Janus Bioparticles: Asymmetric Nucleosomes and Their Preparation Using Chemical Biology Approaches

Abstract

The 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.

Figure 1.

Nucleosome structure and symmetry. (a) Ribbon diagram structure of the nucleosome, composed of two copies of each histone (color-coded) (PDB 1kx5). (b) Comparison of symmetric vs. asymmetric nucleosomes demonstrated by highlighting sister H3 histones in a given nucleosome. Symmetric nucleosomes bear two identical copies of histone H3 (blue), whereas asymmetric nucleosomes bear two different copies (blue and red). (c) Factors contributing to asymmetric nucleosome generation in the cell. Adapted with permission from ref . Copyright (2019) Springer Nature.

Figure 2.

Affinity tag-based methods for generating asymmetric nucleosomes and associated biochemical discoveries. (a) Method utilized by Li and Shogren-Knaack involving the incorporation of a single affinity tag. (b) Method of Voigt et al. in which two affinity tags are used and tandem-affinity purification yields a singular product. (c) SAGA enzymatic cooperativity is mediated by the complex recognition of unmodified lysines on histone H3 tails. (d) H3K4me3 and H3K36me3 inhibit PRC2 deposition of H3K27me3 in cis (on the same histone) but not in trans (on the sister histone).

Figure 3.

Transient cross-linking strategies for synthesizing asymmetric nucleosomes bearing unique (a) H3 or (c) H4 histones and related biochemical findings. First, the lnc-peptide hydrazide is converted to a thioester (i) which is then ligated to a truncated histone protein (H3 or H4 core, respectively) (ii) and desulfurized in situ (iii). Next, the N-terminal cysteine is deprotected (iv) and modified in situ with DTNB (v). A heterodisulfide is then formed (vi). These linked asymmetric histones are refolded with stoichiometric amounts of the other core histones to form cross-linked asymmetric octamers (vii). Finally, nucleosomes are reconstituted, the disulfide bond is reduced (viii), and cross-links are cleaved by the addition of TEV protease (ix). The final two steps are similar for both H3 and H4 asymmetric nucleosome generation and are therefore depicted only in panel (a). Reported yields are indicated. (b) H3K27me3 stimulates PRC2 methyltransferase activity in trans.

Figure 4.

Bump-hole mutant heterodimerization approach and associated biological discoveries. (a) Bump-hole heterodimeric H3 histones selectively dimerize with each other via an altered H3-H3 interface. Depicted are residues mutated to form the bump (H3Y: L109I, C110W) and hole (H3X: L126A, I130V) heterodimeric human histone H3 pair (PDB 1kx5); native residues are shown in translucent light yellow, and bump-hole residues are displayed in dark colors. Asymmetry may then be introduced via the additional mutation of residues within a single histone of the pair (e.g., mH3X). Additional controls are required because bump-hole histone pairs, by design, form a non-native H3-H3 dimer interface. (b) A single H3K36me2 modification (afforded by H3P38V mutation) is sufficient to promote stress tolerance and block cryptic transcription in S. cerevisiae. (c) H3S10A mutation (as a mimetic for loss of serine phosphorylation) inhibits H3K9 acetylation in cis but not in trans in yeast.

Figure 5.

Oriented hexasome-based asymmetric nucleosome assembly and attendant biochemical findings. (a) Formation of DNA sequence-oriented asymmetric nucleosomes based on hexasome complementation with H2A/H2B dimers. (b) Entry-side H2B ubiquitination increases the rate of Chd1-mediated nucleosome remodeling. (c) Asymmetric acidic patch mutant nucleosomes are remodeled differentially by SNF2h based upon mutant location with respect to the DNA overhang.

Figure 6.

Schematic of asymmetric nucleosome assembly using split inteins and associated biochemical/biophysical discoveries. (a) Mixtures of oriented hexasomes and symmetric nucleosomes are combined with tagged N-intein fragment (IntN)-linked H2A/H2B dimers. These asymmetric nucleosomes are affinity-isolated and then tracelessly eluted by on-resin thiolysis via the addition of catalytically dead C-intein fragment (IntC*). Adapted with permission from ref . Copyright (2021) Springer Nature. (b) Asymmetric mutant (H2BE71K or H2BE76K) nucleosomes exhibit increased dimer exchange and decreased thermal stability, albeit to a lesser extent that their symmetric mutant counterparts.