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

Abstract

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. 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) 1The abbreviations used are: PSA, polysialic acid; N-CAM, neural cell adhesion molecule; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; nt, nucleotide(s); the abbreviated nomenclature for cloned sialyltransferases follows the system of Tsuji et al.(31Tsuji S. Datta A.K. Paulson J.C. Glycobiology. 1996; 6: v-viiCrossref PubMed Google Scholar). is a linear homopolymer of α2,8-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 (1Troy F.A. Glycobiology. 1992; 2: 5-23Crossref PubMed Scopus (322) Google Scholar). In the late embryonic and early postnatal stages, neurons mainly express the highly polysialylated form of N-CAM (2Finne J. Finne U. Deagpstomo-Bazin H. Goridis C. Biochem. Biophys. Res. 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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 (6Cremer H. Lange R. Christoph A. Plomann M. Vopper G. Roes J. Brown R. Baldwin S. Kraemer P. Scheff S. Barthels D. Rajewsky K. Wille W. Nature. 1994; 367: 455-459Crossref PubMed Scopus (911) Google Scholar, 7Tomasiewicz H. Ono K. Yee D. Thompson C. Goridis C. Rutishauser U. Magnuson T. Neuron. 1993; 11: 1163-1174Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 8Takamatsu K. Auerbach B. Gerardy S.R. Eckhardt M. Jaques G. Madry N. Cancer Res. 1994; 54: 2598-2603PubMed Google Scholar). In 1995, Eckhardt et al. (9Eckhardt M. Muhlenhoff M. Bethe A. Koopma J. Gerardy-Schahn M.F.R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar) 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 α2,8-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 (10Yoshida Y. Kojima N. Tsuji S. J. Biochem. (Tokyo). 1995; 118: 658-664Crossref PubMed Scopus (80) Google Scholar). On the other hand, we demonstrated that ST8Sia II/STX is another PSA synthase (12Kojima N. Yoshida Y. Kurosawa N. Lee Y.C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar, 13Kojima N. Yoshida Y. Tsuji S. FEBS Lett. 1995; 373: 119-122Crossref PubMed Scopus (79) Google Scholar, 14Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 16Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 17Kojima N. Tachida Y. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). 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 (10Yoshida Y. Kojima N. Tsuji S. J. Biochem. (Tokyo). 1995; 118: 658-664Crossref PubMed Scopus (80) Google Scholar, 12Kojima N. Yoshida Y. Kurosawa N. Lee Y.C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar). Expression of the mST8Sia II/STX gene in the brain was strictly regulated during development (12Kojima N. Yoshida Y. Kurosawa N. Lee Y.C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar). 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 (10Yoshida Y. Kojima N. Tsuji S. J. Biochem. (Tokyo). 1995; 118: 658-664Crossref PubMed Scopus (80) Google Scholar). Our recent results indicated that mST8Sia II/STX and IV/PST synthesize PSA of different sizes in vitro and in vivo (17Kojima N. Tachida Y. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 18Kojima N. Tachida Y. Tsuji S. J. Biochem. (Tokyo). 1997; 122: 1265-1273Crossref PubMed Scopus (32) Google Scholar). 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 (19Yoshida Y. Kurosawa N. Kanematsu T. Kojima N. Tsuji S. J. Biol. Chem. 1996; 271: 30167-30173Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). 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 GC-rich 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 In the we the genomic structure of the mST8Sia IV/PST gene and its promoter sequence involved in the regulation of transcription mouse genomic was and as Y. Kurosawa N. Kanematsu T. A. M. Kojima N. Tsuji S. Glycobiology. 1996; 6: PubMed Scopus Google Scholar). The of the of the mST8Sia IV/PST gene were with or cDNA to the The region of the mST8Sia IV/PST cDNA was isolated from a mouse brain cDNA Y. Kurosawa N. Kanematsu T. A. M. Kojima N. Tsuji S. Glycobiology. 1996; 6: PubMed Scopus Google Scholar) and used in to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to in a of the of mST8Sia IV/PST cDNA was as Y. Kurosawa N. Kanematsu T. A. M. Kojima N. Tsuji S. Glycobiology. 1996; 6: PubMed Scopus Google Scholar). cDNA was transcription of of mouse brain a to mST8Sia IV positions to the cDNA two were with two nested of the and the was the was as but the and the was The cDNA was for of a and 60 The were and was a from which contains the 5′-flanking region of the mST8Sia IV/PST the The was with The was with of from mouse brain or of as a to extension and with as (19Yoshida Y. Kurosawa N. Kanematsu T. Kojima N. Tsuji S. J. Biol. Chem. 1996; 271: 30167-30173Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The extension were a with a chain of as the To of the 5′-flanking region of the mST8Sia IV with polymerase was two and a with as the The mST8Sia gene expression were the from the a and a respectively. of deletion were the the of and the enzyme site The and used were as a the of a the of a the of a the of and a the of with as the the a from was with the the luciferase gene the and the luciferase were of each cell The luciferase activity to each luciferase reporter was to the activity an a gene under the of the In the cell was to the expression of luciferase activity, whereas caused a of and cells were at cells in with fetal to respectively. the neural of P19 cells neuronal cells, the cells were and in in the of acid at the cell of the were and were in fetal to The luciferase used as the reporter and the used as an for were the cells of the cells were with and with cell activity was a and a activity were in and to the activity to for the activity was a The used for the of mutants and deletion mutants of the and binding sites are in were as the The Sp1 binding was with the of and an with the of The Sp1 binding and were as was with the of and an with the of was with the of and an with the of of NF-Y binding and was the the of and was two with the of and as for the amplification in the of the of for and were as the The binding was with the of and an with the of The binding was an with the of and a with the of were and The gene was cloned from P19 cells and D. H. C. D. J. PubMed Scopus Google Scholar). were cloned and the gene D. H. C. D. J. PubMed Scopus Google Scholar) was cloned from an adult mouse brain cDNA and In of the and genes was an to the The resulting were to a system for in vitro The from to was from with and and with were with a in the of of and of a nuclear extract or an of the in were for at in and or were The from to which was from and and the from to were used as and respectively. The positions from to and to nucleotide positions from −60 to were used as the two the were a in The was in the at and and the was with a The of an cell with mST8Sia IV/PST cDNA in the of genomic of the region the mST8Sia IV/PST gene is in The of the mST8Sia IV/PST were and blot a of to the mST8Sia IV/PST cDNA indicated that and not of genomic from to in was with a of and a of the of as However, no were blot of the genomic the two showed that the between the of and was over not We the to their sizes and the The sequences of the to the P. Res. PubMed Scopus Google Scholar). The mST8Sia IV/PST cDNA was from to bp, with sizes of kb, and than 60 of genomic the entire region and the of the region to amino acid a a and a of the the putative domain of the and a large We reported mST8Sia IV/PST cDNA sequences the to the of the mST8Sia IV/PST the mouse brain cDNA was the of the gene. of the revealed that the of the was bp, it a large region of nucleotides of the sequence at the of the of the mST8Sia IV/PST in a The transcription initiation site was extension with from mouse in which the mST8Sia IV/PST gene was expressed Northern blot analysis indicated that the mST8Sia IV/PST gene a whose was The extension with were a The of the extension were with a sequence from the genomic and the The was to be a which to a 333 nucleotides upstream from the initiation we mouse brain to the of the mST8Sia IV/PST and the to the transcription initiation site in the extension mST8Sia IV/PST was at a 333 nucleotides upstream from the initiation at in mouse brain and a of the sequence upstream of the transcription initiation site revealed that the mST8Sia IV/PST gene promoter of a sequence a TATA box In an apparent region from to (GC content, The region of the mST8Sia IV/PST gene promoter is and its content is than of the mST8Sia II/STX gene promoter to N. Tachida Y. Tsuji S. J. Biochem. (Tokyo). 1997; 122: 1265-1273Crossref PubMed Scopus (32) Google Scholar). The mST8Sia IV/PST gene promoter contains an inverted Sp1 binding site at positions to of an inverted NF-Y (CCAAT binding protein) binding site at positions to of and an binding at positions to of The 5′-flanking sequence of the mST8Sia IV/PST gene not exhibit extensive homology with the upstream region of the mST8Sia II/STX gene. To the regions the transcription activity of the we a of different of the 5′-flanking region of the mST8Sia IV/PST gene fused to the luciferase gene in of the was for promoter activity cell at these cell embryonal carcinoma P19 cells showed the highest promoter activity, neuroblastoma F11 cells showed a of promoter activity, and cells showed a very of promoter The of mST8Sia IV/PST gene expression in P19 cells was to that in F11 cells, but cells not express the mST8Sia IV/PST gene at not we to and cells for the for of the promoter promoter activities of types of promoter in a deletions of the region between nucleotide positions and the luciferase deletions from nucleotide positions to the promoter activity in P19 cells as well as in P19 and F11 cells. Although deletions from nucleotide positions to the activity, deletions from nucleotide positions to caused a in the promoter activity from to that of in the cells. On the initiation site to nucleotide the promoter activity of was to the Deletion to nucleotide the promoter activity to the with the On the other hand, from nucleotide positions to the activity in cells. we that the region between and was for of expression in mST8Sia IV/PST cells. We that P19 cells express the mST8Sia IV/PST gene and that the of the gene expression during neural acid N. M. Yoshida Y. Tachida Y. M. Tsuji S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). be that the promoter activities to of the were higher than in P19 cells. This with the mST8Sia IV/PST expression in P19 cells during neural and the of a putative Sp1 binding site to a putative NF-Y binding site to and a putative site to which the To the of the putative and binding sites in mST8Sia IV/PST we two deletion mutants of one of which lacked the inverted Sp1 binding site and its upstream sequence to and the other lacked the Sp1 binding site and its sequence −60 to in which the inverted NF-Y binding site was also In we a the inverted Sp1 binding site nucleotides to which to bind to Sp1 not The deletion upstream of the inverted Sp1 binding site the promoter activity to as compared with that of the The promoter activity of the was also to In the deletion of the inverted Sp1 binding site caused a in the promoter activity to the in cells shift a nuclear extract of P19 cells revealed that one in the of the to nucleotide positions to suggesting the nuclear of P19 cells in region shift of the mST8Sia IV/PST proximal promoter region with a nuclear extract of P19 cells. The from to was with the nuclear extract of P19 cells or with the of the from to the of the from to and of the from to the and to the shift the results of a shift of Sp1 of the nuclear extract of P19 To the of the Sp1 binding we a of deletion mutants of and their promoter deletion from nucleotide positions to of and the promoter activity In the deletion of the sites from to and from to and to a reduction of the promoter activity to the of F11 the of P19 and the of P19 as compared with that of the Deletion of the site from to the promoter activity to that of the the site from to to the inverted NF-Y binding was for the promoter activity in both and P19 cells and F11 cells. the deletion of the site the promoter activity to the but the deletion of the or site it both the inverted Sp1 binding site at and the inverted NF-Y binding site at are for the of the promoter in these cells. The of the binding site the promoter On the other hand, the deletion of the region between nucleotide positions and the promoter activity To or not the inverted Sp1 binding site between nucleotide positions and is we a shift In the shift the from to Sp1 to the a nuclear extract of P19 cells was used for the the as were not abolished the from to but completely in the of the from to the as the indicating that the nuclear extract of P19 cells that bind to The which was in the of in the of the nuclear extract of P19 cells This the of the The results were with nuclear of P19 cells and F11 cells. the inverted Sp1 site at was in the cells. the inverted which to the NF-Y binding was in the site from to we or not was In the shift the from to NF-Y in vitro to the whose to that of a nuclear extract of P19 cells was used and The was not abolished the from to but completely in the of the from to and the inverted at was results that Sp1 and NF-Y are involved in the transcription of mST8Sia IV/PST We recently reported the genomic organization and promoter activity of the mST8Sia II/STX a PSA synthase whose expression is highly regulated during brain development (19Yoshida Y. Kurosawa N. Kanematsu T. Kojima N. Tsuji S. J. Biol. Chem. 1996; 271: 30167-30173Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In the we showed that the genomic organization of the mST8Sia IV/PST another PSA synthase is highly to that of the mST8Sia II/STX whereas the sequence of the 5′-flanking region of the mST8Sia IV/PST gene not exhibit extensive homology with the upstream region of the mST8Sia II/STX gene. We showed that the proximal promoter region of the mST8Sia IV/PST gene has the ability to express the activity, which correlated with the mST8Sia IV/PST gene expression in cell the genomic of other genes reported (19Yoshida Y. Kurosawa N. Kanematsu T. Kojima N. Tsuji S. J. Biol. Chem. 1996; 271: 30167-30173Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, Y. Kurosawa N. Kanematsu T. A. M. Kojima N. Tsuji S. Glycobiology. 1996; 6: PubMed Scopus Google Scholar, B. Paulson J.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Glycobiology. PubMed Scopus (80) Google Scholar, Glycobiology. 1995; PubMed Scopus Google Scholar, H. Paulson J.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, N. M. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the genomic structures of the mST8Sia II/STX gene is to that of the mST8Sia IV/PST gene. In are the regions for the putative of the mST8Sia II/STX and IV/PST The entire amino acid sequence of mST8Sia IV/PST 56% identity with that of mST8Sia and its putative domain exhibits higher to that of mST8Sia II/STX However, the amino acid sequences of and in mST8Sia IV/PST are not in the of mST8Sia On the other hand, both the genomic organization and the amino acid sequence of the mST8Sia IV/PST gene showed no in the to other and that the mST8Sia II/STX and IV/PST genes are and from other In we a highly promoter region of the mST8Sia IV/PST gene of a of promoter sequences in P19 and F11 cells Deletion demonstrated that the promoter sequence from to is for the of the its the reporter gene The promoter activity of the in and cells was correlated with the gene expression in each cell The proximal promoter region of the mST8Sia IV/PST gene be of expression of the gene. an in vitro neural system with P19 cells, we revealed that the promoter activity of the mST8Sia II/STX gene to was in P19 cells but that it during neuronal However, in the of the promoter region of the mST8Sia IV/PST a increase in the activity was not during the the proximal promoter regions of both mST8Sia II/STX and IV/PST are differently regulated during at P19 cell In mST8Sia II/STX expression during but mST8Sia IV/PST expression a N. M. Yoshida Y. Tachida Y. M. Tsuji S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the proximal promoter regions regulatory The proximal promoter region of the mST8Sia IV/PST gene an inverted Sp1 binding site to and an inverted binding to shift showed that the inverted Sp1 binding site was in the cells The results of deletion and of the Sp1 binding site that the Sp1 binding site is involved in transcription regulation in P19 and F11 cells We also showed the of NF-Y in the NF-Y binding to the inverted in the region from to in shift be that deletion of the −60 to region abolished the promoter activity whereas deletion of the inverted Sp1 site or the inverted NF-Y site the promoter activity as compared with the and deletion of the site from to the promoter the two different sites, the inverted Sp1 site and the inverted NF-Y binding are for the promoter activity in P19 and F11 cells. the of Sp1 and NF-Y is essential for the transcription of mST8Sia IV/PST Sp1 and NF-Y are to be transcription In cells express Sp1 and NF-Y at the to P19 and F11 cells not the promoter activity in cells is very the minimal promoter region in to be an essential transcription and other transcription factors be involved in the promoter activity in P19 and F11 cells. The shift indicated the of other nuclear that bind to the proximal promoter region of mST8Sia This the of other sites that are for the regulation of the mST8Sia IV/PST gene in the proximal promoter However, we not sites at of sites is We recently demonstrated that the minimal promoter region of the mST8Sia 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 of the promoter regions of the mST8Sia II/STX and IV/PST genes revealed no extensive sequence homology However, both the of these two genes Sp1 binding but TATA This of promoter is associated with genes but has also found in a of genes, the neural of the and and genes J. 1995; PubMed Scopus Google Scholar). 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 In to the that NF-Y is involved in the regulation of the expression of the mST8Sia IV/PST but not in that of the mST8Sia II/STX of other factors that with the proximal promoter regions of the mST8Sia II/STX and IV/PST genes of the differential regulation of the two there is a putative binding site in the proximal promoter regions of both we are to regulatory that with the proximal promoter regions of the mST8Sia II/STX and IV/PST We are to of the and of the of the of and for their and