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Dynamic coupling of R-loops with transcriptional pausing modulates tissue development in Drosophila

ABSTRACT: The R-loop in the genome participates in modulating transcription, replication and DNA damage repair, yet its regulation and biologic impact on development in vivo remain to be explored. Here, we modified CUT&Tag strategy and established R-loop profiles of Drosophila across multiple developmental stages. While lack of GC preference, the broad class of R-loops in Drosophila all highly follow the RNAPII pausing, which is also a conserved R-loop regulatory mechanism in mammals. Meanwhile, RNAPII-regulated R-loop may be regulated by multiple developmental specificity and universality transcription factors. To test the specificity and universality of transcription factor-mediated R-loop involvement in development, we knocked out the transcription elongation factor Spt6, upregulated R-loop and increased RNAPII pausing, and altered the expression of critical genes and cutin genes, thereby affecting Drosophila lifespan and cutin development. Interestingly, complementation of rnh1 could partially rescue the transcription factor deletion phenotype. We conclude that RNAPII is a conserved mechanism of R-loop regulation across species and organism specific damage caused by R-loop dysregulation is mediated by the functions of transcription factors.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE246878 | GEO | 2024/11/02

REPOSITORIES: GEO

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Project description:Work from our mouse models showed that COBRA1, the B subunit of NELF, could facilitate R-loop (DNA:RNA hybrid) accumulation in the BRCA1-deficient mouse mammary epithelium. Since NELF could promoter RNA PolII pausing at promoter proximal regions, we asked whether COBRA1-mediated RNA Pol II pausing could contribute to accumulated R-loops. To answer this question, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) for RNA Pol II, as well as two NELF subunits, NELF-A and NELF-B (COBRA1) in mouse primary mammary epithelium cells. To locate R-loops in the same cell population, we performed DNA:RNA immunoprecipitation-sequencing (DRIP-seq) with or without treatment of RNase H, a nuclease that specifically degrades RNA in the R-loop structure. Our sequencing results showed that RNA Pol II, NELF and R-loops are all enriched at transcription start sites (TSSs). Notably, genes with TSS R-loop accumulation are significantly enriched for COBRA1 binding. Taken together, the genomic data lent additional support to the notion that COBRA1-mediated RNAPII pausing contributes to R-loop dynamics in mammary epithelium.

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