Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time

DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.

Methods
For this dataset, force spectroscopy (optical tweezers) experiments were performed with dual-beam single-trap instruments equipped with force feedback using fluidics chambers constructed in house.

For each trajectory file in this dataset, a 6.2kb DNA tether was formed between an anti-dig-coated bead held in the optical trap and a streptavidin-coated bead that was held in place by a suction micropipette. Force and extension data were then recorded at 1 kHz.. First, a force-extension curve was obtained by ramping the force from ~2 to ~40pN and back. Xenopus high-speed extract (HSE) was then flown through the chamber via a shunt for ~5 min while the tether was maintained at a high (15 pN) constant force. The constant force was then changed to ~1.5 pN to allow DNA compaction, and again to 15 pN for decompaction, with several cycles performed for each tether. DNA contour at each time point was obtained by fitting the force-extension curve to the extensible worm-like chain model.

Please see the accompanying publication for further details of data collection and processing: https://www.pnas.org/doi/10.1073/pnas.2221309120