| Popis: |
The Notch pathway is an indispensable signaling mechanism, in which contacts between neighboring cells result in changes in gene regulation that dictate cellular development, proliferation, and differentiation. The necessity for proper signaling is self-evident, as mutation of Notch pathway components has been shown to result in cancer, development defects, and congenital diseases. Notch signals are transduced into transcriptional outputs by the pathway’s sole transcription factor, CSL (CBF-1, Su(H), Lag-1), which regulates expression from Notch responsive genes. In the absence of a Notch signal, CSL functions as a repressor by forming complexes with transcriptional corepressor proteins. This dual functionality of CSL highlights its importance in signaling and the need to understand at the molecular level its interactions with coactivators and corepressors. The work described herein aims to characterize the thermodynamic, structural, and functional details of CSL mediated transcription complexes. In Chapter II, using ITC and X-ray crystallography, we provide a quantitative description of the Notch RAM domain and its interaction with CSL. Based on these findings, we propose an allosteric model, in which RAM binding facilitates ternary complex formation. In Chapter III, ITC and X-ray crystallography are also used to investigate the molecular details of CSL recognizing consensus and nonconsensus DNA binding sites. These experiments give a detailed thermodynamic and structural explanation for CSL binding to two known in vivo binding sites, highlighting the differences between the two sites and the overall moderate affinity of CSL for DNA. In Chapter IV, we investigated the binding of the corepressor MINT to CSL, which is the first quantitative study of a CSL-corepressor complex. This study provided molecular insights into the proposed competition between coactivators and corepressors for binding sites on CSL. Finally, in Chapter V, knowledge gleaned from our structural studies of CSL mediated transcription complexes was used to develop CSL mutants that were tested in cellular transcriptional reporter assays. While the aim of these studies was to disrupt transcriptional activation, interestingly, some of these CSL mutants were also defective in transcriptional repression as well. These data seem to indicate that both coactivators and corepressors bind similar interfaces on CSL. In sum, the dissertation work presented here provides substantially new insights into the structure and function of Notch pathway transcription complexes - details that could aid in the development of therapeutics designed to target Notch signaling related diseases. |
