Molekulare Mechanismen der STAT1-vermittelten Erkennung und Aktivierung von Zielgenen

Signal transduction mediated by STAT1 is the best characterized one in the family of STAT transcription factors. Nevertheless, many questions concerning the mechanism of gene activation by STAT1 remain unanswered. This concludes molecular steps in target gene finding, recruitment of coactivators and regulation of DNA-binding. In this thesis we have generated STAT1-binding mutants, with either higher affinity towards their palindromic GAS sites or prefered cooperative DNA binding, respectively. These DNA-binding mutants were used as valuable tools for studying the activation of IFNγ-sensitive genes. Mutation of two critical glutamyl residues within the DNA-binding domain adjacent to the phosphodiester backbone of DNA identified a molecular mechanism that efficiently releases phospho-STAT1 dimers from DNA and thereby positively controls transcriptional activity. These amino acids act as a molecular off-switch that liberates STAT1 sequence-independently from DNA. A defect in this switch mechanism inhibits the dissociation from DNA, broadens the repertoire of putative binding sites on DNA and also enhances binding to high-affinity GAS sites. Despite elevated levels of tyrosine phosphorylation and a prolonged nuclear accumulation period, the DNA-binding mutants displayed a significantly reduced transcriptional activity upon stimulation of cells with IFNγ. This reduced transcriptional response is explained by the deposition of oligomerized STAT1 molecules outside from GAS sites and a hindered renewal of promoter-bound STAT1 during cytokine stimulation. Thus, a high dissociation rate from DNA is a key feature of STAT1 signal transduction and explains why hyper-phosphorylated STAT1 mutants with conserved GAS recognition and enhanced DNA binding activate target genes less efficiently than the wild-type protein. Additionally, we demonstrate here that nuclear accumulation is dispensable for gene induction. Furthermore, in the course of this work, we identified two mutations located in the N-domain which resulted in an improved cooperative DNA-binding. This second group of DNA binding mutants with conserved GAS recognition exhibited differential gene expression, depending on the number and affinity of GAS sites located in the promoter of target genes. This observation allowed, for the first time, to investigate the impact of preferential cooperative DNA binding on transcriptional activity. We found that gene activation was enhanced for target genes harbouring multiple binding sites, but was indistinguishable from the wild-type molecule on target genes with only a single discernable GAS site. In human samples from patients with dilated cardiomyopathy (n=235) and leukemia (n=25) we found no clinical evidence suggesting the pathophysiological significance of such DNA-binding mutants.