The underlying causes of cancer are often linked to signaling pathways, such as the Notch pathway, which through mutation become deregulated and signal abnormally. Aberrant Notch signaling underlies the pathogenesis of many types of cancer, most notably T-cell acute lymphoblastic leukemia - a common childhood leukemia. Additionally, the oncogenic viruses EBV and KSHV co-opt Notch signaling to maintain viral persistence in vivo and cause cancer primarily in immunocompromised individuals. Therefore, medicinal modulation of the Notch pathway holds great promise for novel anti-cancer chemotherapeutics. Canonical Notch signaling results in changes in gene expression, which is regulated by the DNA binding transcription factor CSL. CSL regulates both repression and activation of transcription from Notch target genes by forming complexes with different transcriptional coregulators. The centrality of CSL in the transcriptional regulation of Notch target genes makes it an attractive target for therapeutic intervention. Our long-term goal is to understand at the molecular level how transcription is regulated in the Notch pathway and to determine whether these processes can be manipulated for therapeutic benefit. While progress has been made in characterizing the structure and function of active Notch transcription complexes, our understanding at the molecular level for how CSL functions as transcriptional repressor and how viral proteins subvert CSL function is incomplete. The objective of this proposal is to determine structures of CSL in complex with negative and viral regulators of transcription, define their thermodynamic binding parameters using ITC, and characterize their function in cellular assays. We hypothesize that there are generally two modes by which transcription is regulated at Notch target genes - one, at the protein level, whereby coregulators directly compete for binding surfaces on CSL;and two, at the chromatin level, whereby CSL-coregulator complexes recruit the histone modification machinery. To achieve our objective and test our hypothesis we will pursue the following three aims: (1) Characterize how negative regulators bind CSL to repress transcription;(2) Characterize how CSL recruits the chromatin remodeling machinery;and (3) Characterize the structure and function of CSL-viral protein complexes. Completion of this proposal will increase our understanding of how transcription is regulated in the Notch pathway, thereby advancing the field. Additionally, completion of these studies will provide new sites of clinical intervention and facilitate the development of small molecules that target Notch signaling for anti-cancer therapeutics.
The abnormal action of a cellular signaling pathway, termed Notch, results in human cancers. In this proposal, we are using biophysical approaches to (1) determine three-dimensional models of Notch pathway components and the complexes they form, and (2) quantitatively characterize the affinities of these complex interactions. It is expected that these studies will lead to a better understanding of pathway function, biology and tumorigenesis, which will have an impact on disease diagnosis, prevention, and treatment.
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|Oswald, Franz; Rodriguez, Patrick; Giaimo, Benedetto Daniele et al. (2016) A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes. Nucleic Acids Res 44:4703-20|
|Contreras, Ashley N; Yuan, Zhenyu; Kovall, Rhett A (2015) Thermodynamic binding analysis of Notch transcription complexes from Drosophila melanogaster. Protein Sci 24:812-22|
|Torella, Rubben; Li, Jinghua; Kinrade, Eddie et al. (2014) A combination of computational and experimental approaches identifies DNA sequence constraints associated with target site binding specificity of the transcription factor CSL. Nucleic Acids Res 42:10550-63|
|Collins, Kelly J; Yuan, Zhenyu; Kovall, Rhett A (2014) Structure and function of the CSL-KyoT2 corepressor complex: a negative regulator of Notch signaling. Structure 22:70-81|