The Notch pathway regulates numerous cell fate decisions throughout the development of a multicellular organism. One well-characterized Notch-dependent differentiation process is the development of T cells. Notch functions as the T lineage commitment factor at the early stages of differentiation and is required for survival, proliferation and further developmental progression later on. Although the importance of Notch signaling in T cell differentiation is long-established and some information on Notch targets important in this process exists, no systematic attempt to characterize the molecular program downstream of Notch signaling in developing T cells was made so far. In fact, due to the lack of appropriate tools, until today, a systematic search for Notch targets in any system was largely restricted to studies in cell lines and conditions of artificially overactivated Notch signaling. We plan to dissect the molecular program downstream of Notch throughout T cell development by a combination of ChIP-Seq, RNA-Seq, and RNAi approaches. First, we will utilize the unique tools developed in the laboratory – biotin-tagged murine knock-in alleles of Notch1 and its DNA-binding cofactor Rbpj – to map the genome-wide binding pattern of these factors by ChIP-Seq. We will then identify the Notch-regulated program at different stages of T cell development by an RNA-Seq approach. Finally, we will take advantage of collaborations and cutting-edge infrastructure available in the institute to identify functionally relevant Notch targets in a small-scale multiplexed RNAi screen. These experiments will result in the first systematic characterization of the Notch-induced molecular program in primary cells and identification of novel regulators of T cell development. The approaches pioneered in this study will allow characterization of Notch target genes in other primary cell types as well as in cancer models.
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