ConspectusThe fundamental repeating unit of chromatin, the nucleosome, is composed of DNA wrapped around two copies each of four canonical histone proteins. Nucleosomes possess 2-fold pseudo-symmetry that is subject to disruption in cellular contexts. For example, the post-translational modification (PTM) of histones plays an essential role in epigenetic regulation, and the introduction of a PTM on only one of the two "sister"histone copies in a given nucleosome eliminates the inherent symmetry of the complex. Similarly, the removal or swapping of histones for variants or the introduction of a histone mutant may render the two faces of the nucleosome asymmetric, creating, if you will, a type of "Janus"bioparticle. Over the past decade, many groups have detailed the discovery of asymmetric species in chromatin isolated from numerous cell types. However, in vitro biochemical and biophysical investigation of asymmetric nucleosomes has proven synthetically challenging. Whereas symmetric nucleosomes are readily formed via a stochastic combination of their histone and DNA components, asymmetric nucleosome assembly demands the selective incorporation of a single modified/mutant histone copy alongside its wild-type counterpart.Herein we describe the chemical biology tools that we and others have developed in recent years for investigating nucleosome asymmetry. Such approaches, each with its own benefits and shortcomings, fall into five broad categories. First, we discuss affinity tag-based purification methods. These enable the assembly of theoretically any asymmetric nucleosome of interest but are frequently labor-intensive and suffer from low yields. Second, we detail transient cross-linking strategies that are amenable to the preparation of histone H3- or H4-modified/mutant asymmetric species. These yield asymmetric nucleosomes in a traceless fashion, albeit through the use of more complicated synthesis techniques. Third, we describe a synthetic biology technique based on the generation of bump-hole mutant H3 histones that selectively heterodimerize. Although currently developed only for H3 and a related isoform, this method uniquely allows for the interrogation of nucleosome asymmetry in yeast. Fourth, we outline a method for generating H2A- or H2B-modified/mutant asymmetric nucleosomes that relies on the differential DNA-histone contact strength inherent in the Widom 601 DNA sequence. This technique involves the initial formation of hexasomes which are then complemented with distinct H2A/H2B dimers. Finally, we review an approach that utilizes split intein technology to isolate asymmetric H2A- or H2B-modified/mutant nucleosomes. This method shares steps in common with the former but exploits tagged, intein-fused dimers for the facile purification of asymmetric products.Throughout the Account, we highlight various biological questions that drove the development of these methods and ultimately were answered by them. Though each technique has its own shortcomings, collectively these chemical biology tools provide a means to biochemically interrogate a plethora of asymmetric nucleosome species. We conclude with a discussion of remaining challenges, particularly that of endogenous asymmetric nucleosome detection.
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