Abstract
Structural characterization of chromatin is challenging due to conformational and compositional heterogeneity in vivo and dynamic properties that limit achievable resolution in vitro. Although the maximum resolution for solving structures of large macromolecular assemblies by electron microscopy has recently undergone profound increases, X-ray crystallographic approaches may still offer advantages for certain systems. One such system is compact chromatin, wherein the crystalline state recapitulates the crowded molecular environment within the nucleus. Here we show that nucleosomal constructs with cohesive-ended DNA can be designed that assemble into different types of circular configurations or continuous fibers extending throughout crystals. We demonstrate the utility of the method for characterizing nucleosome compaction and linker histone binding at near-atomic resolution but also advance its application for tackling further problems in chromatin structural biology and for generating novel types of DNA nanostructures. We provide a library of cohesive-ended DNA fragment expression constructs and a strategy for engineering DNA-based nanomaterials with a seemingly vast potential variety of architectures and histone chemistries.
Highlights
The eukaryotic genome is packaged into chromatin by an approximately equal mass of histone proteins, which provide a foundation for modulating gene expression, maintaining genomic stability and regulating DNA transactions in general
240 DNA base pairs into nucleosomes, the repeating units of chromatin [1,2]. This chromatin fiber, which can consist of more than one million nucleosomes in tandem for a given chromosome, can in turn be further compacted into higher order structures through the action of a variety of chromatin architectural factors [3,4,5]
We have just scratched the surface of the vast condition and design space available, as we have so far tested this approach for 24 mononucleosome and dinucleosome constructs in the context of either no additional chromatin factors or in the presence of linker histone
Summary
The eukaryotic genome is packaged into chromatin by an approximately equal mass of histone proteins, which provide a foundation for modulating gene expression, maintaining genomic stability and regulating DNA transactions in general. 240 DNA base pairs (bp) into nucleosomes, the repeating units of chromatin [1,2] This chromatin fiber, which can consist of more than one million nucleosomes in tandem for a given chromosome, can in turn be further compacted into higher order structures through the action of a variety of chromatin architectural factors [3,4,5]. These include linker histones, cohesins, condensins, HP1 and CTCF, amongst other factors, which appear to act at different levels of structural hierarchy in condensing nucleosome fiber. Chromatin higher order structure at the level of the organization of interacting/proximal nucleosomes has remained unclear because of conformational and compositional heterogeneity in the cell and dynamic properties that limit achievable resolution in vitro
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