Department of Clinical Chemistry, School of Pharmaceutical Sciences, Toho University, 221, Miyama, Funabashi, Chiba 2748510, Japan
Received on November 1, 1999; revised on December 26, 1999; accepted on January 3, 1999.
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Abstract |
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Key words: sialyltransferase/gene expression/transcriptional regulation/sialyl-Lex/HL-60
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Introduction |
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Among sialyltransferases, hST6Gal I is the most thoroughly studied. Multiple hST6Gal I mRNA forms, differing only in the 5'-untranslated region, have been described (Aasheim et al., 1993; Wang et al., 1993
; Lo and Lau, 1996a
,b). A short mRNA form (Form 1) has been isolated from liver. A large transcript (Form 3), containing two 5'-untranslated exons (exon Y + Z), has been isolated from several human cell types. A distinct transcript (Form 2) containing exon X, but not exon Y + Z, has been isolated from human B cell lymphoblastoma cell lines. Transcription of the three mRNA forms is initiated by promoter regions P1, P2, and P3.
The leukemia cell line HL-60 consists predominantly of promyelocytes which can be induced to differentiate into myelocytes, metamyelocytes, and granulocytes by the addition of dimethyl sulfoxide (DMSO) to the growth medium. Cell surface glycoprotein and glycolipid profiles are specifically altered during differentiation (Gahmberg et al., 1979; Fukuda et al., 1981
; Nojiri et al., 1984
; Nakamura et al., 1992
). Therefore, this cell line is utilized as a useful model for studying the regulation of carbohydrate expression. Previous studies have demonstrated that DMSO-induced differentiation of HL-60 cells resulted in an increase in cell surface expression of sialyl-Lex. This model of differentiation has been used to investigate glycosyltransferase activities (Skacel et al., 1991
; Clarke and Watkins, 1996
). These results suggest that the increased expression of sialyl-Lex is related to
2,6-sialyltransferase activity. Therefore, the regulation of hST6Gal I may be important for the expression of sialyl-Lex in HL-60 cells.
In myeloid cells, as in other cell types, the expression of most genes is regulated at the transcriptional level by a class of proteins called transcription factors. Several transcription factors that are involved in myeloid cells-specific gene regulation have been characterized, such as MZF-1 (Morris et al., 1995; Robertson et al., 1998
), Sp1 (Rao et al., 1998
; Hauses et al., 1998
), and Oct-1 (Kim et al., 1995
; Lopez-Rodriguez et al., 1997
; Corbi and Lopez-Rodriguez, 1997
; Tenen et al., 1997
). The Sp1 protein is ubiquitously expressed in proliferating cells and contributes to the activation of many growth-promoting genes (Deng et al., 1986
; Kadonaga et al., 1987
; Swick et al., 1989
; Dynan and Tjian, 1983
; Melton et al., 1984
).
We have previously shown that DMSO-, all trans-retinoic acid (ATRA) and phorbol myristate acetate (PMA)induced differentiation elicits negative regulation of hST6Gal I at the mRNA level in the HL-60 cell line and that transcriptional regulation of hST6Gal I depends on the P3 promoter that exists 5'-upstream of exon Y (Taniguchi et al., 1998). We report here transcriptional regulation of the hST6Gal I gene during HL-60 cell differentiation. To elucidate the molecular basis of hST6Gal I gene expression, the genomic region containing the P3 promoter of hST6Gal I was isolated and functionally characterized. The present results suggest that the Sp1 binding site (GC-box) and Oct-1 binding sites (octamer sequence) of the P3 promoter may play a critical role in transcriptional regulation of the hST6Gal I gene during HL-60 cell differentiation.
The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the accession number AB030576.
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Results |
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A mutation of the Sp1-1 (mutS1) and Sp1-2 (mutS2) sites reduced luciferase activity about 60% (Figure 4). Double mutants at of Sp1-1 and Sp1-2 (mutS1/2) sites reduced expression to ~20% of that of the pGL-317-P3 (wt). A mutation of the Oct-1 (mutO) site resulted in about 70% reduction in promoter activity. A triple mutation of the two Sp1 sites and one Oct-1 site (mutS1/2/O) resulted in about 90% reduction in promoter activity. These results suggest that Sp1-1, Sp1-2, and Oct-1 sites are necessary for full enhancer activity.
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Discussion |
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There are three known Sp1-related proteins, Sp2, Sp3, and Sp4 (Hagen et al., 1992; Hagen et al., 1994
), but because Sp2 binds to GT rather than GC box (Verrijzer and van der Vliet, 1993
), Sp4 expression is restricted to the nervous system (Hagen et al., 1992
), and the level of Sp3 expression is considerably lower than Sp1 abundance in HL-60 cells (Rao et al., 1998
), only Sp1 is likely to contribute to GC box binding in HL-60 cell extracts. The Sp1 protein is ubiquitously expressed in proliferating cells and contributes to the activation of many growth-promoting genes (Deng et al., 1986
; Kadonaga et al., 1987
; Swick et al., 1989
; Dynan and Tjian, 1983
; Melton et al., 1984
). However, little is known about the regulation and mechanisms of Sp1-mediated transcriptional regulation during HL-60 differentiation. Rao et al. (1998)
observed the truncation of Sp1 by myeloblastin in undifferentiated HL-60 cells. This result suggested that Sp1 truncation is important for GC-box mediate gene regulation during myeloid cell differentiation.
Expression of the H2B histone mRNA is high in undifferentiated HL-60 cells and suppresses during differentiation of the HL-60 cells (Kim et al., 1995), which is similar to the case of hST6Gal I gene. Kim et al. (1995)
also demonstrated that the level of Oct-1 decreased during HL-60 cell differentiation. This result suggested that the transcriptional repression of hST6Gal I gene during HL-60 cell differentiation may be mediated by reduced level of Oct-1.
Sialyl-Lex is highly expressed on the surface of mature granulocytes and is believed to be a major ligand recognized by E- and P-selectin (Philips et al., 1990; Walz et al., 1990
; Tiemeyer et al., 1991
). Previous studies demonstrated that DMSO-induced differentiation of HL-60 cells resulted in an increase in cell surface expression of sialyl-Lex, and have been performed on
2,3-,
2,6-sialyltransferase and
1,3-fucosyltransferase activities (Skacel et al., 1991
). These results suggest that the reduced expression of sialyl-Lex in undifferentiated HL-60 cells is related to the presence of the strong
2,6-sialyltransferase, which uses the precursor at the expense of the
1,3-fucosyltransferase and competes the synthesis of Lex and sialyl-Lex. We have previously shown that differentiation elicits negative regulation of hST6Gal I at the mRNA level in the HL-60 cell line and that transcriptional regulation of hST6Gal I depends on the P3 promoter that exists 5'-upstream of exon Y (Taniguchi et al., 1998
). Therefore, transcriptional regulation of hST6Gal I P3 promoter during the differentiation of HL-60 cells may play an important role in the expression of sialyl-Lex.
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Materials and methods |
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Cloning of the 5'-flanking region of the hST6Gal I P3 promoter
Cloning and isolation of 5'-flanking region of P3 promoter was performed using GenomeWalker kit (Clonetech, USA) according to the manufacturers instructions. EcoRV 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, GSP1(5'-CTATTATCCATGGGAGGGAAGGTTTAT-3') for exon Y of hST6Gal I. Secondary PCR using the provided adapter primer (AP2) and gene specific primer (GSP2, 5'-CCAAGGCCCATTTTTCTCAGGATGATC-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).
PCR Amplification of the 5'-cDNA end (5'-RACE)
Amplification of the 5' end of hST6Gal I cDNA was performed according to the manufacturers instructions (5'-full RACE core set, Takara, Japan) (Maruyama et al., 1995; Taniguchi and Matsumoto, 1998
). First-strand cDNA was synthesized from 3 mg of total RNA using the gene specific primer, 5'-pTCTGAGACTGGCCGA-3'. After digestion of template mRNA with RNase H at 30°C for 1 h, cDNA was precipitated with ethanol and ligated using T4 RNA ligase at 16°C for 16 h. A 1 in 10 dilution of the ligation mixture was used as the template for the first PCR amplification using 5'-TTCTTTTCCTTCCACACACAGATG-3' as the sense primer and 5'-AAAGGGAGTTACTATGATTCCTTT-3' as the anti-sense primer. Twenty-five cycles of denaturation at 94°C for 30 s, annealing at 56°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, and amplified under the same conditions using 5'-TGCAAACAGAAGAAAGACCAGGAC-3' as the sense primer and 5'-CCATGGGGTCTGATTCCCAGTCTG-3' as the anti-sense primer. The final PCR products were subcloned into the pCR2.1 vector using the Original TA cloning kit (Invitrogen, CA), and then sequenced.
Construction of plasmids for luciferase assay
The oligonucleotides primers designed and used were: pGL-876-P3; 5'-AGCTCGAGCAGCTTGGTTAACCTCTTT-3', pGL-pGL-676-P3; 5'-ATCTCGAGTCATGCCTCAATGTTAGGA-3', pGL-460-P3; 5'-ATCTCGAGGCGTTTAGGGGTCCTT-3', pGL-317-P3; 5'-ACCTCGAGGCCGGTTTGGGGCGCGGTG-3', pGL-174-P3; 5'-ATCTCGAGTCCCTTCCACTGTGCGTCT-3', pGL-84-P3; 5'-ATCTCGAGATCCTGCCCTTGCCGA-3' and 5'-GCAAGCTTAACGCCGGGCAGTCGC-3', and (pGLPRO-317-174 and pGLPRO-174-317; 5'-ACTCTGAGGCCGGTTTGGGGCGCGGTG-3', and 5'-CTCTCGAGCTGGAGAAGGAAGGAAGAAGA-3'), respectively. The underlined nucleotides indicate restriction sites that were incorporated into the primers. Twenty-five cycles of PCR amplification consisting of denaturation at 98°C for 20 sec, and annealing and extension at 68°C for 1 min was carried out in a Parkin Elmer/Cetus thermal cycler. A single band was obtained by agarose gel electrophoretic analysis. PCR products were digested using the XhoI and HindIII restriction enzymes and cloned into the XhoI and HindIII sites of the pGL3-Basic Vector or into the XhoI site of the pGLPRO vector (Promega, USA). The identity of the amplification products was verified by sequence analysis.
Luciferase assay
Transient transfection of HL-60 cells was performed using Effectene Transfection Reagent (Qiagen, Germany). Cells were plated at a density of approximately 13 x 105 cells per 35 mm dish, and then transfected with 1 mg of pGL constructs and 0.1 mg of pRL-CMV (Promega, USA), containing the CMV promoter located downstream of the Renilla luciferase gene, as an internal control for variations in transfection efficiency. After 24 h, uninduced HL-60 cells or HL-60 cells induced to differentiate by treatment with 1.35% of DMSO, cells were harvested and cell lysates were prepared. Firefly and Renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega, USA).
Mutagenesis of the Sp1 and Oct-1 binding sites
Mutations with base substitutions were constructed for each Sp1 and Oct-1 motif using the GeneEditor in vitro site-directed mutagenesis system (Promega, USA) according to the manufacturers protocol. The oligonucleotides used for site-directed mutagenesis were 5'-AGAGGTGGAGTGGGAACGGAGACGACC-3' (for pGL-317-P3 mutS1, mutS1/2, and mutS1/2/O), 5'-ACTCGCTTCCTTCCCGAACCTCCGCCG-3' (for pGL-377-P3mutS1/2, mutS2, and mutS1/2/O) and 5'-AGTCTTCGCAACTCTATAAGCATACGG-3' (for pGL-377-P3mutO and mutS1/2/O). Mutation sites of these primers are underlined.
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Footnotes |
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2 Present address: Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo, 1608582, Japan
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