Biophysical Society Thematic Meeting| Les Houches 2019

Multiscale Modeling of Chromatin: Bridging Experiment with Theory

Friday Speaker Abstracts

CHROMATIN HIGHER ORDER FOLDING IN IRREGULAR FIBERS: A CRITICAL ROLE OF LINKER DNA John Van Noort 1 ; 1 Leiden University, Institute of Physics, Leiden, Zuid-Holland, The Netherlands Nucleosome-nucleosome stacking interactions drive nucleosomal arrays into dense fibers though resolving the structure and dynamics of these fibers remains challenging. The linker DNA that connects stacked nucleosome plays an important role in fiber folding due to its limited bending and twisting flexibility. Using single-molecule force spectroscopy, we recently measured the unfolding pathway of regular chromatin fibers with 1 bp increments in linker length and complemented these with rigid-bp Monte Carlo simulations. Both studies reveal a periodic variation of stacking energies. It appears that twist of the linker DNA is much more restrictive for nucleosome stacking than bend, yielding the most stable stacking interactions for linker lengths of multiples of 10 bps. In fibers with short linker DNA (20-30 bps) stacking is compromised, whereas longer linkers (45-55 bps) provide enough flexibility for optimal orientation of stacked nucleosomes. To get a better understanding of the folding of chromatin in vivo, we developed a protocol to pull down specific gene fragments from nuclear extracts, while keeping their native structure and composition intact. Subsequent force spectroscopy yields unfolding curves that allow for detailed comparison with regular reconstituted fibers. Unfolding native fibers of the 18S RNA gene featured larger compositional heterogeneity and signatures of tetrasomes next to full nucleosomes. Though we observed reduced unfolding forces, the native fibers showed similar stiffness and unfolding pathways. Our systematic single-molecule analysis of a large variety 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.

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