During transcription DNA supercoils accumulate ahead and behind of the transcriptional machinery. These topological barriers can block further movement of RNA polymerase 2 (RNAP2) and pose a challenge for gene expression. Therefore, the removal of supercoils by topoisomerases is crucial for transcription of highly expressed genes. We showed that RNAP2 directly controls Topoisomerase 1 (TOP1) by physical interaction and stimulation above basal activity, thereby regulating DNA topology during the transcriptional cycle (Baranello et al. Cell 165,357-371, 2016). To further dissect the cellular function of this regulatory interaction, we generated a HCT116 cell line, which expresses a catalytically active TOP1 variant that cannot bind RNAP2 (HCT116 KI). To investigate how transcriptional bursts are affected by this deregulation, we are performing SLAMseq (as described in Herzog et al. 2017 Nat Methods. 2017 14(12):1198-1204). To detect non-full-length RNA with this method as well, we used random primers instead of oligodTs. We have been working on modifying the SLAMdunk pipeline for SLAMseq analysis in accordance with the changed output. To monitor nascent transcription under different conditions in HCT116 WT and HCT116 KI. Recently we have developed CAD-seq, a novel high-throughput sequencing technique based on the rapid approach to DNA adduct recovery (RADAR) that efficiently separates DNA-protein covalent complexes using a combination of chaotropic salts and detergents combined with antibody immunoprecipitation and sequencing to select only TOP-DNA cleavage complexes from all DNA-protein covalent complexes (Kuzin et al. 2022 STAR protocols 2022, 3(3), 101581). By exploiting the TOP inhibitors’ ability to trap the TOP cleavage complex CAD-seq enables to map TOP sites of activity. Overlaying the profiles of TOP activity along with ChIP-seq profiles of transcription factors, can reveal the details of TOP regulation during transcription. We have applied this method together with other genomic methods to interrogate the interplay of TOP1 and RNA Polymerase II (RNAPII) transcription during mitosis (Wiegard et al., 2021 Mol. Cell 81, 5007-5024.e9) and to show how transcription factor MYC assembles TOP1 and TOP2 in a “topoisome" (Das et al., 2022 Mol. Cell 82, 140-158.e12). Recently, we have investigated a novel combination therapy (TOP and transcription inhibitors) in PDX models of colon cancer using next generation sequencing methods including RNA-seq, SLAMseq, ChIP-seq, CAD-seq and ATAC-seq as well as how the therapy affects alternative gene splicing and selective repression of long genes (Cameron et al., 2023 Science Advances, 9(41), eadg5109.). Currently, we are working on further optimization of CAD-seq and its application to determine specific TOP2A sites of action and supercoiling accumulation in the genome. With this we aim to test our hypothesis of regulatory supercoiling "sinks" in the genome with increased TOP2A activity. We also aim to develop a probe to map positive supercoiling and investigate its role in genome folding using ChIP-seq and Micro-C. We will then test our hypothesis that topoisomerases work with cohesin to relieve supercoils in topological closed domains.