Defining the Budding Yeast Chromatin Associated Interactome

Title: Defining the Budding Yeast Chromatin Associated Interactome
Authors: Lambert, Jean-Philippe
Date: 2011
Abstract: The identification of protein-protein as emerged as a powerful tool to characterize biological systems. In budding yeast in particular, systematic mapping of protein-protein interactions for most open reading frames drastically improve our understanding of this model organisms. Still, some classes of proteins, such as DNA binding proteins, remain poorly studied due to a lack of proper tools for their study. In this thesis, I describe the development of a novel affinity purification approach for the characterization of DNA binding proteins and of their associated proteins. The modified chromatin immunopurification (mChIP) approach consist of a single affinity purification step whereby chromatin-bound protein networks are isolated from mildly sonicated and gently clarified cellular extracts using magnetic beads coated with antibodies. The mChIP method was characterized in details demonstrating significant gain in sensitivity toward the determination of DNA binding protein interactome. Moreover, I showed that mChIP purification were successful for multiple class of proteins including numerous non-histone DNA binding proteins. Following the successful development of the mChIP method, I embarked on a large-scale characterization of chromatin associated proteins in budding yeast. In this way, 102 different DNA binding proteins were characterized by mChIP to determine their chromatin associated protein networks. This effort resulted in the detection of 3576 high confidence protein associations with 724 distinct preys. Approximately 75% of the baits had significantly improved interaction coverage using mChIP compared to the classical affinity purification methodologies. I also utilized the mChIP approach to perform targeted study of histone chaperones and unravel their interconnection. In summary, the mChIP method that I developed now makes it possible to systematically study chromatin associated proteins in budding yeast, breaking down a technical barrier that existed in the field of chromatin research for too long.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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