Probing the Function of Long Non-Coding RNAs in Differentiation

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PROJECT SUMMARY The over-arching focus of my research program has been to identify spatial and temporal aspects of regulating gene expression. Our recent emphasis has incorporated mouse embryonic stem cells (ESCs) as a model system to understand gene dynamics and various aspects of gene expression, including the role of long non- coding RNAs (lncRNAs). LncRNAs represent an exciting class of tens of thousands of RNAs among which very few have thus far been functionally characterized. A number of lncRNAs have been shown to exhibit cell- type specific expression, localization to subcellular compartments, and association with human diseases suggesting that they have critical roles in many cellular processes. The long-term goal of this project is to identify the mechanism of action of several lncRNAs identified in an RNA-seq screen that have roles in pluripotency and/or differentiation, and potentially represent different functional classes, Such analysis will provide insight into how these lncRNAs regulate differentiation through their impacts on gene expression and/or nuclear organization. Here, we propose to characterize an exciting nuclear-retained lncRNA, Platr4, that is highly expressed in pluripotent ESCs and is significantly down-regulated upon ESC differentiation. We will examine the impact of Platr4 knockout and over-expression on global gene expression in ESCs. In addition, Platr4 expression will be examined throughout embryonic development and the impact of Platr4 knockout will be examined in developing mouse embryos to identify critical points of function and candidate genes and pathways regulated by Platr4 in early mouse development. Based upon preliminary studies, the role of Platr4 in mesodermal and endodermal lineage commitment will be an immediate focus of investigation. The functional interactions of Platr4 will be identified by an RNA-tagging strategy combined with RNA-seq (ChIRP-seq) to identify genomic interactions of Platr4, and with mass spectrometry (ChIRP-MS) in order to identify proteins interacting with Platr4. Together, the proposed studies will provide important insights into the functional role of Platr4 in lineage commitment and differentiation, and will provide new insights as to how regulatory signals instructed by a long non-coding RNA can impact differentiation and potentially reprogramming.