Abstract
The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg2+-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.
Highlights
The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown
We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated
This observation, based on analysis of relatively short 20and 25-bp linkers typical for yeast genome, poses new questions: Whether the effect on DNA topology could be extended to larger DNA linkers such as those found in eukaryotic cells and whether the nucleosomes in topologically constrained circular nucleosome arrays would adopt a folded structure substantially different from those found in linear arrays
Summary
L, linker DNA length; NRL, nucleosome repeat length; EMANIC, EM-assisted Nucleosome Interaction Capture; DNP, deoxynucleoprotein; AUC, analytical ultracentrifugation; TEM, transmission EM; Lk, linking number; CQ, chloroquine; sc, supercoils; MC, Monte Carlo; FINI, frequency of internucleosome interactions; Topo I, topoisomerase I. Using precisely positioned clone 601– based circular nucleosome arrays, we have conducted topological analysis showing that a 5-bp difference leading to rotational change of half turns between nucleosomes could strongly affect DNA topology in the circular arrays [27] This observation, based on analysis of relatively short 20and 25-bp linkers typical for yeast genome, poses new questions: Whether the effect on DNA topology could be extended to larger DNA linkers such as those found in eukaryotic cells and whether the nucleosomes in topologically constrained circular nucleosome arrays would adopt a folded structure substantially different from those found in linear arrays. Based on our experiments and modeling, we suggest that the {10n} DNA linker length may play an important role in formation of folded chromatin fiber structures and DNA topology typical of heterochromatin, whereas {10nϩ5} linkers support the more flexible and amenable chromatin structures, providing new implications for regulation of chromatin accessibility to DNA binding factors and RNA transcription machinery
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