Genome-wide mapping of DNA supercoiling in human cells shows that transcription-driven negative supercoiling accumulates around active genes — because topoisomerases preferentially relax positive supercoils — and that this accumulation represses local transcription, creating a topology-mediated negative feedback on gene expression (Zhu et al. 2026, bioRxiv). The molecular step at which negative supercoiling inhibits transcription is, however, undefined; candidates include transcription factor binding, chromatin remodeling, RNA polymerase recruitment, promoter melting, and promoter clearance. This problem asks for mechanistic identification of the step(s) mediating the repression, together with how topoisomerase-SMC (cohesin/condensin) interplay modulates the feedback across genomic contexts. Only partial, context-specific mechanisms have been reported to date, so the genome-wide human mechanism remains open.

Background

DNA supercoiling is the over- or underwinding of the double helix relative to its relaxed B-form state, conventionally quantified as a linking difference; negative supercoiling means underwinding. Because an elongating RNA polymerase (RNAP) cannot rotate freely around the helical DNA it tracks, transcription generates 'twin-supercoiled domains': positive supercoils ahead of the polymerase and negative supercoils behind it. DNA topoisomerases dissipate this torsional stress — TOP1 by transient single-strand swiveling, TOP2A and TOP2B by ATP-dependent double-strand passage. SMC ('structural maintenance of chromosomes') complexes such as cohesin and condensin are ring-shaped ATPases that extrude DNA loops and organize the interphase and mitotic genome, respectively; TADs (topologically associating domains) are self-interacting genomic intervals whose boundaries coincide with cohesin/CTCF binding sites. Genome-wide supercoiling became measurable through psoralen-based assays such as ATMP-seq, in which the intercalator trimethylpsoralen crosslinks preferentially into underwound DNA and sequencing of the crosslinked fragments yields genome-scale supercoiling maps; this was first applied across the human genome by Yao et al. (2025). Building on that assay, the source preprint (Zhu et al. 2026, bioRxiv) showed that human topoisomerases relax positive supercoils faster than negative ones, so negative supercoiling accumulates around active genes and extends to transcriptionally active regions including TAD boundaries; in parallel, cohesin shapes interphase topology and condensin establishes an overall positively supercoiled mitotic genome. Functionally, acutely reducing negative supercoiling around genes (simultaneous TOP1 depletion and TOP2 inhibition) increased nascent transcription, measured by transient-transcriptome sequencing (TT-seq), genome-wide and most strongly at gene 5' ends — evidence for a supercoiling-mediated negative feedback that constrains transcriptional output and acts at the level of initiation. Which molecular step the feedback attacks is unknown. The canonical transcription cycle proceeds through transcription-factor binding, chromatin remodeling, RNAP recruitment (assembly of the pre-initiation complex), promoter melting (open-complex formation), and promoter clearance (escape into elongation). The bacterial literature generally finds that negative supercoiling promotes promoter melting, which makes the human repression effect mechanistically non-obvious. Partial mechanisms exist in specific contexts: in budding-yeast GAL genes, supercoil accumulation destabilizes binding of the transcription factor Gal4 (Patel et al. 2023); at promoters containing potential G-quadruplex-forming sequences, negative supercoiling drives cooperative G-quadruplex and R-loop formation that impedes RNAP loading in biophysical settings (Hwang et al. 2025); and single-molecule and cryo-electron tomography work has characterized how torsion stalls elongating polymerases (Qian et al. 2026; Zhang et al. 2026). None of these establishes the mechanism operating genome-wide in human cells.

Problem Statement

Determine the molecular mechanism by which accumulated negative DNA supercoiling represses transcription in human cells. Specifically: (i) identify which step(s) of the transcription cycle causally mediate the repression — candidate steps include transcription factor binding, chromatin remodeling, RNA polymerase recruitment, promoter melting, and promoter clearance — with evidence that generalizes across genomic contexts rather than a single locus or sequence class; and (ii) establish how the interplay between topoisomerases (TOP1, TOP2A, TOP2B) and SMC complexes (cohesin in interphase, condensin in mitosis) modulates this supercoiling-mediated feedback across loci. These are two deliverables of one objective: a mechanistic account of the supercoiling-to-transcription negative-feedback loop documented by Zhu et al. (2026), in which the step identification is the core result and the topoisomerase-SMC interplay defines how the feedback's strength varies with genomic context. The source's further suggestion that genome-wide supercoiling may act in DNA replication and mitotic chromosome condensation is an exploratory adjacent direction and is not part of the required completion of this problem.

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