7.P.36 NOVEL FUNCTIONAL SP1 BINDING SITES IN EGFR GENE INTRON 1

Abstract

Tumorgenesis is often accompanied by transcriptional deregulation of oncogenes, such as the Epidermal Growth Factor Receptor (EGFR). Transcriptional activation of a gene requires the binding of transcription factors (TF) to regulatory DNA elements at specific transcription binding sites (TFBS). A better understanding of these interactions and regulation mechanisms is essential for the development of improved therapeutic applications.

ChIP was carried out to prove the existence of four new SP1 binding sites within intron 1 of the egfr gene. Site-directed Mutagenesis was performed on plasmids carrying the regulative sequence of the egfr gene in order to alter these binding sites. Activity of these sites and their influence on the transcriptional regulation were analysed by in vitro transcription and quantification using Ribonuclease Protection Assay (RPA) and qRT PCR.

Using ChIP, four novel SP1 binding sites could be confirmed to be active at the egfr gene intron 1 locus. Expression of the egfr gene was found to be highly dependent of these sites. Consequently, their mutation led to a 50% decrease of the transcriptional activity of the egfr gene.

The four new SP1 binding sites in the egfr intron 1 have a functional role in the egfr gene regulation, leading to a higher transcription rate. As so far only little is known about egfr gene activation, more TFs and TFBSs have to be analysed in order to gain a comprehensive understanding about the regulation of this important oncogene.