Nucleosomes sterically occlude their wrapped DNA from interaction with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of the nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; but rates for access to the nucleosome interior have not been measured. Here we show that for a selected high affinity nucleosome positioning sequence the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position-dependence to the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics; and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.

The packaging of eukaryotic DNA into chromatin sterically occludes polymerases, recombinases and repair enzymes. How chromatin structure changes to allow their actions is unknown. We constructed defined fluorescently labeled trinucleosome arrays, allowing analysis of chromatin conformational dynamics via fluorescence resonance energy transfer (FRET). The arrays undergo reversible Mg2+-dependent folding like that of longer arrays studied previously. We define two intermediate conformational states in the reversible folding of the nucleosome arrays, and characterize the microscopic rate constants. Nucleosome arrays are highly dynamic even when compact, undergoing conformational fluctuations on seconds to microseconds timescales. Compact states of the arrays allow binding to DNA within the central nucleosome via site exposure. Protein binding can also drive decompaction of the arrays. Thus, our results reveal multiple modes by which spontaneous chromatin fiber dynamics allows for the invasion and action of DNA processing protein complexes.

Cooperativity in transcription factor (TF) binding is essential in eukaryotic gene regulation, and arises through diverse mechanisms. Here we focus on one mechanism, collaborative competition, which is of interest because it arises both automatically, with no requirement for TF co-evolution, and spontaneously, with no requirement for ATP-dependent nucleosome remodeling factors. Previous experimental studies of collaborative competition analyzed cases in which target sites for pairs of cooperating TFs were contained within the same side of the nucleosome. Here we utilize new assays to measure cooperativity in protein binding to pairs of nucleosomal DNA target sites. We focus on the cases that are of greatest in vivo relevance, in which one binding site is located close to the end of a nucleosome, and the other binding site is located at diverse positions throughout the nucleosome. Our results reveal energetically significant positive (favorable) cooperativity for pairs of sites on the same side of the nucleosome, but, for the cases examined, energetically insignificant cooperativity between sites on opposite sides of the nucleosome. These findings imply a special significance for TF binding sites that are spaced within one half nucleosome length (74 bp) or less along the genome, and may prove useful for prediction of cooperatively acting TFs genome-wide.

Macromolecular assemblies and machines undergo large-scale conformational changes as essential features of their normal function. Modern stopped-flow instrumentation and biotechnology combine to provide a powerful tool for characterizing the rates and natures of these conformational changes. Standard commercially available instruments provide extraordinary sensitivity and speed, allowing analysis of millisecond or longer timescale processes, with concentrations as low as a few nanomolar and volumes of just a few hundred microliters. One can now place specific dyes anywhere desired on a nucleic acid, and often on a protein as well. This ability allows the use of fluorescence resonance energy transfer experiments for detailed conformational analyses, even as the system is evolving rapidly over time following the initiation of a reaction. This approach is ideally suited for analysis of intrinsic properties of chromatin and of the machines that control chromatin assembly, disassembly, and function.

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