DNA supercoiling and DNA topoisomerase in chromatin structure and function

Division Of Basic Sciences - Nci

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Abstract

We have developed ATMP-seq, a novel assay that quantitatively measures both negative and positive DNA supercoiling across the genome. Using ATMP-seq, we characterized DNA supercoiling throughout the human genome, visualized twin-supercoiled domains around actively transcribed genes, and identified large-scale supercoiling domains (SDs). These SDs are modulated by DNA topoisomerases in human cells and colocalize with features of 3D chromatin structure, particularly A/B compartments, but not with topologically associating domains (TADs). Through targeted perturbation of human topoisomerases, we found that supercoiling levels within SDs can influence global transcription activity. Our ongoing research aims to: (1) Elucidate the mechanisms underlying the formation of DNA supercoiling, both locally around genes and across large genomic regions leading to SDs; (2) Dissect the propagation kinetics of DNA supercoiling along chromatin in living human cells; (3) Understand DNA supercoiling relaxation by topoisomerase activity, by probing both topoisomerase binding and catalytic activity throughout the human genome. In parallel, we have developed single-cell sequencing assays capable of profiling both the regular transcriptome and the newly synthesized nascent transcriptome, providing a genome-wide readout of transcription activity in single cells. Using these tools, we aim to investigate how DNA supercoiling shapes gene expression dynamics during chromatin regulation in human cells. In particular, we will examine the role of supercoiling in transcriptional bursting and temporal transcription kinetics in human cells, and explore how DNA supercoiling, topoisomerase activity, and chromatin structure interact during chromatin state transitions and gene regulation. One specific focus is on topoisomerase-dependent acute gene activation in response to estrogen receptor stimulation in breast cancer cells.