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First identification and functional analysis of the human xylosyltransferase II promoter

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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.

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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

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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.

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Correspondence to Benjamin Müller.

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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

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  • DOI: https://doi.org/10.1007/s10719-012-9439-5

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