Abstract
Recently, we demonstrated that the human xylosyltransferase II (XT-II) has enzymatic activity and is able to catalyze the initial and rate-limiting step in the biosynthesis of glycosaminoglycans (GAGs) like chondroitin and dermatan sulfate, as well as heparan sulfate and heparin. Therefore, this enzyme also very likely assumes a crucial regulatory role in the biosynthesis of proteoglycans (PGs). In this study, we identified and characterized for the first time the XYLT2 gene promoter region and transcription factors involved in its regulation. Several binding sites for members of the Sp1 family of transcription factors were identified as being necessary for transcriptional regulation of the XYLT2 gene. This was determined by mithramycin A treatment, electrophoretic mobility shift and supershift assays, as well as numerous site-directed mutagenesis experiments. Different 5′ and 3′ deletion constructs of the predicted GC rich promoter region, which lacks a canonical TATA and CAAT box, revealed that a 177 nts proximal promoter element is sufficient and indispensable to drive the constitutive transcription in full strength in HepG2 hepatoma cells. In addition, we also detected the transcriptional start site using 5′-RACE (rapid amplification of cDNA ends). Our results provide an insight into transcriptional regulation of the XYLT2 gene and may contribute to understanding the manifold GAG-involving processes in health and disease.
Abbreviations
- AP-1/2:
-
Activator protein 1/2
- HNF4α:
-
Hepatocyte nuclear factor 4α
- Sp1/3:
-
Specificity protein 1/3
- XT-I/II:
-
Xylosyltransferase I/II (mRNA and protein)
- XYLT1/2:
-
Xylosyltransferases 1/2 (human gene)
- Xylt2:
-
Xylosyltransferases (murine gene)
- PGs:
-
Proteoglycans
- GAGs:
-
Glycosaminoglycans
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- EMSA:
-
Electrophoretic mobility shift assay
- RT-PCR:
-
Reverse transcriptase polymerase chain reaction
- 5′–RACE:
-
5′ rapid amplification of cDNA ends
- TFBS:
-
Transcription factor binding sites
References
Iozzo, R.V.: Matrix proteoglycans: from molecular design to cellular function. Annu. Rev. Biochem. 67, 609–652 (1998)
Iozzo, R.V., Danielson, K.G.: Transcriptional and posttranscriptional regulation of proteoglycan gene expression. Prog. Nucleic Acid Res. Mol. Biol. 62, 19–53 (1999)
Kjellen, L., Lindahl, U.: Proteoglycans: structures and interactions. Annu. Rev. Biochem. 60, 443–475 (1991)
Götting, C., Kuhn, J., Kleesiek, K.: Human xylosyltransferases in health and disease. Cell Mol. Life Sci. 64, 1498–1517 (2007)
Schön, S., Prante, C., Bahr, C., Kuhn, J., Kleesiek, K., Götting, C.: Cloning and recombinant expression of active full-length xylosyltransferase I (XT-I) and characterization of subcellular localization of XT-I and XT-II. J. Biol. Chem. 281, 14224–14231 (2006)
Kearns, A.E., Campbell, S.C., Westley, J., Schwartz, N.B.: Initiation of chondroitin sulfate biosynthesis: a kinetic analysis of UDP-D-xylose: core protein beta-D-xylosyltransferase. Biochemistry (Mosc). 30, 7477–7483 (1991)
Götting, C., Kuhn, J., Zahn, R., Brinkmann, T., Kleesiek, K.: Molecular cloning and expression of human UDP-d-Xylose:proteoglycan core protein beta-d-xylosyltransferase and its first isoform XT-II. J. Mol. Biol. 304, 517–528 (2000)
Pönighaus, C., Ambrosius, M., Casanova, J.C., Prante, C., Kuhn, J., Esko, J.D., Kleesiek, K., Götting, C.: Human xylosyltransferase II is involved in the biosynthesis of the uniform tetrasaccharide linkage region in chondroitin sulfate and heparan sulfate proteoglycans. J. Biol. Chem. 282, 5201–5206 (2007)
Cuellar, K., Chuong, H., Hubbell, S.M., Hinsdale, M.E.: Biosynthesis of chondroitin and heparan sulfate in chinese hamster ovary cells depends on xylosyltransferase II. J. Biol. Chem. 282, 5195–5200 (2007)
Condac, E., Silasi-Mansat, R., Kosanke, S., Schoeb, T., Towner, R., Lupu, F., Cummings, R.D., Hinsdale, M.E.: Polycystic disease caused by deficiency in xylosyltransferase 2, an initiating enzyme of glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 104, 9416–9421 (2007)
Roch, C., Kuhn, J., Kleesiek, K., Götting, C.: Differences in gene expression of human xylosyltransferases and determination of acceptor specificities for various proteoglycans. Biochem. Biophys. Res. Commun. 391, 685–691 (2009)
Quandt, K., Frech, K., Karas, H., Wingender, E., Werner, T.: MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res. 23, 4878–4884 (1995)
Blume, S.W., Snyder, R.C., Ray, R., Thomas, S., Koller, C.A., Miller, D.M.: Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J. Clin. Invest. 88, 1613–1621 (1991)
Condac, E., Dale, G.L., Bender-Neal, D., Ferencz, B., Towner, R., Hinsdale, M.E.: Xylosyltransferase II is a significant contributor of circulating xylosyltransferase levels and platelets constitute an important source of xylosyltransferase in serum. Glycobiology 19, 829–833 (2009)
Schön, S., Schulz, V., Prante, C., Hendig, D., Szliska, C., Kuhn, J., Kleesiek, K., Götting, C.: Polymorphisms in the xylosyltransferase genes cause higher serum XT-I activity in patients with pseudoxanthoma elasticum (PXE) and are involved in a severe disease course. J. Med. Genet. 43, 745–749 (2006)
Schön, S., Huep, G., Prante, C., Müller, S., Christ, R., Hagena, F.W., Kuhn, J., Kleesiek, K., Götting, C.: Mutational and functional analyses of xylosyltransferases and their implication in osteoarthritis. Osteoarthr. Cartil. 14, 442–448 (2006)
Hendig, D., Tarnow, L., Kuhn, J., Kleesiek, K., Götting, C.: Identification of a xylosyltransferase II gene haplotype marker for diabetic nephropathy in type 1 diabetes. Clin. Chim. Acta. 398, 90–94 (2008)
Schön, S., Prante, C., Müller, S., Schöttler, M., Tarnow, L., Kuhn, J., Kleesiek, K., Götting, C.: Impact of polymorphisms in the genes encoding xylosyltransferase I and a homologue in type 1 diabetic patients with and without nephropathy. Kidney Int. 68, 1483–1490 (2005)
Gardiner-Garden, M., Frommer, M.: CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282 (1987)
Butler, J.E., Kadonaga, J.T.: The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev. 16, 2583–2592 (2002)
Zhu, J., He, F., Hu, S., Yu, J.: On the nature of human housekeeping genes. Trends Genet. 24, 481–484 (2008)
Goto, T., Matsui, Y., Fernandes, R.J., Hanson, D.A., Kubo, T., Yukata, K., Michigami, T., Komori, T., Fujita, T., Yang, L., Eyre, D.R., Yasui, N.: Sp1 family of transcription factors regulates the human alpha2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells. J. Bone Miner. Res. 21, 661–673 (2006)
Huber, R., Schlessinger, D., Pilia, G.: Multiple Sp1 sites efficiently drive transcription of the TATA-less promoter of the human glypican 3 (GPC3) gene. Gene 214, 35–44 (1998)
Li, H., Melford, K., Judson, A., Bensadoun, A.: Murine glypican-4 gene structure and expression; Sp1 and Sp3 play a major role in glypican-4 expression in 3T3-F442A cells. Biochim. Biophys. Acta 1679, 141–155 (2004)
Monslow, J., Williams, J.D., Fraser, D.J., Michael, D.R., Foka, P., Kift-Morgan, A.P., Luo, D.D., Fielding, C.A., Craig, K.J., Topley, N., Jones, S.A., Ramji, D.P., Bowen, T.: Sp1 and Sp3 mediate constitutive transcription of the human hyaluronan synthase 2 gene. J. Biol. Chem. 281, 18043–18050 (2006)
Vatsyayan, J., Peng, H.L., Chang, H.Y.: Analysis of human UDP-glucose dehydrogenase gene promoter: identification of an Sp1 binding site crucial for the expression of the large transcript. J. Biochem. 137, 703–709 (2005)
Bontemps, Y., Vuillermoz, B., Antonicelli, F., Perreau, C., Danan, J.L., Maquart, F.X., Wegrowski, Y.: Specific protein-1 is a universal regulator of UDP-glucose dehydrogenase expression: its positive involvement in transforming growth factor-beta signaling and inhibition in hypoxia. J. Biol. Chem. 278, 21566–21575 (2003)
Müller, B., Prante, C., Kleesiek, K., Götting, C.: Identification and characterization of the human xylosyltransferase I gene promoter region. J. Biol. Chem. 284, 30775–30782 (2009)
Klingenhoff, A., Frech, K., Quandt, K., Werner, T.: Functional promoter modules can be detected by formal models independent of overall nucleotide sequence similarity. Bioinformatics 15, 180–186 (1999)
Kel, A.E., Kel-Margoulis, O.V., Farnham, P.J., Bartley, S.M., Wingender, E., Zhang, M.Q.: Computer-assisted identification of cell cycle-related genes: new targets for E2F transcription factors. J. Mol. Biol. 309, 99–120 (2001)
Kel-Margoulis, O.V., Romashchenko, A.G., Kolchanov, N.A., Wingender, E., Kel, A.E.: COMPEL: a database on composite regulatory elements providing combinatorial transcriptional regulation. Nucleic Acids Res. 28, 311–315 (2000)
Cartharius, K., Frech, K., Grote, K., Klocke, B., Haltmeier, M., Klingenhoff, A., Frisch, M., Bayerlein, M., Werner, T.: MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21, 2933–2942 (2005)
Fessele, S., Maier, H., Zischek, C., Nelson, P.J., Werner, T.: Regulatory context is a crucial part of gene function. Trends Genet. 18, 60–63 (2002)
Hughes, T.R., Tengku-Muhammad, T.S., Irvine, S.A., Ramji, D.P.: A novel role of Sp1 and Sp3 in the interferon-gamma -mediated suppression of macrophage lipoprotein lipase gene transcription. J. Biol. Chem. 277, 11097–11106 (2002)
Suske, G.: The Sp-family of transcription factors. Gene 238, 291–300 (1999)
Bouwman, P., Philipsen, S.: Regulation of the activity of Sp1-related transcription factors. Mol. Cell. Endocrinol. 195, 27–38 (2002)
Li, L., He, S., Sun, J.M., Davie, J.R.: Gene regulation by Sp1 and Sp3. Biochem. Cell Biol. 82, 460–471 (2004)
Lania, L., Majello, B., De Luca, P.: Transcriptional regulation by the Sp family proteins. Int. J. Biochem. Cell Biol. 29, 1313–1323 (1997)
Martinez-Jimenez, C.P., Gomez-Lechon, M.J., Castell, J.V., Jover, R.: Underexpressed coactivators PGC1alpha and SRC1 impair hepatocyte nuclear factor 4 alpha function and promote dedifferentiation in human hepatoma cells. J. Biol. Chem. 281, 29840–29849 (2006)
Müller, B., Prante, C., Kleesiek, K., Götting, C.: Identification and characterization of the human xylosyltransferase I gene promoter region. J. Biol. Chem. (2009)
Müller, B., Prante, C., Gastens, M., Kuhn, J., Kleesiek, K., Götting, C.: Increased levels of xylosyltransferase I correlate with the mineralization of the extracellular matrix during osteogenic differentiation of mesenchymal stem cells. Matrix Biol. 27, 139–149 (2008)
Prante, C., Bieback, K., Funke, C., Schön, S., Kern, S., Kuhn, J., Gastens, M., Kleesiek, K., Götting, C.: The formation of extracellular matrix during chondrogenic differentiation of mesenchymal stem cells correlates with increased levels of xylosyltransferase I. Stem Cells. 24, 2252–2261 (2006)
Prante, C., Milting, H., Kassner, A., Farr, M., Ambrosius, M., Schön, S., Seidler, D.G., Banayosy, A.E., Körfer, R., Kuhn, J., Kleesiek, K., Götting, C.: Transforming growth factor beta1-regulated xylosyltransferase I activity in human cardiac fibroblasts and its impact for myocardial remodeling. J. Biol. Chem. 282, 26441–26449 (2007)
Pfaffl, M.W.: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001)
Acknowledgements
We thank Sarah L. Kirkby for her linguistic advice.
Funding
This work was supported by the Forschungsförderung an der Medizinischen Fakultät der Ruhr-Universität Bochum (FoRUM), Grant F609-2007.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Müller, B., Prante, C., Knabbe, C. et al. First identification and functional analysis of the human xylosyltransferase II promoter. Glycoconj J 30, 237–245 (2013). https://doi.org/10.1007/s10719-012-9439-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10719-012-9439-5