Dimerization of the transcription factor STAT3 analyzed by single molecule fluorescence spectroscopy and advanced microscopy

STAT3 (signal transducer and activator of transcription-3) is activated by numerous cytokines and growth factors. As a transcription factor, STAT3 plays important roles in many processes including embryonic development, immunity and different types of cancer progressions. In response to cytokine stimulation, STAT3 is activated through phosphorylation of a single tyrosine residue. The phosphorylated STAT3 molecules dimerize via a reciprocal interaction between the phosphorylated tyrosine of one and the SH2 (Src homology 2)-domain of the other monomer, accumulate in the nucleus and bind to specific DNA sequences, which is followed by the target gene expression. In addition, STAT3 is able to dimerize in the absence of cytokine treatment. However the structural background and the function of these preformed dimers are not known. We analyzed the structural organizations and requirements of the nonphosphorylated and phosphorylated STAT3 dimer associations using fluorescence based microscopy techniques as dual-focus fluorescence correlation spectroscopy (2f-FCS) and Förster resonance energy transfer (FRET). Results presented in this thesis reveal the similar parallel associations of the non-activated and activated STAT3 dimer forms, however the dimers are stabilized by diverse interdomain interactions. We demonstrate the importance of the N-terminal domain in dimerization of latent STAT3, moreover we showed that this dimer is mostly stabilized by homotypic interactions between the N-terminal fragments, similar to other STAT family members. In addition, we identified a single residue (L78) in the N-terminal domain of STAT3 promoting this dimerization, but also the tetramerization of the molecules on specific DNA sequences. We showed that inhibition of latent STAT3 dimers positively affects the phosphorylation of STAT3, demonstrating the importance of preassociation in the negative regulation of STAT3 activation. These structural findings reveal the unique structural characteristics of STAT3 in the STAT family and partially explains the different biochemical features, functions and the constitutive activation in various diseases of this very important transcription factor