Genomic Structure and Promoter Activity of the Mouse Polysialic Acid Synthase (mST8Sia IV/PST) Gene*

The mouse gene encoding ST8Sia IV/PST, one of two polysialic acid synthases, was isolated and character-ized. The mST8Sia IV/PST gene was found to comprise over 60 kilobases and to be composed of five exons. Primer extension analysis revealed that transcription started from 333 nucleotides upstream of the transla-tional initiation site. Transfection with nested deletion mutants of the 5 * -flanking region fused to the luciferase reporter gene revealed that the promoter activity of the 2 107/ 1 145 region was correlated with the gene expression of mST8Sia IV/PST in embryonal carcinoma P19 and neuroblastoma F11 cells. This proximal promoter region lacks an apparent TATA box but has putative binding sites for transcription factors Sp1 and NF-Y (CCAAT binding protein) at nucleotide positions 2 66/ 2 57 and 2 47/ 2 37, respectively. Individual deletions and mutations of the inverted Sp1 binding site or inverted NF-Y binding site caused significant reduction of the promoter activity, indicating that each binding site was involved in essential transcription control. Mobility shift assaying also revealed that Sp1 and NF-Y in a nuclear extract of P19 cells bind to the promoter region of the mST8Sia IV/PST gene. Deletion of the region from 2 60 to 2 40, which contains parts of both the Sp1 and NF-Y binding sites, completely abolished the promoter activity, suggesting that both Sp1 and NF-Y are syner-getically involved in transcription regulation of the mST8Sia IV/PST gene in P19 and F11

The mouse gene encoding ST8Sia IV/PST, one of two polysialic acid synthases, was isolated and characterized. The mST8Sia IV/PST gene was found to comprise over 60 kilobases and to be composed of five exons. Primer extension analysis revealed that transcription started from 333 nucleotides upstream of the translational initiation site. Transfection with nested deletion mutants of the 5-flanking region fused to the luciferase reporter gene revealed that the promoter activity of the ؊107/؉145 region was correlated with the gene expression of mST8Sia IV/PST in embryonal carcinoma P19 and neuroblastoma F11 cells. This proximal promoter region lacks an apparent TATA box but has putative binding sites for transcription factors Sp1 and NF-Y (CCAAT binding protein) at nucleotide positions ؊66/ ؊57 and ؊47/؊37, respectively. Individual deletions and mutations of the inverted Sp1 binding site or inverted NF-Y binding site caused significant reduction of the promoter activity, indicating that each binding site was involved in essential transcription control. Mobility shift assaying also revealed that Sp1 and NF-Y in a nuclear extract of P19 cells bind to the promoter region of the mST8Sia IV/PST gene. Deletion of the region from ؊60 to ؊40, which contains parts of both the Sp1 and NF-Y binding sites, completely abolished the promoter activity, suggesting that both Sp1 and NF-Y are synergetically involved in transcription regulation of the mST8Sia IV/PST gene in P19 and F11 cells. Although the overall structures of the two polysialic acid synthase genes (ST8Sia II/STX and IV/PST) are very similar, there is no extensive sequence homology between the 5-flanking regions of the ST8Sia II/STX and IV/PST genes, suggesting that these two genes are expressed under different regulatory systems.
Polysialic acid (PSA) 1 is a linear homopolymer of ␣2, 8-sialic acid residues mainly associated with the neural cell adhesion molecule (N-CAM) in mammalian cells and is implicated in the reduction of N-CAM adhesion through its large negative charge (1). In the late embryonic and early postnatal stages, neurons mainly express the highly polysialylated form of N-CAM (2,3). However, in the course of neural development, the content of PSA associated with N-CAM decreases, resulting in an increase in the adhesive ability of the N-CAM itself (3)(4)(5). Recent data imply important functions of PSA in the pathfinding and targeting in the innervation of axons, migration of neuronal cells and tumor cells, and spatial learning and memory (6 -8).
In 1995, Eckhardt et al. (9) cloned the cDNA of a sialyltransferase, which is the key enzyme for PSA expression in Chinese hamster ovary cells, and named the enzyme polysialyltransferase-1 (PST-1). We independently cloned a mouse cDNA encoding an ␣2,8-sialyltransferase, ST8Sia IV, whose amino acid sequence exhibits 99.8% identity to that of hamster PST-1, and showed that ST8Sia IV is a PSA synthase (10). On the other hand, we demonstrated that ST8Sia II/STX is another PSA synthase (12)(13)(14)(15)(16)(17). In the mouse, the amino acid sequences of the two types of PSA synthases, ST8Sia II/STX and IV/PST, exhibit 56% identity, which is the highest score among the sialyltransferases cloned so far. Northern blot analysis indicated that expression of the mST8Sia II/STX gene was restricted to the brain and testis, whereas the mST8Sia IV/PST gene was expressed strongly in the lung, spleen, and heart, rather than the brain (10,12). Expression of the mST8Sia II/STX gene in the brain was strictly regulated during development (12). Expression of the mST8Sia IV/PST gene was also higher in fetal than adult brain but was less regulated during brain development as compared with that of the mST8Sia II/STX gene (10). Our recent results indicated that mST8Sia II/STX and IV/PST synthesize PSA of different sizes in vitro and in vivo (17,18). However, it is not clear why two types of PSA synthases exist and how they are differently expressed.
To elucidate the mechanisms underlying the differential expression of the mST8Sia II/STX and IV/PST genes, it is important to know the structures and activities of their promoters. We recently reported the entire genomic organization and the promoter structure of the mST8Sia II/STX gene (19). We demonstrated that the minimal promoter region of the mST8Sia II/STX gene conferred the cell type-specific expression in the reporter gene. The minimal promoter was embedded in a GCrich domain (GC content, 74%), in which two Sp1 binding motifs as well as a long purine-rich region were found, but it lacked TATA and CAAT boxes. In the present study, we describe the genomic structure of the mST8Sia IV/PST gene and its promoter sequence involved in the regulation of transcription activity.

EXPERIMENTAL PROCEDURES
Isolation of Genomic and cDNA Clones Encoding mST8Sia IV/ PST-A mouse genomic cosmid library was constructed and screened as described previously (20). The locations of the exons of the mST8Sia IV/PST gene were determined by PCR (GeneAmp XL PCR Kit, Perkin-Elmer) with specific oligonucleotide primers or by hybridizing radiolabeled sialyltransferase cDNA to the same blots.
PCR Amplification of the 5Ј-cDNA End (RACE)-Amplification of the 5Ј end of mST8Sia IV/PST cDNA was performed as described previously (20). cDNA was synthesized by reverse transcription of 5 g of mouse brain poly(A) RNA using primer O5-EX2, a 32-mer oligonucleotide complementary to mST8Sia IV mRNA (nucleotide positions ϩ325 to ϩ293, Table I). After the cDNA had been A-tailed, two consecutive PCRs were performed with two nested sets of primers. For pair 1, the forward primer was NotI-(dT) 18 (Pharmacia Biotech Inc.), and the reverse primer was O5-EX2. For pair 2, the forward primer was as above but without the T-tail, 5Ј-AACTGGAAGAATTCGCGGCCGCAGGAA-3Ј, and the reverse primer was O5-N5 (Table I). The cDNA was amplified for 35 cycles of a step program (94°C, 40 s; 55°C, 40 s; and 72°C, 60 s). The amplified products were subcloned into pUC119 and then sequenced.
Primer Extension Analysis-pO5-22E1.5 was constructed by subcloning a 1.5-kb EcoRI fragment from COS O5-22, which contains the 1030-bp 5Ј-flanking region of the mST8Sia IV/PST gene, into the pUC118 plasmid. The O5-EX2 primer was end-labeled with [ 32 P]ATP using T4 polynucleotide kinase. The radiolabeled O5-EX2 was hybridized with 5 g of poly(A) RNA prepared from mouse brain or 5 g of yeast tRNA, as a control to extension specificity, and then extended with Superscript II (Life Technologies, Inc.) as described previously (19). The primer extension products were separated on a 6% sequencing gel along with dideoxy-mediated a chain termination sequencing reaction of pO5-22E1.5, using O5-EX2 as the primer.
Analysis of Promoter Activity-To obtain various lengths of the 5Јflanking region of the mST8Sia IV gene, PCR with Tth DNA polymerase (GeneAmp XL PCR Kit, Perkin-Elmer) was performed using two primers, O5-ATGNco (Table I) and a reverse sequencing primer, with pO5-22E1.5 as the template. The mST8Sia IV/PST-luciferase fusion gene expression plasmids were constructed by subcloning the following restriction fragments from the PCR products into pPicaGene-Basic II (pPGBII, Toyo-ink, Japan): pBO5-BN0.87 carries a 0.87-kb BamHI-NcoI fragment and pBO5-SaN0.16 carries a 0.16-kb SacI-NcoI fragment, respectively. Other series of deletion plasmids were constructed by subcloning the restriction enzyme-digested PCR products amplified using the primer set of O5-ATGNco and the restriction enzyme site introducing mutagenic primers into pPGBII. The primers and template plasmids used were as follows: pBO5-XN0.53 carries a 0.53-kb XhoI-NcoI fragment amplified by using the primer set of O5-530X/O5-ATGNco; pBO5-XN0.44 carries a 0.44-kb XhoI-NcoI fragment amplified by using the primer set of O5-440X/O5-ATGNco; pBO5-NhN0.35 carries a 0.35-kb NheI-NcoI fragment amplified by using the primer set of O5-350Nh/O5-ATGNco; pBO5-XN0.31 carries a 0.31-kb XhoI-NcoI fragment amplified by using the primer set of O5-310X/O5-ATGNco; and pBO5-XH0.25 carries a 0.25-kb XhoI-HindIII fragment amplified by using the primer set of O5-440X/O5-150H, respectively, with pO5-22E1.5 as the template (Table I). For the pBO5-NhN3.5 construct, a 2.8-kb NheI-XhoI fragment from COS O5-22 was subcloned into pBO5-XN0.67 digested with the same restriction enzymes. As controls, plasmids pBSV, containing the luciferase gene driven by the SV40 promoter, and pPGBII, containing the promoterless luciferase gene, were transfected into parallel cultures of each cell line. The luciferase activity due to each luciferase reporter plasmid was normalized to the ␤-galactosidase activity by cotransfecting an internal control plasmid, pSR␤-gal, carrying a ␤-galactosidase gene under the control of the SR␣ promoter. In all the cell lines tested, pPGBII was inactive to the expression of luciferase activity, whereas pBSV caused a high level of expression.
differentiation of P19 cells into neuronal cells, the cells were seeded into and aggregated in bacteriological grade dishes in the presence of 1 mM retinoic acid at the cell density of 1 ϫ 10 5 /ml. After 3 days, the aggregates were trypsinized, and then approximately 1 ϫ 10 5 cells per 60-mm diameter dish (tissue culture grade dishes) were plated in Dulbecco's modified Eagle's medium, 10% fetal calf serum 24 h prior to transfection. The luciferase plasmid (5 g) used as the reporter and the pSR␤-gal plasmid (0.5 g) used as an internal control for transfection efficiency were transfected into the cells by means of LipofectAMINE (Life Technologies, Inc.). After 48 h transfection, the cells were washed three times with phosphate-buffered saline and then lysed with cell lysis buffer (PG␤-50, Toyo-ink, Japan). Luciferase activity was measured using a PicaGene Luciferase Assay System (Toyo-ink) and a Luminescencer AB-2000 (ATTO, Japan). Light activity measurements were performed in quadruplicate, averaged, and then normalized to the ␤-galactosidase activity to correct for the transfection efficiency. ␤-Galactosidase activity was measured using a Luminescent ␤-Galactosidase Detection Kit II (CLONTECH).
Cloning of the NF-Y Gene-The NF-YA gene was cloned from P19 cells by reverse transcriptase-PCR using primers 5Ј-GAAGCTTCAG-GACTCTTAAC-3Ј and 5Ј-TGACTGATCAGCTCTGCCACC-3Ј (22). PCR products were cloned into pBluescriptII SKϩ and then sequenced. The NF-YB gene (22) was cloned from an adult mouse brain cDNA library by plaque hybridization and then sequenced.
In Vitro Transcription and Translation-In vitro transcription of the NF-YA and NF-YB genes was performed using an mCAP mRNA capping kit (Stratagene) according to the manufacturer's instructions. The resulting mRNA samples (2 g) were applied to a rabbit reticulocyte lysate system (Amersham Corp.) for in vitro translation.
Gel Shift Assay-The DNA fragment from Ϫ107 to Ϫ16 was prepared from pBO5-XN0.44 by digestion with XhoI and PstI and then endlabeled with [ 32 P]dCTP using Klenow polymerase. Binding assays were performed with a labeled probe (10 -20 k cpm) in the presence of 2 g of poly(dI-dC)⅐poly(dI-dC) (Pharmacia) and 2 g of a nuclear protein extract or an appropriate volume of the products translated in vitro. Binding reactions were carried out for 30 min at 0°C in 25 mM HEPES-KOH (pH 7.9), 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10% glycerol. Competitor fragments or anti-Sp1 antibodies (Santa Cruz Biotechnology) were included where indicated. The DNA fragment from Ϫ339 to Ϫ221, which was prepared from pBO5-XN0.63 by digestion with XhoI and BlnI, and the DNA fragment from Ϫ107 to Ϫ16 were used as nonspecific and specific competitors, respectively. The synthetic DNA fragments, 5Ј-CGC-CCCCTCAGCACGGTGATTGGCTGG-3Ј (nucleotide positions from Ϫ62 to Ϫ36) and 5Ј-AGGCCAGCCAATCACCGTGCTGAGGGGG-3Ј (complementary to nucleotide positions from Ϫ60 to Ϫ33), were used as competitors after the two synthetic DNAs had been annealed. After incubation, the samples were loaded onto a 4% polyacrylamide gel (acrylamide:bisacrylamide, 19:1) in 0.5ϫ TBE. The gel was run in the cold at 200 V and dried, and then the radioactivity was detected with a BAS 2000 image analyzer (Fuji Film, Japan).

Isolation of mST8Sia IV/PST Genomic Clones-
The screening of an NIH3T3 cell cosmid library with mST8Sia IV/PST cDNA resulted in the isolation of three independent genomic clones. A restriction map of the approximately 100-kb region containing the mST8Sia IV/PST gene is shown in Fig. 1. The locations of the mST8Sia IV/PST exons were determined by PCR and Southern blot hybridization using a variety of oligonucleotides designed according to the known mST8Sia IV/PST cDNA sequence. Since cross-hybridization experiments indicated that CosO5-12 and CosO5-19 did not overlap, further screening of 5 ϫ 10 5 genomic clones (inserts ranging from 30 to 40 kb in size) was performed with a 2-kb fragment of CosO5-12 including exon 4 and a 1.3-kb fragment of the T3 primer end of CosO5-19 as probes. However, no overlapping clones were obtained. Southern blot analyses of the NIH3T3 genomic DNA using the above two probes showed that the distance between the termini of CosO5Ϫ12 and Ϫ19 was over 12 kb (data not shown).
We sequenced the exons to determine their exact sizes and the intron/exon junctions (Table II). The sequences of all the intron/exon splice junctions conformed to the GT-AG rule (23). The mST8Sia IV/PST cDNA was divided into 5 exons, ranging from 132 to 5656 bp, with intron sizes of 8 -24 kb, and spanning more than 60 kb of genomic DNA (Fig. 1). Exon 1 contained the entire 5Ј-untranslated region and the beginning of the coding region to amino acid residue 37, containing a cytoplasmic domain, a short hydrophobic signal anchor sequence, and a part of the stem domain. Exons 3-5 encoded the putative active domain of the enzyme, and exon 5 contained a large 3Ј-untranslated region. We previously reported mST8Sia IV/PST cDNA sequences lacking the whole 3Ј-untranslated region. Therefore, to determine the 3Ј end of the 5-kb mST8Sia IV/PST transcript, the mouse brain cDNA library was screened by PCR using primers distributed along the 3Ј part of the gene. Sequence analyses of the PCR products revealed that the size of the transcribed RNA was 6786 bp, thus it included a large 3Јuntranslated region of 5372 bp. Poly(A) addition occurred 23 nucleotides downstream of the sequence at the T residue of the polyadenylation signal (AATAAA).
Mapping of the Transcription Initiation Site-The transcription initiation site was determined by primer extension with RNA recovered from 1-day-old mouse brain, in which the mST8Sia IV/PST gene was expressed (Fig. 2). Northern blot analysis indicated that the mST8Sia IV/PST gene gave a single transcript, whose size was about 5 kb. The primer extension products obtained with primer O5-EX2 were analyzed on a 6% sequencing gel. The end points of the extension were determined by comparison with a sequence ladder derived from the same genomic DNA template and the original primer, O5-EX2. The end point was determined to be a guanine (ϩ1), which corresponded to a position 333 nucleotides upstream from the initiation codon, ATG. Moreover, we performed 5Ј-RACE-PCR on newborn mouse brain poly(A) RNA to identify the 5Ј end of the mST8Sia IV/PST gene, and the longest RACE-PCR product corresponded to the transcription initiation site determined in the primer extension experiments. Therefore, mST8Sia IV/PST mRNA was transcribed at a single position 333 nucleotides upstream from the initiation codon at least in mouse brain and gave a single transcript.
Analysis of the 5Ј-Flanking Region-Analysis of the sequence immediately upstream of the transcription initiation site re-vealed that the mST8Sia IV/PST gene promoter consists of a G ϩ C-rich sequence lacking a canonical TATA box (Fig. 3). In this promoter, an apparent G ϩ C-rich region extends from Ϫ100 to ϩ150 (GC content, 64%). The G ϩ C-rich region of the mST8Sia IV/PST gene promoter is shorter and its GC content is lower than those of the mST8Sia II/STX gene promoter (nt Ϫ175 to ϩ168, 74%) (18). The TATA-less mST8Sia IV/PST gene promoter contains an inverted Sp1 binding site at positions Ϫ66 to Ϫ57 (8 of 10 matching), an inverted NF-Y (CCAAT binding protein) binding site at positions Ϫ47 to Ϫ37 (9 of 11 matching), and an AP-2 binding site, CC(G/C)C(A/G)GGC, at positions ϩ84 to ϩ91 (7 of 8 matching). The 1-kb 5Ј-flanking sequence of the mST8Sia IV/PST gene does not exhibit extensive homology with the upstream region of the mST8Sia II/STX gene.
Demonstration of Promoter Activity-To characterize the regions regulating the transcription activity of the gene, we constructed a series of chimeric plasmids containing different lengths of the 5Ј-flanking region of the mST8Sia IV/PST gene fused to the promoterless luciferase gene in pPGBII (Fig. 4). One of the constructs, pBO5-NhN3.5, was assayed for promoter activity by transient transfection into several cell lines at first FIG. 2. Identification of the transcription initiation sites of the mST8Sia IV/PST gene of mouse brain. The transcription initiation site has been mapped by means of primer extension analysis. For the primer extension reaction, the O5-EX2 primer was [␥-32 P]ATP-endlabeled, hybridized with 5 g of poly(A) RNA from 1-day-old mouse brain or 5 g of yeast tRNA, and then reverse-transcribed. The primer extended products were run on a sequencing gel along with a sequencing reaction of pO5-22E1.5, using O5-EX2 as the primer. Lane 1, primer extension with yeast tRNA; lane 2, with 1-day-old mouse brain mRNA. The arrow indicates the position of the transcription initiation site.

TABLE II Exon/intron junctions of the mST8Sia IV/PST gene
The nucleotide sequences at the intron (lowercase letters) and exon (uppercase letters) junctions are shown. The derived amino acid sequence is displayed below the nucleotide sequence. Exons are numbered from the 5Ј end, as described in Fig. 1 (Table III). Of these cell lines, embryonal carcinoma P19 cells showed the highest promoter activity, neuroblastoma F11 cells showed a moderate level of promoter activity, and NIH3T3 fibroblast cells showed a very low level of promoter activity. The level of endogenous mST8Sia IV/PST gene expression in P19 cells was similar to that in F11 cells, but NIH3T3 cells do not express the mST8Sia IV/PST gene at all (data not shown). Thus, we decided to use P19, F11, and NIH3T3 cells for the following study for comparison of the promoter activities. Sequential deletions of the region between nucleotide positions Ϫ3140 and Ϫ541 had little effect on the luciferase activity. Further deletions from nucleotide positions Ϫ541 to Ϫ200 increased the promoter activity in differentiated P19 cells as well as in undifferentiated P19 and F11 cells. Although further deletions from nucleotide positions Ϫ200 to Ϫ107 (pBO5-XN0.44) had little effect on the activity, deletions from nucleotide positions Ϫ200 to Ϫ15 (pBO5-NhN0.35) caused a drastic decrease in the promoter activity from one-fourth to one-fifth that of pBO5-XN0.44 in the examined cells. On further truncation beyond the transcriptional initiation site to nucleotide position ϩ26, the promoter activity of pBO5-XN0.31 was reduced to the basal level. Deletion to nucleotide position ϩ173 reduced the promoter activity to the level seen with the pro-moterless control vector, pPGBII. On the other hand, truncation from nucleotide positions ϩ333 to ϩ145 (pBO5-XH0.25) had little effect. All the constructs exhibited barely detectable activity in NIH3T3 cells. Taken together, we concluded that the region between Ϫ107 and ϩ145 was responsible for high levels of expression in mST8Sia IV/PST mRNA-expressing cells. We have shown that P19 cells express the endogenous mST8Sia IV/PST gene and that the level of the gene expression slightly increases during neural differentiation on retinoic acid treatment (24). It should be noted that the promoter activities due to most of the constructs were approximately 1.5-fold higher than those in undifferentiated P19 cells. This observation correlates with the mST8Sia IV/PST mRNA expression in P19 cells during neural differentiation.
Mapping of the Functional Regions in mST8Sia IV/PST Transcription- Figs. 3 and 4 indicate the presence of a putative Sp1 binding site (inverted form, nt Ϫ66 to Ϫ57), a putative NF-Y binding site (inverted form, nt Ϫ47 to Ϫ37), and a putative AP-2 site (nt ϩ84 to ϩ91) within pBO5-XN0.53, which gave the maximum transcriptional activity. To examine the involvement of the putative Sp1, NF-Y, and AP-2 binding sites in mST8Sia IV/PST mRNA transcription, we first constructed two internal deletion mutants of pBO5-XN0.53, one of which lacked the inverted Sp1 binding site and its upstream sequence (nt Ϫ75 to Ϫ56; pBO5-XN0.53 (⌬Ϫ75/Ϫ56)), and the other lacked the Sp1 binding site and its downstream sequence (nt Ϫ60 to Ϫ40; pBO5-XN0.53 (⌬ Ϫ60/Ϫ40), in which the inverted NF-Y binding site was also deleted) (Fig. 5). In addition, we introduced a mutation into the inverted Sp1 binding site by replacing four nucleotides (pBO5-XN0.53 (Sp1*); cctccgcccc changed to cctccgaatt), which failed to bind to recombinant Sp1 (data not shown). The deletion upstream of the inverted Sp1 binding site reduced the promoter activity to 30 -40% (construct pBO5-XN0.53 (⌬Ϫ75/Ϫ56)) as compared with that of the wild-type construct. The promoter activity of the Sp1-mutated construct (pBO5-XN0.53 (Sp1*)) was also reduced to 40 -50% (Fig. 5). In contrast, the deletion downstream of the inverted Sp1 binding site (construct pBO5-XN0.53 (⌬Ϫ60/Ϫ40)) caused a drastic decrease in the promoter activity to the basal level in all cells examined (Fig. 5). A mobility shift experiment involving a nuclear protein extract of P19 cells revealed that only one shifted band (Fig. 6, band C) disappeared in the presence of the synthetic DNA fragment corresponding to nucleotide positions Ϫ62 to Ϫ33, suggesting the nuclear protein of P19 cells bound in this region (Fig. 6, lane 5).
To clarify the involvement downstream of the Sp1 binding site, we constructed a series of internal deletion mutants of pBO5-XN0.53 and analyzed their promoter activities. Each 3-base deletion from nucleotide positions Ϫ53 to Ϫ45 of pBO5-XN0.53 (constructs pBO5-XN0.53 (⌬Ϫ53/Ϫ51), pBO5-XN0.53 (⌬Ϫ50/Ϫ48), and pBO5-XN0.53 (⌬Ϫ47/Ϫ45)) had little effect on the promoter activity (Fig. 5). In contrast, the deletion of the sites from Ϫ41 to Ϫ39 and from Ϫ39 to Ϫ37 (constructs pBO5-XN0.53 (⌬Ϫ41/Ϫ39) and pBO5-XN0.53 (⌬Ϫ39/Ϫ37)) led to a reduction of the promoter activity to 42% (in the case of F11 cells), 15% (in the case of undifferentiated P19 cells), and 20% (in the case of differentiated P19 cells) as compared with that of the wild-type construct (Fig. 5). Deletion of the site from Ϫ44 to Ϫ42 moderately reduced the promoter activity to 50 -70% that of the wild-type construct. Therefore, the site from Ϫ44 to Ϫ37, corresponding to the inverted NF-Y binding site, was required for the maximal promoter activity in both differentiated and undifferentiated P19 cells and F11 cells. Since the deletion of the Ϫ60/Ϫ40 site reduced the promoter activity to the basal level, but the deletion of the Ϫ75/Ϫ56, Ϫ41/Ϫ39, or Ϫ39/Ϫ37 site reduced it only partly, both the inverted Sp1 binding site at Ϫ65/Ϫ57 and the inverted NF-Y binding site at Ϫ44/Ϫ37 are critical for the function of the promoter in these cells.
The mutation of the AP-2 binding site (pBO5-XN0.53 (Ap2*)) had little effect on the promoter activity. On the other hand, the deletion of the region between nucleotide positions ϩ42 and ϩ87 increased the promoter activity (Fig. 5).
Involvement of Sp1 and NF-Y in Transcription of the mST8Sia IV/PST Gene-To determine whether or not the inverted Sp1 binding site between nucleotide positions Ϫ65 and Ϫ57 is recognized by Sp1, we performed a mobility shift assay. In the mobility shift experiments involving the DNA fragment from Ϫ107 to Ϫ16, recombinant Sp1 bound to the DNA fragment (Fig. 6, lane 7). When a nuclear protein extract of undifferentiated P19 cells was used for the experiment, the labeled DNA fragment appeared as several shifted bands (Fig.  6, bands A-D). These shifted bands were not abolished by the nonspecific competitor (DNA fragment from Ϫ339 to Ϫ221) but completely disappeared in the presence of the non-labeled spe-cific competitor (DNA fragment from Ϫ107 to Ϫ16, the same as the labeled probe), indicating that the nuclear extract of P19 cells contained some proteins that specifically bind to this fragment. The corresponding band, which was observed in the presence of recombinant Sp1, appeared (lane 2) in the presence of the nuclear protein extract of P19 cells (band B). This shifted band disappeared on the addition of the anti-Sp1 polyclonal antibodies (Fig. 6, lane 6). The same results were obtained with nuclear protein extracts of differentiated P19 cells and F11 cells. Thus, the inverted Sp1 site at Ϫ65/Ϫ57 was functional in the examined cells.
Since the inverted CCAAT motif, which corresponds to the NF-Y binding site, was included in the site from Ϫ44 to Ϫ37, we analyzed whether or not this motif was recognized by NF-Y. In the mobility shift experiment involving the DNA fragment from Ϫ107 to Ϫ16, NF-Y translated in vitro bound to the DNA fragment (Fig. 7, lane 3), whose mobility corresponded to that of band C observed when a nuclear extract of P19 cells was used (Fig. 6, lane 2, and Fig. 7, lane 2). The shifted band was not abolished by the nonspecific competitor (DNA fragment from Ϫ339 to Ϫ221) but completely disappeared in the presence of the non-labeled specific competitor (synthetic DNA fragment from Ϫ62 to Ϫ33; Fig. 6, lane 5, and Fig. 7, lane 5). Thus, the inverted CCAAT motif at Ϫ44/Ϫ37 was recognized by NF-Y. These results suggested that Sp1 and NF-Y are involved in the transcription of mST8Sia IV/PST mRNA. DISCUSSION We recently reported the genomic organization and promoter activity of the mST8Sia II/STX gene, a PSA synthase gene, whose expression is highly regulated during brain development (19). In the present study, we showed that the genomic organization of the mST8Sia IV/PST gene, another PSA synthase gene, is highly similar to that of the mST8Sia II/STX gene, whereas the sequence of the 5Ј-flanking region of the mST8Sia IV/PST gene does not exhibit extensive homology with the upstream region of the mST8Sia II/STX gene. We showed that TABLE III Relative promoter activities of several types of cells pBO5-NhN3.5 was used to assay the promoter activity as luciferase activity. Luciferase activity was normalized as to the ␤-galactosidase activity of a cotransfected internal control plasmid, pSR␤-Gal, and expressed as a percentage of the SV40 promoter activity in that cell type.

FIG. 4. mST8Sia IV/PST gene promoter activity and identification of the regulatory regions.
A schematic representation of DNA constructions containing various lengths of the mST8Sia IV/PST promoter linked to the luciferase gene is presented. Each DNA fragment subcloned into the luciferase reporter plasmid is defined to its position in the mST8Sia IV/PST gene promoter relative to the transcription initiation site (ϩ1). 5 g of each construct was transfected into NIH3T3, F11 (F11), undifferentiated P19 (P19), or neural-differentiated P19 (P19(Dif)) cells. Luciferase activity was normalized to the ␤-galactosidase activity of a cotransfected internal control plasmid, pSR␤-Gal, and expressed as a percentage of the SV40 promoter activity in that cell type. Each datum is the average of four or five experiments. the proximal promoter region of the mST8Sia IV/PST gene has the ability to express the transcriptional activity, which correlated with the endogenous mST8Sia IV/PST gene expression in several cell lines.
So far, the genomic organizations of six other sialyltransferase genes have been reported (19,20,(25)(26)(27)(28)(29). Among them, the genomic structures of the mST8Sia II/STX gene is fairly similar to that of the mST8Sia IV/PST gene. In particular, three introns are inserted into the regions coding for the putative active domains of the enzymes, mST8Sia II/STX and IV/PST (Fig. 8A). The entire amino acid sequence of mST8Sia IV/PST shows 56% identity with that of mST8Sia II/STX, and its putative active domain exhibits higher similarity to that of mST8Sia II/STX (Fig. 8B). However, the amino acid sequences of exons 1 and 2 in mST8Sia IV/PST are not conserved in the corresponding exons of mST8Sia II/STX. On the other hand, both the genomic organization and the amino acid sequence of the mST8Sia IV/PST gene showed no similarity, except in the sialyl motifs, to other known ␣2,3and ␣2,6-sialyltransferase genes. These observations suggest that the mST8Sia II/STX and IV/PST genes are evolutionarily related and distant from other sialyltransferase genes.
In this study, we mapped a highly active promoter region of the mST8Sia IV/PST gene by transient transfection of a series of deleted promoter sequences in P19 and F11 cells (Fig. 4). Deletion analyses demonstrated that the promoter sequence from Ϫ107 to ϩ15 is critical for the function of the promoter, because its removal effectively reduced the reporter gene expression. The promoter activity of the pBO5-XN0.53 construct in F11, P19, and NIH3T3 cells was correlated with the endogenous gene expression in each cell line. The proximal promoter region of the mST8Sia IV/PST gene may be capable of directing specific expression of the gene. By using an in vitro neural differentiation model system with P19 cells, we previously revealed that the promoter activity of the mST8Sia II/STX gene (nt Ϫ158 to ϩ167) was low in undifferentiated P19 cells but that it increased about 10 times during neuronal differentiation. However, in the case of the promoter region of the mST8Sia IV/PST gene, a drastic increase in the activity was not observed during the differentiation. Therefore the proximal promoter regions of both mST8Sia II/STX and IV/PST are differently regulated during at least P19 cell differentiation. In fact, mST8Sia II/STX mRNA expression increased 20-fold during differentiation, but mST8Sia IV/PST mRNA expression increased only a few times (24). Therefore, the proximal promoter regions may possess specific regulatory elements.
The proximal promoter region of the mST8Sia IV/PST gene contained an inverted Sp1 binding site (nt Ϫ64 to Ϫ57) and an FIG. 5. mST8Sia IV/PST gene promoter activity on mutant analysis. A schematic representation of pO5-XN0.53 mutants with deletion or mutation of the Sp1, NF-Y, and AP-2 binding sites in the mST8Sia IV/PST promoter, respectively. The sequences of the putative inverted Sp1 binding site (Sp1; nt Ϫ66 to Ϫ57), the putative inverted NF-Y binding site (NF-Y; nt Ϫ47 to Ϫ37), and the putative AP-2 binding site (AP-2; nt ϩ84 to ϩ91) are shown in the figure. The deleted parts are indicated by the notch marks. The mutational sequences are shown as underlined italics. The relative promoter activity was measured as luciferase activity, which was normalized to the ␤-galactosidase activity of a cotransfected internal control plasmid, pSR␤-Gal. The values are presented as percentages of the promoter activity due to pO5-XN0.53, from which was subtracted the basal activity due to pO5-XN0. 31. The activities of the promoter of pO5-XN0.53 relative to those of pBSV in F11, undifferentiated P19, and differentiated P19 cells were 93.6 Ϯ 13.6, 109.1 Ϯ 13.4, and 126.3 Ϯ 10.9%, respectively. The basal activities due to pO5-XN0.31 relative to those of pBSV in F11, undifferentiated P19, and differentiated P19 cells were 15.0 Ϯ 3.7, 21.4 Ϯ 1.4, and 19.5 Ϯ 5.2%, respectively.
inverted CCAAT motif (NF-Y binding site, nt Ϫ47 to Ϫ37). A mobility shift assay showed that the inverted Sp1 binding site was functional in the examined cells (Fig. 6). The results of deletion and mutation of the Sp1 binding site suggested that the Sp1 binding site is partly involved in transcription regulation in P19 and F11 cells (Fig. 5). We also showed the involvement of NF-Y in the transcriptional regulation, NF-Y binding to the inverted CCAAT motif located in the region from Ϫ44 to Ϫ37 in mobility shift assays (Fig. 7). It should be noted that deletion of the Ϫ60 to Ϫ40 region (pBO5-XN0.53(⌬Ϫ60/Ϫ40)) abolished the promoter activity almost completely, whereas deletion of either the inverted Sp1 site (Ϫ64/Ϫ57) or the inverted NF-Y site (Ϫ44/Ϫ37) reduced the promoter activity only partly (about 40% as compared with the wild type), and deletion of the site from Ϫ53 to Ϫ45 had little effect on the promoter activity. Thus, the two different sites, the inverted Sp1 site and the inverted NF-Y binding site, are required for the promoter activity in P19 and F11 cells. Probably, the synergetic effect of Sp1 and NF-Y is essential for the transcription of mST8Sia IV/PST mRNA. Sp1 and NF-Y are thought to be ubiquitous transcription factors. In fact, NIH3T3 cells express Sp1 and NF-Y at almost the same levels to P19 and F11 cells (data not shown), although the promoter activity in NIH3T3 cells is very low. Therefore, the minimal promoter region identified in this study seems to be an essential transcription unit, and some other transcription factors may be involved in the specific promoter activity in P19 and F11 cells. The mobility shift experiment indicated the occurrence of other nuclear proteins that specifically bind to the proximal promoter region of mST8Sia IV/PST. This may suggest the existence of other sites that are required for the transcriptional regulation of the mST8Sia IV/PST gene in the proximal promoter region. However, we could not identify such additional sites at this stage. Identification of such sites is required. We recently demonstrated that the minimal promoter region of the mST8Sia II/PST gene conferred cell type-specific expression in the reporter gene. The minimal promoter was embedded in a GC-rich region (GC content, 74%), in which two Sp1 binding motifs as well as a long purine-rich region were found, but it lacked TATA and CAAT boxes. Comparison of the promoter regions of the mST8Sia II/STX and IV/PST genes revealed no extensive sequence homology (Fig. 9). However, both the promoters of these two genes have functional Sp1 binding site(s) but lack canonical TATA boxes. This type of promoter is usually associated with housekeeping genes but has also been found in a number of tissue-specific genes, including the neural cellspecific promoters of the neuron-specific enolase, type II sodium channel, syanpsins I and II, and D 1A dopamine receptor genes (30). Although Sp1 binding sites are found in the proximal promoter regions of the mST8Sia II/STX and IV/PST genes, NF-Y binding sites are not found in the proximal promoter region of the mST8Sia II/STX gene (Ϫ158/ϩ167). In addition to the difference that NF-Y is involved in the regulation of the expression of the mST8Sia IV/PST gene, but not in that of the mST8Sia II/STX gene, identification of other factors that interact with the proximal promoter regions of the mST8Sia II/STX and IV/PST genes may facilitate understanding of the differential regulation of the two genes. For example, there is a putative cAMP-responsive element-binding protein binding site in the proximal promoter regions of both genes. Now, we are trying to identify regulatory factors, including cAMP-responsive element-binding protein, that interact with the proximal promoter regions of the mST8Sia II/STX and IV/PST genes.