Asymmetric Unwrapping of Nucleosome Revealed by Single Molecule Fluorescence-Force Spectroscopy

Abstract

The nucleosome is the fundamental unit of eukaryotic chromatin structure which compacts DNA in the nucleus. It is a stable yet dynamic complex which is significant to transcriptional regulation. In cells, DNA is likely to be under tension to varying extents. For example enzymes such as RNA polymerase and chromatin remodelers can generate force on nucleosomes. Thus it is important to understand the dynamics of nucleosomes under mechanical constraints. We are utilizing a cutting-edge single-molecule optomechanical technology which combines fluorescence with optical tweezers (aka ‘fleezers') to probe the conformational transitions of nucleosomes under force. Our hybrid fleezers instrument provides fluorescence readouts with nm resolution under pN levels of force applied in order to locally monitor conformational dynamics of nucleosome at various coordinates. Here, we report the first observation of force-dependent gradual unwrapping and rewrapping of the nucleosome using fluorescence reporters. Moreover, we observed force-induced two-state hopping corresponding to the opening and closing of the outer DNA wrap as well as more complex transitions for the inner wrap. Interestingly, our fluorescence probes at various locations report significant asymmetric unwrapping behavior. In our pulling experiments, it takes less than 6 pN to unwrap the outer DNA at the 3' end of the 601 positioning sequence but takes up to ∼15-17 pN to unwrap the DNA from the 5' end. Asymmetric unwrapping may have implications for how enzymes such as chromatin remodelers interact with nucleosome substrates.