Aberrant activation from the Notch pathway is a causative factor in the leukemogenesis of T-cell acute lymphoblastic leukemia (T-ALL), the most common leukemia found in children. Oncogenic Notch signaling is also observed in other blood-borne cancers, solid tumor malignancies, and virally induced tumors. The considerable extent to which aberrant Notch signaling is associated with human neoplasias emphasizes the need for developing novel therapeutics that pharmacologically manipulate the pathway. Our long-term goal is to gain a molecular understanding of how transcriptional regulation is achieved in the Notch pathway. The conserved transcription factor CSL is the primary regulator of transcription in the Notch pathway, and its centrality makes it an attractive target for therapeutic intervention. While progress has been made in identifying binding partners of CSL, the structural details as to how these complexes regulate transcription are poorly understood. The objective of this proposal focuses on characterizing CSL-mediated transcription complexes with corepressors and viral proteins. We hypothesize that allosteric changes in CSL and/or overlapping coregulator binding sites on CSL underlie the conversion of CSL from a repressor to an activator of transcription. To test our hypothesis and accomplish our objective the following three aims will be pursued. (1) We will analyze the CSL binding surfaces utilized by transcriptional coregulators by creating a library of CSL interfacial mutants based on the CSL-Notch-Mastermind active transcription complex. (2) We will determine the minimal domains necessary for the corepressors CIR, SMRT, and SHARP to interact with CSL and pursue X-ray structures for these complexes. (3) We will delineate the domains of the herpesvirus proteins EBNA2, RTA, and LANA necessary for binding CSL and determine X-ray structures for these complexes. Completion of this proposal will illuminate the molecular details that comprise CSL transcription complexes with corepressors and herpesvirus proteins, thereby fundamentally advancing the field, and provide a structural basis for rationally developing drugs that target CSL-mediated transcription complexes. ? ? """"""""Lay language"""""""" - The abnormal action of the cellular Notch pathway results in human cancers. We are determining structural models for Notch pathway components in order to better understand pathway function, biology and tumorigenesis, which will have an impact on disease diagnosis, prevention, and treatment. ? ? ?
