Identification of novel multicomponent protein complexes of the critical transcription factors Steroidogenic factor 1 and Liver Receptor Homolog 1 essential to embryonic stem cells and induced pluripotent stem cells
Final Report Abstract
Multi-domain transciption factors underlie controls in many fundamental cellular processes by targeting both chromatin modifying protein complexes and the transcription machinery to their specific response elements on the DNA of target genes. The human nuclear receptor steroidogenic factor 1 (SF-1) is a protein that is a component of a transcription complex involved inter alia in sex differentiation and in the maintenance of pluripotency in embryonic carcinoma cells. Understanding the precise molecular mechanism underlying the regulation of SF-1 transcription complexes is essential for clinical applications in regenerative medicine to treat diseases like cancer. A severe limitation for the detailed study of SF-1 in vitro for example for cystallographic studies, is the difficulty of expressing stable full length SF-1 and SF-1-protein complexes. I generated SF-1 full length constructs in high yield with appropriate DNA response elements showing remarkably enhanced protein stability. Using Differential scanning fluorimetry I could show a clear relationship between protein stability and DNA length. With the stabilized full length SF-1 as a starting point I showed the interaction of SF-1 with several important coregulator molecules. Biochemical techniques that were used to determine the binding affinity of SF-1 to this cofactors included differential scanning fluorimetry and pull-down experiments. Co-Crystallographic studies of the identified protein binding partners in complex with SF-1 are still ongoing. One coregulator protein of SF-1 is called Sox-9. Mutations in the gene Sox-9 result in the syndrome of campomelic dysplasia, which includes sex-reversal. More than 60 mutations involving Sox-9 have been found to cause campomelic dysplasia. 15 of these mutations occur in the DNA binding domain of Sox-9. Surpisingly, I could show that three of the 15 mutations cause a significant stabilization of the Sox-9 DNA binding domain. This stabilization effect could be even enhanced when the Sox-9 mutant is bound to an approriate DNA binding element. Co-crystallization of these three stabized Sox-9 mutants alone or in combination with SF-1 is very promising and will help to further understand and treat campomelic dysplasia. The importance of protein interactions is in contrast to the limited stability of multi-component assemblies. Post-translational modifications (PTMs) provide an important additional site for receptor regulation and might increase the overall protein stability. SF-1 possesses a large hinge region that is subject to PTMs. The semi-synthesis of full length SF-1 featuring a phosphorylated Ser203 was successfully established and permits to study the crosstalk between SF-1 and coregulator proteins on the molecular level in the future. First in vitro analyses have shown that the semi-synthetic construct has the same binding properties as native SF-1 indicating that semi-synthesis does not affect the structure of SF-1 and leads to a well folded protein. Further studies of phosphorylated SF-1 will help to understand the exact role of phosphorylation in the interaction of SF-1 with other proteins on the molecular level. Considering the short period of the sponsorship, the progress of the results is in accordance to the proposed time line or is even above the proposed time. The preliminary results helped to drive future studies into the right direction and are an optimal starting point for further investigation to fully understand the mechanism of gene regulation by transcription factors.
