Dissecting long non-coding RNA function using in vivo synthetic biology

Precise spatiotemporal regulation of gene expression is essential for healthy embryonic development. Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression. However, our current understanding of lncRNAs derives mostly from in vitro studies, with little known about their function in vivo. The core aim of this action is to dissect mechanisms of gene regulation by lncRNAs in vivo. The lncRNA Maenli is a perfect model to gain insight into lncRNA function as it activates a specific target gene in a tissue-specific manner during mouse embryonic development. This enables a quantitative in vivo read-out without compounding genome-wide effects. My hypothesis is that Maenli function is restricted to its 3D chromatin domain where it activates its target gene through transcription-associated processes, and not via the mature lncRNA transcript. To test this, I require methods that enable manipulation of the entire ~25kb locus and site-specific insertion in the genome. I will use cutting-edge synthetic biology to re-construct and manipulate Maenli, insert it in specific genomic regions, and examine its effect on target gene expression and phenotype in the mouse embryo. I will use low-input CUT&RUN to assess the functional chromatin state associated with Maenli. This interdisciplinary approach will go beyond the state of the art to illuminate specific mechanisms of cis-acting lncRNA function in vivo. This has not been possible until now due to technical limitations in conventional gene editing and challenges associated with studying lncRNAs in vivo. I anticipate that SynLncRNAs will set a new standard to study lncRNA function in vivo; impacting fundamental, disease and evolutionary research on gene regulation, as well as society and education. With expertise in the non-coding genome and in vivo synthetic biology, the Mundlos laboratory is the perfect environment for me to execute this action and train to become an independent group leader in the future.