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.
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Abstract |
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Key words: sialylytansferase/Sp1/USF/transcriptional regulation
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Introduction |
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Several studies have examined the transcriptional regulation of some sialyltransferases genes (Lo and Lau, 1996; Taniguchi and Matsumoto, 1998
, 1999; Taniguchi et al., 1998
, 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., 1998
, 2000a). Moreover, AP2 may contribute to the epithelium cellspecific transcriptional regulation of the hST3Gal IV gene (Taniguchi and Matsumoto, 1998
, 1999). The structure and chromosomal location of hST3Gal I gene has been determined (Chang et al., 1995
). 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.
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Results |
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Discussion |
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Previous studies have shown that hST3Gal I mRNA is overexpressed in colorectal cancer tissues compared with nonmalignant mucosa (Ito et al., 1997) and hST3Gal I is elevated in primary breast carcinoma when compared to normal or benign tissue (Burchell et al., 1999
), 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., 1986
; Dynan and Tjian, 1983
; Kadonaga et al., 1987
; Melton et al., 1984
; Swick et al., 1989
). 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., 1990
; Kaulen et al., 1991
; Kozlowski et al., 1991
; Sirito et al., 1994
). In mammals, there are two ubiquitously expressed genes, USF1 and USF2 (Lin et al., 1994
; Sirito et al., 1998
; Vallt et al., 1997
). USF proteins and Myc oncoproteins share a similar polypeptide structure and similar DNA-binding specificity (Bendall and Molloy, 1994
; Blackwell et al., 1990
; Kerkholf et al., 1991
; Murre et al., 1989
), 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, 1996; Taniguchi and Matsumoto, 1998
, 1999; Taniguchi et al., 1998
, 1999, 2000a). In the case of hST6Gal I and hST3Gal IV, the cell typespecific regulation of this gene is controlled by both of specific promoter utilization and cell typespecific transcriptional factors (Kitagawa et al., 1996
; Lo and Lau, 1996
; Taniguchi and Matsumoto, 1998
, 1999; Taniguchi et al., 1998
, 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., 1996
; Lo and Lau, 1996
; Taniguchi and Matsumoto, 1998
, 1999; Taniguchi et al., 1998
, 1999, 2000a,b) and is not controlled by cell typespecific transcriptional factors in colon adenocarcinoma and leukemia cell lines. The expression of hST3Gal I mRNA occurs in a cell typespecific manner (Chang et al., 1995
). 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., 1996
; Gillespie et al., 1993
; Priatel et al., 2000
). Sp1 and USF are thought to be ubiquitous transcriptional factors (Lin et al., 1994
; Saffer et al., 1990
, 1991; Sirito et al., 1998
; Vallt et al., 1997
). 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.
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Materials and methods |
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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 Vdigested 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 Vdigested 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., 2000b). Cells were plated at a density of approximately 13 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.
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Appendix |
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Footnotes |
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References |
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