The co-repressor Groucho limits progression through the early transcription elongation checkpoint in vivo

Promoter-proximal pausing of RNA polymerase II (RNAP II) at the early elongation checkpoint is a key regulatory step in developmental and stimulus-responsive gene expression. How pausing is established and modulated in a gene-specific manner during animal development remains unclear. The Groucho (Gro)/Transducin-like Enhancer of split (TLE) family of co-repressors is widely used by transcription factors to repress transcription, yet the mechanism of Gro-mediated repression in vivo is unresolved. Here, we combine genome-wide chromatin profiling with in vivo genetic analysis in Drosophila melanogaster to test whether Gro regulates RNAP II pausing. Analysis of ChIP-seq data across distinct cell types shows that Gro recruitment is largely cell-type specific but consistently occurs as discrete peaks within accessible, enhancer-associated chromatin. Gro occupancy frequently overlaps promoters enriched for pausing regulators, including Negative Elongation Factor (NELF), GAGA Factor (GAF), and components of Positive Transcription Elongation Factor b (P-TEFb), without excluding their recruitment. Using a sensitised wing-specific knockdown assay, we demonstrate that partial depletion of NELF subunits, GAF, and 7SK snRNP components synergistically enhances gro phenotypes beyond additive effects. These genetic interactions support a shared role in regulating transcription during development. Our findings support a model in which Gro attenuates transcription by modulating progression through the P-TEFb-dependent early elongation checkpoint in vivo.