Identification and Characterization of the Human Xylosyltransferase I Gene Promoter Region

Benjamin Müller,Christian Prante,Knut Kleesiek,Christian Götting

doi:10.1074/jbc.m109.016592

Abstract

Human xylosyltransferase I catalyzes the initial and rate-limiting step in the biosynthesis of glycosaminoglycans and proteoglycans. Furthermore, this enzyme has been shown to play a major role in the physiological development of bone and cartilage as well as in pathophysiological processes such as systemic sclerosis, dilated cardiomyopathy, or fibrosis. Here, we report for the first time the identification and characterization of the XYLT1 gene promoter region and important transcription factors involved in its regulation. Members of the activator protein 1 (AP-1) and specificity protein 1 (Sp1) family of transcription factors are necessary for the transcriptional regulation of the XYLT1 gene, which was proven by curcumin, tanshinone IIA, mithramycin A, and short interference RNA treatment. A stepwise 5' and 3' deletion of the predicted GC-rich promoter region, which lacks a TATA and/or CAAT box, revealed that a 531-bp core promoter element is able to drive the transcription on a basal level. A binding site for transcription factors of the AP-1 family, which is essential for full promoter activity, was identified by site-directed mutagenesis located 730 bp 5' of the translation initiation site. The ability of this site to bind members of the AP-1 family was further verified by electrophoretic mobility shift assays. A promoter element containing this binding site was able to drive the transcription to about 79-fold above control in SW1353 chondrosarcoma cells. Our findings provide a first insight into the regulation of the XYLT1 gene and may contribute to understanding the processes taking place during extracellular matrix formation and remodeling in health and disease.

Highlights

The gene coding for human XT-I is located on the short arm of chromosome 16 at the region 16p12.3
The enzyme can be used as a diagnostic marker for systemic sclerosis [11, 12], participates in bone development [13], and was recently proven to exhibit a key role in carticin A in the cell culture supernatant was sufficient to reduce the XT-I mRNA expression to 14% (Ϯ0.45%)
TGF␤1 Treatment—To elucidate a putative TGF␤1-mediated induction of the XT-I promoter activity, cells transfected with the 1638-bp, 797-bp, and the 797-bp activator protein 1 (AP-1) site mutated

Summary

Introduction

The gene coding for human XT-I is located on the short arm of chromosome 16 at the region 16p12.3. The XYLT1 cDNA was already cloned in 2000 [6] and the involvement of this enzyme in many physiological and pathophysiological processes is well established, little is known about the transcriptional activation and regulation of XT-I. The aim of this work was to identify and characterize the promoter region of the XYLT1 gene. We determined that a 531-bp promoter element is able to drive the transcription on a basal level. We present evidence that members of both the Sp1 and the AP-1 family of transcription factors are necessary to drive the transcription of the XYLT1 gene at full strength. This work extends our knowledge of the transcriptional regulation of XT-I and may contribute to a better understanding of extracellular matrix formation and remodeling in tissue development and during pathophysiological processes

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