Uncharged Components of Single-Stranded DNA Modulate Liquid-Liquid Phase Separation With Cationic Linker Histone H1.

Masahiro Mimura,Shunsuke Tomita,Sayaka Ishihara,Yoichi Shinkai,Ryoji Kurita,Hiroka Sugai

doi:10.3389/fcell.2021.710729

Abstract

Liquid–liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

Highlights

Liquid–liquid phase separation (LLPS) triggered by interactions between biomacromolecules has increasingly been recognized as a possible mechanism governing the formation of biomolecular condensates, such as germline P granules (Brangwynne et al, 2009) and stress granules (Boeynaems et al, 2018)
We used a model system in which liquid droplets are formed by mixing linker histone H1 (H1) and single-stranded DNA (ssDNA) (Figure 1A; Shakya and King, 2018b; Turner et al, 2018; Mimura et al, 2021)
The C-terminal intrinsically disordered region (IDR) has been proposed to play an important role in the organization of chromatin domains via a LLPS process involving interactions with inter-nucleosome linker DNAs (Gibson et al, 2019; Shakya et al, 2020)

Summary

Introduction

Liquid–liquid phase separation (LLPS) triggered by interactions between biomacromolecules has increasingly been recognized as a possible mechanism governing the formation of biomolecular condensates, such as germline P granules (Brangwynne et al, 2009) and stress granules (Boeynaems et al, 2018) These condensates are often referred to as ‘liquid droplets’ due to their fluidic properties (Brangwynne et al, 2009; Uversky, 2017) and are distinct from the solid-like aggregates (Patel et al, 2015). DNA binding is essential for LLPS of the transcriptional repressor VRN1 (Zhou et al, 2019) and the chromatin component histone proteins (Gibson et al, 2019; Shakya et al, 2020) In many of these examples, the interactions of DNA with nonstructural cationic polypeptide chains are subject to LLPS. Given the potential diverse involvement of DNA in current and primitive biological LLPS, it is crucial to understand which properties of DNA are most involved in the process in order to elucidate the molecular mechanisms of biological LLPS

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Discussion

Conclusion