Charakterisierung des Transkriptionsfaktors SIX2 und seiner Bindungsstellen im GDNF- und Six2-Promotor

Abstract

Early kidney development in mammals is a classical model of organogenesis. Although morphogenetical processes are well characterised during this time, not much is known about the genetical mechanisms of nephrogenesis. Oneimportant factor involved in this process is the protein of glial cell line derived neurotrophic factor gene, (GDNF), which is secreted from the metanephric mesenchyme. GDNF binds to the nephric duct, where it initiates the formation of the uretric bud and kidney development can continue. Many of the factors that regulate the expression of GDNF are still unknown. Knock out experiments of genes, which are expressed in metanephric mesenchyme during the stages of development when GDNF is expressed give some hint in respect of the factors, that could be involved in regulation of GDNF. Some of this factors include Pax2, Six2 and Eyal. The fact, that these genes are involved in regulation of GDNF leads to the establishment of a possible regulatory network during kidney development, since other members of these gene families are known to act in such way during development of other organs. The regulation of expression of Pax2, Six2 and Eyal is also mostly unknown. One aim of this work was to identify factors, that are directly involved in regulation of the expression of GDNF. Such a direct effect of PAX2, SIX2, EYA1 and WT1 on the GDNF-promoter can be characterised by transient transfection assays. Only SIX2 has shown an effect on the promoter fragment used and two binding sites of SIX2 have been identified by electromobility shift assays and footprint assays. In accordance with a regulatory model containing Pax2-Eyal-Six2-GDNF pathway, factors, that are necessary for expression of Six2 were characterised. I cloned a 920 bp fragment of the murine Six2-promoter and examined possible transactivation effects and binding abilities with the same genes used in the GDNF promoter studies. I have found, that SIX2 activates its own promoter and similarly to the GDNF-promoter, there are two binding sites of SIX2 in the Six2-promoter. Both pairs of binding sites contain palindrome sequences. To show an in vivo activity of the used promoter fragments, transgenic mice were generated. Expression of a reporter gene, under the control of the Six2- or GDNF promoter fragments in transgenic mice, was found in tissues, where endogenous expression of Six2 or GDNF in wildtype mice is also seen. The function of SIX2 as transcription factor is not well characterised. In this study I demonstrate that the C-teminus of SIX2 contains a transactivation domain. I also showed, that SIX3 and SIX6 also have an effect on the Six2-promoter and that these members of the Six-family can interact with members of the Pax-gene family.