PROJECT SUMMARY Although multiple epigenetic processes contribute to developmental gene regulation, the mechanisms by which they rewire gene regulatory networks during lineage specification are often unclear. Defects in epigenetic gene regulation contribute to developmental disorders and disease. Nuclear RNAs, including multiple classes of non-coding RNAs and nascent transcripts of coding genes help regulate the epigenetic landscape and facilitate developmental gene regulation. We have focused on the functions of R-loops—RNA/DNA hybrids that most often arise when nascent transcripts hybridize to their DNA templates—in the control of epigenetic gene regulation in mouse embryonic stem cells (mESCs). We previously discovered that R-loops modulate binding of the Tip60-p400 chromatin remodeling complex and partial depletion of R-loops from mESCs impairs the fidelity of differentiation. However, the precise roles of R-loops in differentiation are unclear. We have recently taken systematic and unbiased approaches to identify how R-loops impact the epigenome of mESCs and uncover how depletion of R-loops disrupts differentiation. In addition, we uncovered R-loop binding proteins that likely mediate some of their regulatory functions. We further developed new, single-cell genomic tools necessary to uncover how R-loops and other classes of nuclear RNAs meditate their effects in different lineages. Here, we propose to characterize the effects of one novel R-loop binding protein that functions in gene regulation and is essential for cell proliferation. In addition, we will examine the roles of R-loops in transcription elongation, fidelity, and RNA processing. Finally, we will leverage novel tools we developed for simultaneous profiling of epigenetic features to identify the locations of R-loops and epigenetic marks during gastrulation, where the three primary germ layers are established from pluripotent progenitors. Drawing on this in vivo atlas, we will use an inducible RNaseH1 mESC line to disrupt R-loops in an ESC differentiation model, uncovering their cell type-specific targets and mechanisms by which they control developmental gene expression. Together, these studies will uncover the functions of a key mediator of R-loop dependent gene regulation, the mechanisms by which R-loops modulate epigenetic marks critical for transcriptional fidelity, and the developmental targets of R-loops. These studies will significantly expand our understanding of RNA- mediated gene regulation and its roles in establishment of the mammalian body plan.