Global coupling of R-loops with transcription dynamics modulates early development and survival of Drosophila

R-loops are involved in many biological processes in cells, yet the regulatory principles for R-loops in vivo and their impact on development remain to be explored. Here, we modified the CUT&Tag strategy to profile R-loops in Drosophila at multiple developmental stages. While high GC content promotes R-loop formation in mammalian cells, it is not required in Drosophila. In contrast, RNAPII abundance on chromatin appears to be a universal inducing factor for R-loop formation in the Drosophila genome, including active promoters and enhancers, and H3K27me3 decorated repressive regions and intergenic repeat sequences. Importantly, such a regulatory relationship is dynamically maintained throughout development and many development-related transcription factors that may regulate RNAPII activation and R-loop dynamics were uncovered. By ablating the elongation factor Spt6, we further showed the global R-loop induction coupled with RNAPII pausing, genome instability, and defects in larvae survival and cuticle development. Overexpression of rnh1 largely reversed the upregulation or downregulation of gene expression depending on gene length, DNA damage, and defects in survival and cuticle development. Altogether, our findings indicate that dynamic R-loop regulation is dictated by RNAP pausing and transcription activity, and plays a feedback role in gene regulation, genome stability maintenance, and Drosophila development. Overall design: To explore the regulatory mechanism of R-loop in Drosophila and embryonic development, we constructed a multi-temporal R-loop map of embryonic development. As well as pol2-chip-seq and cut&tag before and after interfering transcription factor spt6. RNA-seq experiments were performed on da-GAL4, da-GAL4>Spt6-RNAi, da-GAL4>rnh1 and da-GAL4>rnh1, Spt6-RNAi Drosophila in 14-16h embryos