DarkCellFader · Uncovering the role and regulation of 3D DNA-RNA nuclear dynamics in controlling cell fate decisions

At the onset of mammalian life, the first lineage specification decision is made where embryonic cells opt tobecome either part of the placenta or the future body. Proper regulation of this choice is crucial for subsequentdevelopment. However, we dont know how the transcription program and 3D genome architecture arise,interconnect, and are controlled to determine the fate of each cell in a developing embryo. Recent studies,including my work, suggest that in addition to canonical mRNA-coding genes, RNAs from the dark parts ofthe genome (e.g., transposons, repeats, long-non-coding RNAs) play a significant role during these events, yethow specific classes of RNA regulate gene expression and nuclear architecture post fertilization remainselusive. The main aim of my proposal is to understand the interrelationship between 3D genome organizationand the transcriptome across early development and to identify novel factors that lead to the first cell-fatedecision and concomitant decrease in cell potency.Recent technical advances, which I co-developed, enabled simultaneous measurement of 3D genomeorganization and the transcriptome, and facilitated large-scale functional screens in early mammalian embryos.Thus, I now propose to generate spatiotemporal maps of 3D DNA and RNA organization from early mouseembryos at single-cell resolution (Obj.1) to build a complete picture of the relationships between nucleararchitecture and emerging cell-type specific transcriptome that drive early cell fate choices. I will combinethese data with large-scale in vivo perturbations targeting protein coding genes (Obj.2) and dark genomeRNAs (Obj.3) to identify key inducers/regulators of lineage specification and to determine molecularmechanisms governing cell-state transitions. The proposed research will help us to control, correct, andeventually employ early stages of embryonic development and their high cell potency in vitro in reproductivemedicine and stem cell research.