While it is now clear that lincRNAs are functionally important, the mechanisms by which they carry out their regulatory role is currently unknown. Our lab aims to understand the role of lincRNAs, rather than other protein regulators, in controlling cell states and regulating gene expression programs. We aim to systematically define the proteins that interact with lincRNAs and determine how lincRNAs interact with and assemble diverse macromolecular complexes (Aim 1), how lincRNAs recruit complexes to target sites and how lincRNAs achieve regulatory specificity (Aim 2), and how lincRNAs, through these interactions, give rise to phenotypic states and control cell state decisions (Aim 3).
Through a systematic understanding of these lincRNA-protein interactions, how they get assembled, and the functional role of these interactions, we will be able to understand how regulatory specificity can be imparted by lincRNAs. A detailed understanding of this process will allow us to understand the full complexity of how lincRNAs can interact with both nucleic acid and protein modules to carry out regulatory roles and will provide an understanding of how large ncRNAs can regulate gene expression in normal and disease states.
It is now clear that many lincRNAs carry out their functional roles through their physical interaction with diverse regulatory proteins. As such, understanding how lincRNAs control cell states requires a detailed understanding of lincRNA-Protein complexes. To date, our understanding has been limited because most studies of lincRNA-Protein complexes have used protein-immunoprecipitation to identify the RNAs associated with a specific protein complex; this requires knowledge of the specific proteins that may interact with a lincRNA. We will explore three paths that together will allow us to define the complexity of lincRNA-Protein complexes in embryonic stem cells. We will comprehensively identify protein-complexes with which lincRNAs interact, determine where these protein interactions occur on RNA, and determine how these lincRNA-Protein complexes assemble. Together, this will provide a map of lincRNA-Protein complexes in mouse ES cells and allow us to determine the regulatory complexes that lincRNAs assemble and the principles of how lincRNA complexes form providing a key component in understanding the regulatory role of lincRNAs.
While it is now clear that lincRNAs have major functional effects in the cell, their direct regulatory targets remain unknown. As many lincRNAs physically interact with diverse regulatory proteins including many chromatin regulators, an attractive hypothesis is that many lincRNAs may work by localizing regulatory proteins to specific genomic DNA regions as has been demonstrated for the XIST ncRNA. Yet, beyond a few examples, these interactions remain largely unexplored. Determining how lincRNAs provide regulatory specificity requires defining the direct targets and the mechanism of lincRNA targeting. We will determine the principles governing lincRNA localization to regulatory target sites. This will provide an understanding of lincRNA-Protein localization to regulatory targets and provide an understanding of the rules governing specificity of lincRNA-Protein complexes to regulatory targets.
Understanding how lincRNAs give rise to specific patterns of gene expression will require determination of the functional contribution of each interaction domain. To date, methods to characterize the function of lincRNAs deplete the entire lincRNA which cannot provide insights into how a lincRNA is functionally assembled. Defining the unique functional role of a lincRNA will require a different approach, one that allows targeting of individual domains and creation of synthetic lincRNA alleles. We will define the function of lincRNA-Protein interaction domains and directly test the modularity and flexibility of lincRNAs. These results will determine the functional role of individual lincRNA-Protein interaction domains, and test whether lincRNAs are functionally modular and flexible as predicted by the 'flexible modular scaffold' model. This data will provide an understanding of how lincRNAs are functionally assembled and how lincRNAs can interact with both nucleic acid and protein modules to carry out regulatory roles.