Final Report Summary - NOTCH IN THYMOCYTES (Genome-wide dissection of the Notch-induced molecular program in developing T cells) 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. In the course of this project we for the first time characterized the molecular program downstream of Notch throughout early T cell development by a combination of ChIP-Seq, RNA-Seq, and RNAi approaches. First, we utilized 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 then identified the Notch-regulated program at different stages of T cell development by an RNA-Seq approach. Finally, we took advantage of collaborations and cutting-edge infrastructure available in the institute to establish multiplexed RNAi screening system that will allow us to identify functionally relevant Notch targets. Several unexpected observations emerged from the analysis of the data:1. In contrast to the textbook view on Notch pathway, which suggests that Rbpj is constantly bound to DNA even in the absence of Notch signaling, our results revealed a Notch-dependent Rbpj binding in the majority of the sites.2. We identified an unexpected group of ‘ephemeral’ Notch targets, which are induced in the earliest thymocytes but rapidly downregulated thereafter. Intriguingly, many of these genes were characteristic for transcriptional signatures of myeloid and/or NK cells.3. We identified prominent Notch1 and Rbpj occupancy in the Tcrb enhancer which is likely to provide a missing molecular explanation of a previously described Tcrb recombination defect in Notch1-deficient mice. Consistently, short-term exposure of uncommitted progenitors to Notch ligands induced germline transcription of the Tcrb locus.4. Although Notch signaling strength is known to drop upon pre-TCR expression, Notch acquires a mitogenic function at this transition, suggesting that a spectrum of its target genes may change. Our analysis suggests, that Notch targets in pre-TCR-expressing cells represent a subset of Notch targets prior to pre-TCR expression and no new Notch targets emerge at this transition.In summary, these experiments resulted in the first systematic characterization of the Notch-induced molecular program in primary cells. The approaches pioneered in this study will allow characterization of Notch target genes in other primary cell types as well as in cancer models.
