Project A.05

Transposable Elements (TEs) are genetic parasites that are reminiscent of viruses and are present in nearly every species on Earth. Across evolutionary time, the potentially mutagenic outcome of TE activity has selected for host mechanisms able to control TEs. Likewise, evolution at the level of TE sequences can enable the evasion of such mechanisms, leading to the increase in frequency of the successful TE alleles in the population. As this mutually antagonistic evolutionary arms-race continues, a plethora of novel genetic variants that are hotspots for various molecular control mechanisms can become substrate for the evolution of genome regulation at all levels.

My project is focused on discovering novel mechanisms of TE control using unbiased genetic and proteomic screens in cell culture models. Thus far, most studies have investigated the role of small RNAs and the epigenetic control of transcription of TE activity, instead I will focus on relatively unexplored parts of the TE life cycle: the control of translation of their messenger RNAs, and the stability of their encoded proteins. I am setting up TE-encoded protein immunofluorescence and detection of its mRNA by RNA-fluorescence in situ hybridization (FISH) to be performed as selective readouts after CRISPR-Cas9 pooled genome-wide loss of function perturbations. In this screening setup, loss of function alleles that lead to increased or decreased output of TE-encoded mRNA and its translation products can be isolated and identified for further study. In addition, I am also performing an unbiased proteomic screen based on antibody-driven proximity biotinylation to identify cellular proteins that interact with TE-encoded proteins.

While we are setting up these strategies using murine cells in culture as a model, both approaches lend themselves to future studies in non-model organisms. Because genetically encoded readouts are hard to achieve in cultured cells of non-model organisms, we predict that using the endogenous TEs as a screen redout may facilitate future genetic screens in cultured cells from diverse non-model systems. Likewise, antibody-based proximity biotinylation proteomics does not rely on transgenes, essentially being a model-agnostic approach to the discovery of protein-protein interactions whose unique requirement is to develop a specific antibody against the target protein of interest. We are excited about developing and providing proof-of-principle of these techniques while discovering novel TE regulators in the mouse, and we are looking forward to applying these approaches to probe the diversity of fast-evolving TE control mechanisms in different species.