Transcription of the vascular endothelial growth factor receptor-3 (VEGFR3) gene is regulated by the zinc finger proteins Sp1 and Sp3 and is under epigenetic control: transcription of vascular endothelial growth factor receptor 3.
BACKGROUND
In the past, the vascular endothelial growth factor receptor-3 (VEGFR-3) has been linked to the regulation of lymphangiogenesis and the lymphatic spread of solid malignancies. The molecular mechanisms controlling VEGFR3 gene expression have, however, remained poorly understood. Here, we aimed at assessing these mechanisms through VEGFR3 gene promoter analysis and the identification of transcription factors binding to it. In addition, we focussed on epigenetic modifications underlying VEGFR3 transcription regulation.


METHODS
5' Deletion analyses for the identification of functional promoter elements, electrophoretic mobility shift assays, chromatin immunoprecipitations, methylation-specific PCRs, and Trichostatin A (TSA) and 5-Aza desoxycytidine (5-Aza dC) treatments were performed in this study.


RESULTS
Following the isolation of a 2 kb stretch of 5'-flanking DNA of VEGFR3, we identified a novel GC-rich element (GRE) spanning -101/-66 sufficient for VEGFR3 transcription and activated by Sp1 and Sp3, respectively. Histone de-acetylase inhibition by TSA led to the accumulation of acetylated histones H3/H4 at the VEGFR3 gene promoter, up-regulation of its mRNA levels, and transactivation of promoter reporter constructs in endothelial cell lines. Similarly, methylation inhibition by 5-Aza dC triggered up-regulation of VEGFR3 mRNA levels and increased promoter activity. TSA and 5-Aza-dC did not influence Sp1/Sp3 binding, but increased the transactivating capacity of both transcription factors, suggesting epigenetic modification as an underlying mechanism.


CONCLUSIONS
Here we describe the identification of regulatory elements controlling human VEGFR3 gene expression and show that histone acetylation and CpG methylation are important determinants of VEGFR3 transcription regulation. These findings may facilitate the development of intervention strategies aimed at targeting VEGFR3-based tumor lymphangiogenesis and/or lymphatic tumor spread.
Laboratory for Angiogenesis and Tumor Metastasis, Campus Mitte, Charité University Hospital Berlin, Charitéplatz 1, 10117, Berlin, Germany.
Johannes
J
Hertel
Christoph
C
Hirche
Christoph
C
Wissmann
Matthias P
MP
Ebert
Michael
M
Höcker
D000107:Acetylation; D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D019175:DNA Methylation / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000378:metabolism; D044127:Epigenesis, Genetic; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000235:genetics*; D016335:Zinc Fingers
D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D040321:Vascular Endothelial Growth Factor Receptor-3