R-loops as Mechanisms Governing Neural Differentiation and Cell Type-Specific Transcription

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PROJECT SUMMARY The neuropsychiatric disorders schizophrenia and autism together affect over 15% of children in the United States, representing a significant public health concern. Development of effective therapeutics and diagnostic tools for these disorders has been hindered by our incomplete understanding of their complex neurodevelopmental etiologies. Gene expression variation is a common attribute of these disorders, thought to arise from developmental stage- and tissue-specific RNA regulation during neurodevelopment. New genome- wide mapping strategies have identified a connection between R-loops (a three-stranded nucleic acid structure containing a DNA/RNA hybrid) and transcriptional regulation, suggesting a possible link between R-loops and gene expression variation. However, R-loops have never before been characterized on a genome-wide scale in the human brain, precluding research studying their role in neurodevelopmental illnesses. Our research has showed that R-loops may poise developmental genes for transcription during neural differentiation, and our immediate goal is to test this hypothesis by leveraging human induced pluripotent stem cell (hiPSC)-based models to functionally manipulate R-loop levels. Specifically, we aim to reduce R-loop levels in hiPSC-derived neural progenitor cells and measure the effect this has on transcription, differentiation, and ability of neural cells to form functional synapses. We aim to identify a causal mechanism of this R-loop-mediated transcriptional poising by exploring the hypothesis that R-loops stall RNA polymerase II, as stalled RNA polymerase II is thought to keep developmental genes transcriptionally inactive but ready for expression upon developmental cues. Importantly, we will map R-loops genome-wide for the first time in the developing human brain, and link R-loop distributional shifts during human neurogenesis with neurodevelopmental disease-associated gene expression signatures. Here, we will use cutting-edge techniques, including DNA/RNA immunoprecipitation followed by next- generation sequencing, to achieve these goals. These first steps toward understanding R-loop function in neural cells will facilitate our long-term objective to uncover the epigenetic mechanisms of aberrant RNA regulation in neuropsychiatric illness, in order to improve diagnostics and identify novel therapeutic targets for these illnesses.