TY  - THES
AB  - The xylosyltransferases I and II (XT-I and XT-II) catalyze the transfer of xylose from UDP-xylose to selected serine residues in the proteoglycan core protein, which is the initial and rate-limiting step in glycosaminoglycan biosynthesis. Both XT isoforms represent key enzymes in extracellular matrix assembly and remodeling and do also play a major role in various pathological processes. The xylosyltransferases show a high degree of homology in structure and function. Nevertheless, they have divergent expression levels in different cell types and tissues as well as during the osteogenic and chondrogenic mesenchymal stem cell differentiation. Furthermore, a different response to growth factors of the TGF[beta] family has been observed. The mechanisms underlying the transcriptional regulation of both XYLT genes have not been characterized yet.
In my thesis, I identified for the first time the promoter regions of XT-I and XT-II as well as important transcription factors involved in their regulation. Both putative regulatory regions lack typical promoter elements like a TATA or CAAT box. In contrast, several GC boxes within a CpG island immediately upstream of the translation initiation sites were observed in both genes. To determine the minimal active promoter regions, different 5'- and 3'-truncated promoter fragments were generated by gene synthesis and PCR and subsequently inserted into a reporter gene vector system. A detailed activity analysis of each promoter reporter gene construct in the chondrosarcoma cell line SW1353 and in the hepatoma cell line HepG2 revealed that a 797 bp fragment is essential for full XYLT1 promoter activity. In contrast, a region encompassing 177 nucleotides immediately upstream of the translation start site was sufficient to drive the constitutive transcription of the XYLT2 gene in full strength. Using an in silico approach numerous putative transcription factor binding sites for transcription factors of the AP-1 and Sp1 protein family were identified. Several site-directed mutagenesis experiments as well as electrophoretic mobility shift and supershift assays using specific antibodies were used to clearly identify the transcription factors involved. The findings of my thesis were that cJun/AP-1 and Sp1 proteins essentially participate in the regulation of the XYLT1 gene. This was confirmed by using specific antibodies and sequence specific siRNA targeting Sp1 and Sp3. Additionally, a crucial role of Sp1 transcription factors in regulating the XYLT2 gene could be ascertained. A cooperative interaction of multiple Sp1 recognition sequences was determined as being necessary for the constitutive expression as well as for the vernier adjustment of the transcriptional regulation. The participation of the transcription factors identified for the activation of the XYLT genes was further verified using inhibitors against AP-1 and Sp1. Moreover, an alignment of the human and murine XYLT promoter regions revealed phylogenetically conserved AP-1 and Sp1 transcription factor binding sites.
In conclusion, I demonstrated significant differences in the transcriptional regulation of both XT isoforms. These results can be used as a starting point for further investigation regarding the divergent cell type and tissue specific expression patterns of both xylosyltransferases. Furthermore, the findings of my thesis may contribute to elucidate the different physiological functions of both enzymes.
DA  - 2009
LA  - ger
PY  - 2009
TI  - Identifizierung und funktionelle Analyse der humanen Xylosyltransferase I- und II-Promotoren
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-16312
Y2  - 2024-11-22T03:55:16
ER  -