Control of RNA polymerase II promoter-proximal pausing by DNA supercoiling [ChIP-seq]

During transcription, DNA supercoiling generated by the advance of RNA polymerase II (Pol II) is resolved by DNA topoisomerases, enzymes that bind chromatin and produce transient breaks to relax DNA. Recently, this idea of mere facilitators of transcription progression is changing, as topoisomerases are being assigned new functions in regulating the expression of specific genes. In fact, mammalian type II topoisomerases, both the [Symbol] (TOP2A) and [Symbol] (TOP2B) paralogs, are enriched at promoter regions, where they have been proposed to trigger transcription through the generation of DNA double-strand breaks (DSBs). However, this is difficult to reconcile with the intrinsic catalytic properties of TOP2 and the high risk of genome instability that continuous production and repair of DSBs implies. Here, we show that TOP2A enforces promoter-proximal pausing of Pol II by removing transcription-associated negative DNA supercoiling. Interestingly, this topological balance and its disruption is essential for the transcriptional control of Immediate Early Genes (IEGs) and their typical bursting behaviour in response to stimulus. We therefore uncover a novel layer of transcriptional regulation that relies on canonical functions of TOP2A that are independent of aberrant DSB formation, providing a topological framework for the control of promoter-proximal pausing and the tight regulation of IEGs. A total of 5 ChIP-seq experiments on human RPE cells were generated: ChIP-seq on POL2 (vehicle treatment, 3 replicates), ChIP-seq on POL2 (no treatment, 3 replicates), ChIP-seq on POL2 (30 min merbarone treatment, 3 replicates), ChIP-seq on POL2 (1 h merbarone treatment, 2 replicates) and ChIP-seq on TOP2A (no treatment, 2 replicates). Additionally, 3 IgG samples were generated (untreated, vehicle treatment and merbarone treatment).