto

Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University, 2-2-1, Miyama, Funabashi, Chiba 274-8510, Japan

Received on September 7, 2000; revised on October 2, 2000; accepted on October 24, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Appendix
 References
 
Previous studies have shown that hST3Gal I mRNA is overexpressed in colorectal cancer tissues and primary breast carcinoma compared with nonmalignant or benign tissue, suggesting that the transcriptional regulation of hST3Gal I gene is altered during malignant transformation. We report transcriptional regulation of the hST3Gal I gene in colon adenocarcinoma and leukemia cell lines. To determine the genomic structure of the 5'-untranslated region, we cloned and identified the 5'-untranslated region of hST3Gal I from a human genome library. The 5'-untranslated region was found to be divided into three exons, namely, exons Y, X, and C1. The transcription initiation sites map at –1035 bp from the translation initiation site. Our results indicate that the transcriptional regulation of hST3Gal I depends on the pI promoter that exists 5'-upstream of exon Y in these cell lines. The results of luciferase assay suggest that the nt –304 to –145 region is important for transcriptional activity of hST3Gal I gene in both cell lines. The nt –304 to –145 region contains two sequences similar to the Sp1 recognition elements (GC-box) and one USF binding site. The results of site-directed mutagenesis indicated that the Sp1 binding sites and USF binding site of the pI promoter are involved in the transcription of hST3Gal I mRNA. However, the triple mutant of these sites still exhibits about 50% transcriptional activity, suggesting that there are other transcription factors involved in the transcription of hST3Gal I mRNA. These results suggest that these factors may play a critical role in the up-regulation of the hST3Gal I gene during malignant transformation.

Key words: sialylytansferase/Sp1/USF/transcriptional regulation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Appendix
 References
 
Malignant transformation is frequently accompanied by marked alteration in surface oligosaccharide expression. Carbohydrate determinants expressed preferentially on cancer cells mostly contain sialylated structures. Synthesis of these sialylated carbohydrate determinants in colorecal cancers is regulated by a set of sialyltransferases (Hansson and Zopf, 1985Go; Holmes et al., 1985Go). Six human {alpha}2,3-sialyltransferase genes, which are hST3Gal I–VI, have so far been cloned (Chang et al., 1995Go; Ishii et al., 1998Go; Kitagawa and Paulson, 1993Go, 1994a,b; Okajima et al., 1999Go; Sasaki et al., 1993Go). Previous studies have showed that the expression of hST3Gal I mRNA is increased in cancer tissues compared with nonmalignant colorectal mucosa (Ito et al., 1997Go). Moreover, in situ hybridization of primary breast tissue showed that hST3Gal I is elevated in primary breast carcinoma when compared to normal or benign tissue (Burchell et al., 1999Go). The marked increase in hST3Gal I mRNA is thought to be related to enhanced expression of sialylated carbohydrate determinants in colon and breast cancer tissues. These findings suggest that the transcriptional regulation of the hST3Gal I gene is altered during malignant transformation. hST3Gal I can transfer sialic acid to core 1 (Galß1–3Gal NAc) on O-linked glycoprotein (Gillespie et al., 1992Go; Kitagawa and Paulson, 1994aGo), thereby terminating chain extension. hST3Gal I uses the same substrate (core 1) as C2GnT. Thus, changes in the expression of hST3Gal I could lead to changes in the structure and length of the O-glycans attached to proteins.

Several studies have examined the transcriptional regulation of some sialyltransferases genes (Lo and Lau, 1996Go; Taniguchi and Matsumoto, 1998Go, 1999; Taniguchi et al., 1998Go, 1999, 2000a). The results of these studies suggested that expression of these genes is regulated at the transcriptional level by a class of proteins called transcription factors. For example, in hST6Gal I gene, Sp1 and Oct-1 may play a critical role in the transcriptional regulation of the hST6Gal I gene during differentiation of HL-60 cells (Taniguchi et al., 1998Go, 2000a). Moreover, AP2 may contribute to the epithelium cell–specific transcriptional regulation of the hST3Gal IV gene (Taniguchi and Matsumoto, 1998Go, 1999). The structure and chromosomal location of hST3Gal I gene has been determined (Chang et al., 1995Go). However, genomic structure of the 5'-untranslated region and the transcriptional regulation of the hST3Gal I gene remains unknown.

We report here the transcriptional regulation of the hST3Gal I gene in colon adenocarcinoma and leukemia cell lines. To elucidate the molecular basis of hST3Gal I gene expression, the genomic region containing the pI promoter of hST3Gal I was isolated and functionally characterized. Our results suggest that the Sp1 binding site (GC-box) and USF binding site of the pI promoter are involved in transcriptional regulation of the hST3Gal I mRNA in these cell lines.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Appendix
 References
 
Genomic structure of the hST3Gal I gene
The structure and chromosomal location of the hST3Gal I gene has been determined elsewhere (Chang et al., 1995Go). Genomic structure of hST3Gal I gene has indicated that the coding region is dispersed among seven discrete exon regions (C1–C7). However, genomic structure of the 5'-untranslated region remains unknown. To determine the genomic structure of the 5'-untranslated region, we cloned and identified the 5'-untranslated region of hST3Gal I from a human genome library. The location of the exons was determined by polymerase chain reaction. Sequencing was performed to determine the exon–intron junctions. The 5'-untranslated region was found to be divided into three exons, namely, exons Y, X, and C1 (Figure 1).



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Fig. 1. Schematic diagram of hST3Gal I genomic and cDNA sequences. The protein coding regions, and the 5'- and 3'-untranslated regions are shown by solid and open rectangles, respectively. Exons are denoted as Y, X, and C1–C7 with the size of each exon shown on top. A representation of the hST3Gal I cDNA is shown in bottom part of the figure. The translation initiation site is designated as nucleotide +1.

 
Analysis of transcriptional starting sites on hST3Gal I mRNA in colon adenocarcinoma and leukemia cell lines
To determine the major hST3Gal I transcript in colon adenocarcinoma and leukemia cell lines, we analyzed the transcriptional starting sites of the hST3Gal I gene in colon adenocarcinoma and leukemia cell lines using 5'-RACE analysis. 5'-RACE analysis of colon adenocarcinoma cell lines (HT-29 cells) and leukemia cell lines (HL-60 cells) resulted in approximately 380-bp major extension products (Figure 2). After subcloning the PCR products and sequencing individual bands, most of the transcription initiation sites were mapped at –1035 bp from the translation initiation site in both cell lines. This result coincides with the transcriptional initiation site using cDNA from the submaxillary gland (Chang et al., 1995Go). The results indicated that the transcriptional regulation of hST3Gal I in these cell lines depends on the pI promoter present 5' upstream of exon Y. These results suggest that the hST3Gal I gene does not have multiple mRNAs in these cell lines, as have been identified in hST6Gal I, hST3Gal IV, and FUT4 genes (Kitagawa et al., 1996Go; Lo and Lau, 1996Go; Taniguchi and Matsumoto, 1998Go, 1999; Taniguchi et al., 1998Go, 1999, 2000a,b).



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Fig. 2. 5'-RACE analysis of hST3Gal I mRNA. mRNAs from HT-29 (lane 1) and HL-60 (lane 2) were subjected to 5'-RACE analysis. The PCR products were run onto 2% agarose gels and stained with ethidium bromide.

 
Cloning of the 5'-flanking region of hST3Gal I promoter (pI promoter)
To clarify the transcriptional regulation of the hST3Gal I gene in colon adenocarcinoma and leukemia cell lines, we cloned and identified the 5'-flanking region of hST3Gal I promoter from a human genome library. The sequence of this region is shown in Figure 3. The transcription start sites of 5'-RACE are indicated by arrows. The 5'-flanking region lacks canonical TATA or CCAAT boxes, but contains several putative transcriptional factor binding sites, such as MZF-1, c-Ets, Oct-1, AP-1, Sp1, USF, and Lyf-1.



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Fig. 3. Nucleotide sequence of hST3Gal I pI promoter. Potential regulatory elements are underlined and indicated below the sequence. The transcription start site is marked with an arrow. Nucleotides are numbered with the transcription-initiation site designated as +1. The nucleotide sequence of the pI promoter has been submitted to the GenBankTM/EMBL Data Bank with the accession number AB046716.

 
Deletion analysis of pI promoter
To identify the cell type–specific enhancer elements of hST3Gal I promoter, we prepared luciferase constructs carrying 5'-deleted pI promoter (Figure 4A) and transfected them into HT-29, SW-48, HL-60, and Jurkat cell lines. Plasmids pGL-1009pI, pGL-843pI, pGL-606pI, and pGL-304pI had very high luciferase activities in all cells tested (Figure 4B). Extension of the 5'-deletion of the pI promoter to nt –145 (pGL-145pI) reduced expression to approximately 10–20% of that of the promoter that was deleted to nt –304 (pGL-304pI) (Figure 4B). These results suggest that the nt –304 to –145 region is important for transcriptional activity of hST3Gal I gene in all cell lines tested.




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Fig. 4. Deletion analysis of pI promoter in hST3Gal I gene. Structure of the 5'-deletion mutants of pI promoter (A). Each firefly luciferase construct was co-transfected into HT-29, SW-48, HL-60, and Jurkat (B) with the Renilla luciferase expression vector (pRL-SV40) as the internal control. Relative luciferase activities were normalized to luciferase activity of the pGL3-Control that contained the SV40 promoter-enhancer sequences upstream of the luciferase gene. Each value represents the mean activity of luciferase detected in three independent experiments. Bars indicate standard deviation of the mean activities.

 
Effect of substitution of Sp1 and USF motifs on transcriptional activity
The nt –304 to –145 region contains two sequences similar to the Sp1 recognition elements (Sp1-1 and Sp1-2) (Briggs et al., 1986Go; Kriwacki et al., 1992Go) and one USF binding site (Bendall and Molloy, 1994Go). To examine the contribution of each Sp1 and USF recognition element to the transcriptional activity of the pI promoter, the Sp1 and USF recognition elements were mutated by introducing base substitutions. These mutations have been shown to abolish the binding of these transcription factors by gel mobility shift assay (Bendall and Molloy, 1994Go; Briggs et al., 1986Go; Kriwacki et al., 1992Go). The mutated pGL-304pI plasmids were generated from pGL-304pI and transfected into HL-60 cells, followed by measurement of luciferase activity. Mutation of the Sp1-1 (mutS1) and Sp1-2 (mutS2) sites reduced luciferase activity about 80%, compared with the wild-type construct (Figure 5). Mutation of the USF (mutU) site resulted in about 40% reduction in promoter activity. Mutation of the Sp1 sites exerted a weaker influence on promoter activity than did mutation of the USF. Triple mutation of the two Sp1 sites and one USF site (mutS1/2/U) resulted in about 50% reduction in promoter activity. These results suggest that Sp1-1, Sp1-2, and USF sites are involved in the transcription of hST3Gal I mRNA. However, the triple mutant (mutS1/2/U) still exhibits about 50% transcriptional activity. This suggests that there are other transcription factors involved in the transcription of hST3Gal I mRNA.



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Fig. 5. Effect of mutations in Sp1 and USF sites on hST3Gal I pI promoter activity. The sites of mutation are denoted by (X). Each firefly luciferase construct was cotransfected into HL-60 cells with pRL-SV40 as the internal control. Following transfection luciferase activity was measured after a 24 hr. Relative luciferase activity was normalized to the luciferase activity of the pGL-304-pI. Data is expressed as the mean ± standard deviation (n = 3).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Appendix
 References
 
In this study, we determined the genomic structure of 5'-untranslated region of hST3Gal I and characterized its promoter region by luciferase assays. Our results suggest that Sp1 and USF binding sites may play a critical role in the transcriptional regulation of the hST3Gal I gene in colon adenocarcinoma and leukemia cell lines.

Previous studies have shown that hST3Gal I mRNA is overexpressed in colorectal cancer tissues compared with nonmalignant mucosa (Ito et al., 1997Go) and hST3Gal I is elevated in primary breast carcinoma when compared to normal or benign tissue (Burchell et al., 1999Go), suggesting that the transcriptional regulation of hST3Gal I gene is altered during malignant transformation. In the present study, we examined the transcriptional regulation of the hST3Gal I gene. Our results suggest that Sp1 and USF binding sites are involved in the transcription of hST3Gal I mRNA. The Sp1 protein is ubiquitously expressed in proliferating cells and contributes to the activation of many growth-promoting genes (Deng et al., 1986Go; Dynan and Tjian, 1983Go; Kadonaga et al., 1987Go; Melton et al., 1984Go; Swick et al., 1989Go). This suggests that Sp1 may be involved in up-regulation of hST3Gal I mRNA during malignant transformation. On the other hand, USF is a family of evolutionarily conserved basic-helix-loop-helix-leucin zipper (bHLH-zip) transcription factors (Gregor et al., 1990Go; Kaulen et al., 1991Go; Kozlowski et al., 1991Go; Sirito et al., 1994Go). In mammals, there are two ubiquitously expressed genes, USF1 and USF2 (Lin et al., 1994Go; Sirito et al., 1998Go; Vallt et al., 1997Go). USF proteins and Myc oncoproteins share a similar polypeptide structure and similar DNA-binding specificity (Bendall and Molloy, 1994Go; Blackwell et al., 1990Go; Kerkholf et al., 1991Go; Murre et al., 1989Go), suggesting that USF and Myc play antagonistic role in the controls of cell proliferation. Therefore, overexpression of c-myc may be involved in regulation of hST3Gal I mRNA during malignant transformation.

Several studies have examined the transcriptional regulation of various sialyltransferases genes (Lo and Lau, 1996Go; Taniguchi and Matsumoto, 1998Go, 1999; Taniguchi et al., 1998Go, 1999, 2000a). In the case of hST6Gal I and hST3Gal IV, the cell type–specific regulation of this gene is controlled by both of specific promoter utilization and cell type–specific transcriptional factors (Kitagawa et al., 1996Go; Lo and Lau, 1996Go; Taniguchi and Matsumoto, 1998Go, 1999; Taniguchi et al., 1998Go, 2000b). Our results suggest that the hST3Gal I gene does not have multiple mRNAs, as have been identified in hST6Gal I, hST3Gal IV, and FUT4 genes (Kitagawa et al., 1996Go; Lo and Lau, 1996Go; Taniguchi and Matsumoto, 1998Go, 1999; Taniguchi et al., 1998Go, 1999, 2000a,b) and is not controlled by cell type–specific transcriptional factors in colon adenocarcinoma and leukemia cell lines. The expression of hST3Gal I mRNA occurs in a cell type–specific manner (Chang et al., 1995Go). Moreover, the hST3Gal I gene has been shown to be responsible for the conversion of Peanut Agglutinin (PNA)+ phenotype in immature cortical thymocytes to PNA phenotype in mature medullary thymocytes, suggesting that the expression of hST3Gal I mRNA is regulated during developing thymocytes (Baum et al., 1996Go; Gillespie et al., 1993Go; Priatel et al., 2000Go). Sp1 and USF are thought to be ubiquitous transcriptional factors (Lin et al., 1994Go; Saffer et al., 1990Go, 1991; Sirito et al., 1998Go; Vallt et al., 1997Go). Our result also suggests that there are other transcription factors involved in the transcription of hST3Gal I mRNA. However, we have not yet identified transcription factors other than Sp1 and USF involved in cell type– and stage-specific regulation of hST3Gal I gene. Identification of such transcription factors may facilitate understanding of the mechanisms for tissue- and stage-specific expression of hST3Gal I mRNA.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Appendix
 References
 
Cell culture
Human colon adenocarcinoma (HT-29 and SW48) and leukemia cell lines (HL-60 and Jurkat) were obtained from the American Type Culture Collection (USA). Cells were maintained in the media containing 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. The media used included RPMI 1640 (Nissui, Japan) for HL-60 and Jurkat and Dulbecco’s modified Eagle medium (Nissui) for HT-29 and SW-48.

Genomic cloning of the 5'-untranslated region of the hST3Gal I gene
Cloning and isolation of 5'-untranslated region of hST3Gal I was performed using a GenomeWalker kit (Clonetech, USA) according to the instructions provided by the manufacturer. EcoR V–digested human genomic DNA was ligated with a double-stranded oligonucleotide containing an anchor sequence, which functioned as a primer binding site for subsequent PCR amplification.

Primary PCR was performed with the provided adapter primer (AP1) and a gene-specific primer, GSP1C1(5'-AAGTAGCCTATTCTCCTGCAATCC-3') for exon C1 and GSP1X (5'-ACGGCTGGAGACACGCTGGAGTTT-3') for exon X of hST3Gal I. Secondary PCR using the provided adapter primer (AP2) and gene-specific primers (GSP2C1, 5'-AGCTGATAATGTCTCTCTGATCAAG-3' and GSP2X, 5'-CGGGATTTGGGCATGAAGGGGTTC-3') was required. The PCR amplicon was ligated into pCR2.1-TOPO (Invitrogen, USA) and sequenced using GeneRapid DNA sequencing system (Amersham Pharmacia Biotech, UK).

Genomic cloning of 5'-flanking region of the hST3Gal I gene.
Cloning and isolation of 5'-flanking region of hST3Gal I promoter was performed using GenomeWalker kit (Clonetech) according to the method recommended by the manufacturer. EcoR V–digested human genomic DNA was ligated with a double stranded oligonucleotide containing an anchor sequence, which functioned as a primer binding site for subsequent PCR amplification. Primary PCR was performed with the provided AP1 and a gene-specific primer, GSP1 (5'-ACGGCTGGAGACACGCTGGAGTTT-3') for exon Y of hST3Gal I. Secondary PCR using the provided AP2 and a gene-specific primer (GSP2, 5'-CGGGATTTGGGCATGAAGGGGTTC-3') was required. The PCR amplicon was ligated into pCR2.1-TOPO (Invitrogen) and sequenced using GeneRapid DNA sequencing system (Amersham Pharmacia Biotech).

5'-RACE analysis
Amplification of the 5' end of hST3Gal I cDNA was performed according to the instructions provided by the manufacturer (5'-RACE System for Rapid Amplification of cDNA ends, Gibco BRL, USA). First-strand cDNA was synthesized from 3 mg of total RNA using the gene-specific primer 5'-ATCTCTGTGACAGTC-3'. After digestion of template mRNA with RNase H at 30°C for 30 min, cDNA was precipitated with spin cartridge. A homopolymeric tail was then added to the 3'-end of the cDNA using TdT and dCTP. The dC-tailed cDNA was used as the template for the first PCR amplification using a bridged anchor primer as the sense primer and 5'-CAGTGGAGTCTCTTAACCTCTCTG-3' as the anti-sense primer. Thirty-five cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min were performed. The resulting PCR products were diluted 100-fold with sterile water, then amplified under the same conditions using anchor primer as the sense primer and 5'-TTCATCTCCATAGGCTGAGTGACC-3' as the anti-sense primer. The PCR amplicon was ligated into pCR2.1-TOPO (Invitrogen) and sequenced using GeneRapid DNA sequencing system (Amersham Pharmacia Biotech).

Construction of plasmids for luciferase assay
The following oligonucleotides primers were designed and used in this protocol. pGL-1009pI; 5'-ATGGTACCCCAAAGGCAAACTGTGCCTC-3', pGL-pGL-843pI; 5'-TTGGTACCTAATCCGCAAACCCCAAGC-3', pGL-606pI; 5'-AAGGTACCTAAATCCCACCTCGAGTCC-3', pGL-304pI ; 5'-AAGGTACCTCTGCCGAGCCCGCTGCGG-3', pGL-145pI; 5'-AAGGTACCGCGCGCAGGGGAGGCGGTG-3' and 5'-TTAAGCTTGCAAAGTGTCGAAACTGTC-3', respectively. The underlined nucleotides represent restriction sites that were incorporated into the primers. In the next step, 25 cycles of PCR amplification consisting of denaturation at 98°C for 20 c, and annealing and extension at 68°C for 1 min was carried out in a programmable thermal cycler (Parkin Elmer Cetus, USA). A single band was obtained by agarose gel electrophoretic analysis. PCR products were digested using the Kpn I and Hind III restriction enzymes and cloned into the Kpn I and Hind III sites of the pGL3-Basic Vector (Promega, USA). The identity of the amplification products was verified by sequence analysis.

Luciferase assay
Transient transfection was performed using Effectene Transfection Reagent (Qiagen, Germany) for HL-60 and Jurkat cells and DMRIE-C reagent (Gibco BRL) for HT-29 and SW-48 cells. Luciferase assays were performed as described previously (Taniguchi et al., 2000bGo). Cells were plated at a density of approximately 1–3 x 105 cells per 35-mm dish, and then transfected with 1 µg of pGL constructs and 0.1 µg of pRL-CMV (Promega), containing the CMV promoter located downstream of the Renilla luciferase gene, as an internal control for variations in transfection efficiency. After 24 h, cells were harvested, and cell lysates were prepared. Firefly and Renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega).

Mutagenesis of Sp1 and USF binding sites
Mutations with base substitutions were constructed for each Sp1 and USF motifs using the GeneEditor in vitro site-directed mutagenesis system based on the protocol provided by the supplier (Promega). The oligonucleotides used for site-directed mutagenesis were 5'-AAGTTGGGGAGAACGGGGCCGAACGGA-3' [for pGL-304pI(mutS1)], 5'-GAGGTCGGGAGGAACGGGCACTGGGCG-3' [for pGL-304pI(mutS2)], and 5'-CGCTGCGGTCCATTTGGCTTGGCAGAG-3' [for pGL304pI(mutU)]. Mutation sites of these primers are underlined.


    Appendix
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Appendix
 References
 
The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the accession number AB046716.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Appendix
 References
 
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