Genome-wide DNA supercoiling arises from transcription and SMC activity and mediates transcriptional negative feedback.

Genome-wide DNA supercoiling is closely linked to chromatin organization and gene expression, yet the mechanisms establishing genome-scale supercoiling in living cells and its functional consequences remain unclear. Here, we show that genome-wide supercoiling arises from transcription-driven asymmetric topological relaxation together with contributions from SMC complexes in human cells. During RNA polymerase elongation, human topoisomerases preferentially relax positive over negative supercoils, leading to the accumulation of negative supercoiling around genes. This imbalance enriches supercoiling at transcriptionally active regions including TAD boundaries, and promotes the emergence of large-scale topology. In parallel, SMC complexes independently shape genome-wide supercoiling, with cohesin contributing to interphase topology and condensin establishing an overall positively supercoiled mitotic genome. Functionally, the accumulation of transcription-driven negative supercoiling represses local transcription, revealing a supercoiling-mediated negative feedback mechanism. Together, these findings define the mechanistic basis of genome-scale supercoiling in human cells and establish DNA topology as an integral regulatory layer of transcription.