Our genome is densely packed in a dynamic, hierarchical fashion, balancing compaction with controlled regulation of DNA accessibility. DNA organization starts with strings of nucleosomes, 147 bp DNA segments that are wrapped twice around 8 histone proteins, and fold into dense chromatin fibers. However, the structure of chromatin fibers is poorly defined and heavily debated. We used single-molecule techniques to probe and manipulate the dynamics of nucleosomes in individual chromatin fibers. These novel methods were initially applied to synthetic, highly homogeneous nucleosomal arrays. We found a strong dependence of the structure of the fibers on the length of the linker DNA, which was corroborated by rigid basepair Monte Carlo simulations. Unfortunately, synthetic chromatin lacks the complexity that provides functionality to our epi-genome. We recently developed a method to purify specific chromatin fragments from yeast without crosslinking the fiber. Magnetic Tweezers based force spectroscopy on intact, native fibers uniquely probes chromatin structure, composition and variations in it at the single-molecule level. Though we observed reduced unfolding forces, the native fibers showed similar stiffness and unfolding pathways as compared to synthetic chromatin. Our systematic single-molecule analysis of a wide range of chromatin compositions supports a general picture of nucleosomes stacking in 1- and 2-start topologies, whose stability is determined by the length of the linker DNA. These experimental results constrain the wide range of chromatin models and bring us closer to ab initio prediction of higher order chromatin folding.
