Unravelling the impact of nucleosome arrays on condensin by implementing an in-vitro chromatin-loop extrusion assay

forthcoming

The three-dimensional organization of chromosomal DNA is essential for critical genomic functions, including chromosome segregation, gene expression, and DNA damage repair. Structural Maintenance of Chromosomes (SMC) complexes, such as cohesin, the Smc5/6 complex, and condensin, are integral to preserving chromosomal architecture and supporting these vital processes. Condensin folds DNA into mitotic chromosomes through DNA loop extrusion, a process critical for proper chromosome segregation. Recent single-molecule studies using naked DNA as template have shed light on how condensin drives DNA loop extrusion through multiple dynamic DNA-protein contacts. However, in cells, DNA is organized into nucleosomes that further compact into chromatin fibers, stabilized by linker histone H1, which restricts DNA accessibility to most DNA-binding proteins. How condensin gets access to DNA and extrudes DNA wrapped into nucleosome arrays remains unclear. Understanding how condensin operates on chromatin fibers is essential, as dysregulation of condensin activity in humans is linked to neurological disorders, cancer, and Alzheimer’s disease. This research aims to explore whether and how condensin extrudes nucleosome arrays by implementing a novel single-molecule chromatin loop extrusion assay. This project aims to (1) examine whether in-vitro-reconstituted densely packed nucleosome arrays affect condensin-mediated DNA loop extrusion; (2) study the impact of internucleosomal linker DNA length on this process; (3) assess the influence of nucleosome compaction by linker histone H1 on condensin-mediated loop extrusion; (4) investigate the enhancement of condensin-mediated DNA loop extrusion by chromatin remodellers. Insights gained from these studies have the potential to significantly advance our understanding of how the condensin complex interacts with chromatin fibers and extrudes these fibers into loops within the cellular environment.