J Biosci (2020)45:5 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-019-9978-z ( 0123456789().,-volV)( 0123456789().,-volV) Perspectives

Phase-separation in chromatin organization

GEETA JNARLIKAR Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA

(Email, [email protected])

The organization of chromatin into different types of compact versus open states provides a means to fine tune gene regulation. Recent studies have suggested a role for phase-separation in chromatin compaction, raising new pos- sibilities for regulating chromatin compartments. This perspective discusses some specific molecular mechanisms that could leverage such phase-separation processes to control the functions and organization of chromatin.

Keywords. Histone; nucleosome; heterochromatin; phase-separation; auto-inhibition

The nucleus is a crowded mixture of macromolecules with promote LLPS in model systems (Hancock and Jeon chromatin comprising a dominant component (Hancock 2014). These studies have identified several key determi- and Jeon 2014). Despite such crowding, distinct sophisti- nants that promote LLPS: (i) the polymeric nature of the cated activities occur within the nucleus with high speci- component macromolecules; (ii) the ability of molecules to ficity. Phase-separation, a long-observed property of form multi-valent interactions; (iii) intrinsically disordered macromolecules at high concentration, provides one van- regions (IDRs) within proteins that can participate in tage point to explain how intra-nuclear organization might multivalent interactions; and (iv) macromolecular crowd- occur in a crowded milieu. In its simplest form, phase- ing. The eukaryotic nucleus has an abundance of these separation refers to the process where a mixture of com- determinants. For example, (i) chromatinized DNA pro- ponents in solution de-mix (separate) into two or more vides long polymers, (ii) intrinsically disordered histone distinct phases with different physical and chemical prop- tails participate in multi-valent interactions between erties (Hyman et al. 2014; Alberti et al. 2019). At one nucleosomes, and (iii) macromolecular concentrations extreme, this process describes the non-functional aggre- within a eukaryotic nucleus can reach up to 400 mg/ml gation of misfolded proteins. At another extreme, this (Hancock and Jeon 2014) (figure 1). Consistent with these process describes the formation of functional liquid-like determinants, a series of studies are showing how LLPS compartments formed by the macromolecules that de-mix processes could drive and regulate chromatin organization from solution. This latter form of phase-separation has been within the nucleus (figure 2). Below, I first summarize the termed liquid–liquid phase-separation (LLPS). In LLPS findings of these studies and then discuss how these find- driven states, nucleation and condensation of the compo- ings suggest ideas for mechanisms that regulate phase- nent macromolecules are proposed to drive the formation separation based chromatin organization. of liquid droplets with differentiated material and chemical micro-environments (Hyman et al. 2014; Alberti et al. 1. Uncovering how phase separation mechanisms 2019). Over the last several decades, chemists, physicists can regulate chromatin organization and material scientists have studied the conditions that A substantial portion of the histones extends out from This artice is part of the Topical Collection: Chromatin the folded octamer core in the form of largely Biology and Epigenetics. unstructured N- or C-terminal tails (Luger et al. 1997) http://www.ias.ac.in/jbiosci 5 Page 2 of 5 Geeta J Narlikar

Figure 1. (A) The structure of a nucleosome. DNA is in black. Histone H2A is in red, H2B in yellow, H3 in green and H4 in blue. The H3K9me3 mark is schematically shown as a red circle. (B) Domain diagram of an HP1 dimer. HP1 has two structured domains, the CD, which binds the H3K9me3 mark and the CSD, which forms a dimer that serves as binding interface for various protein ligands. HP1 has three intrinsically disordered regions (IDRs), an N-terminal extension (NTE), a hinge (H) and a C-terminal extension (CTE). Interactions made by the NTE and hinge mediate higher-order HP1 oligomerization.

(figure 1A). Previous work has shown that these tails, chromatin to enable its further compaction (Grewal and which are largely positively charged, participate in Elgin 2002; Canzio et al. 2014; Eissenberg and Elgin inter-nucleosomal interactions that drive the com- 2014). HP1 proteins further recognize chromatin that is paction of long stretches of nucleosomes. Recent work methylated on histone H3 at lysine 9 (figure 1A). On has shown that the interactions mediated by the histone their own HP1 proteins can form long oligomers tails enable chromatin to form phase-separated droplets through interactions mediated by IDRs present within under physiological salt conditions (Gibson et al. HP1 proteins (figure 1B) (Larson et al. 2017). The 2019). This phase-separation process further results in presence of IDR-mediated interactions and the ability substantial condensation of the chromatin. Even more to form multi-valent interactions in the form of oligo- interesting is what the authors find in terms of how mers makes HP1 proteins candidates for phase-sepa- chromatin phases are regulated. They find that the ration. Indeed, work with the human HP1 protein nature of the DNA spacing between the nucleosomes HP1a, showed this protein can phase-separate on its plays a key role in regulating the critical concentration own, but only under conditions that relieved an auto- of chromatin needed for phase-separation. The com- inhibited conformation of the HP1a protein (Larson monly found 10n ? 5 bp spacing is more favorable for et al. 2017). Complementary findings with Drosophila the phase-separation of chromatin compared to the 10n HP1 proteins indicated that HP1-mediated heterochro- bp spacing. Additionally, the authors find that the vis- matin could adopt phase-separated states in vivo and cosity of chromatin phases depends strongly on the in vitro (Strom et al. 2017). Based on these behaviors presence of the linker histone H1, which binds to the identified for HP1 proteins, a simple model can be outside of the nucleosomal unit. proposed wherein the assembly of HP1 proteins on Beyond the genome packaging mediated by chro- chromatin promotes condensation of chromatin into matin formation, additional levels of packaging enable liquid droplets by adding an extra layer of polymeric formation of repressive chromatin states called hete- organization and multi-valent interactions. Yet, recent rochromatin. A conserved mechanism for heterochro- work with the S. pombe HP1 protein, Swi6, shows that matin formation involves the use of proteins, termed HP1 proteins do not just add to the multi-valency of HP1 proteins, that assemble and oligomerize across chromatin. Instead, Swi6 proteins were found to Phase-separation in chromatin organization Page 3 of 5 5 deform individual nucleosomal units to expose buried is thought to be mediated by interactions between the histone interfaces that promoted LLPS of chromatin hinge region of HP1 proteins and the C-terminal (Sanulli et al. 2019). These results have suggested that extension (figure 1). Two types of cues are suggested the buried core of the histone octamer can directly to relieve this autoinhibition: phosphorylation of the participate in the biochemical interactions that drive N-terminal extension (NTE) or binding of the hinge by nucleation and condensation of chromatin. DNA. It is proposed that by binding the positively Some insight into the material properties of chro- charged hinge, the negatively charged phosphorylated matin-based phases formed in cells was provided by a NTE displaces the CTE-hinge contacts and promotes study that used CRISPR-based approaches to target the contacts between HP1a dimers. These interactions are processes of nucleation and condensation by various then proposed to drive multivalent contacts and phase- phase-separating protein fragments to telomeric DNA separation. In the context of DNA binding it is pro- sequences (Shin et al. 2018). This study found that posed that hinge-DNA interactions displace the inhi- phase-separated droplets nucleated at spatially distinct bitory CTE contacts and allow the opened up HP1a regions of the genome could result in the coalescence dimer to form interactions with other HP1 dimers of these regions. The authors propose a mechanism that eventually leading to the multivalency required for involves the fusion of the phases nucleated at different phase-separation. Relief of auto-inhibition could pro- genomic sites. These synthetically engineered phases vide an important control point in the biological reg- thus highlight the potential for phase-separation ulation of HP1a phase-separation. For example, mechanisms to organize genome packaging. The study specific non-chromatin HP1 ligands could either pro- further found that the synthetically generated phases mote or relieve the auto-inhibited state based on could mechanically exclude surrounding regions of developmental cues. Analogously, changes in nuclear chromatin. Interestingly, the type of chromatin most localization of the NTE kinase in response to extra- resistant to mechanical disruption by the engineered cellular signals could switch HP1 between soluble and phase-separating droplets was heterochromatin (Shin phase-separated states. et al. 2018). These results raise the possibility that HP1 and other heterochromatin proteins impart specialized physico-chemical and mechanical properties to this 2.2 Chemical and conformational modifications chromatin state. of chromatin

The work with Swi6 indicates that deformation of the 2. Emerging regulatory mechanisms that control histone core can be a control point for regulating phase- phase-separation of chromatin separation of chromatin (Sanulli et al. 2019). If mole- cules other than Swi6 also deform the nucleosome The recent work highlighted above describes how phase- core, these could regulate chromatin folding by separation-based mechanisms can play a role in genome enabling nucleosome conformations that either pro- organization (figure 2). In the context of these findings it mote or inhibit chromatin compaction. Additionally, becomes relevant to ask the following question: if the qualitative differences in the type of nucleosome conditions in the nucleus are highly conducive to phase- deformation could result in different types of packing separation, how is this fundamental physico-chemical architectures within the compacted chromatin. Similar process controlled to carry out the diverse processes to proteins that bind nucleosomes, one can imagine within the nucleus? The same studies that have provided how chemical modifications on the core histone resi- experimental evidence for chromatin-based phase-sep- dues could directly regulate the conformational state of aration have also uncovered some potential regulatory the nucleosome and have effects that propagate across mechanisms as discussed below. scales to impact the folding of chromatin domains.

2.1 Control through autoinhibition 2.3 Control through nucleosome arrangement

Work with the human HP1a protein showed that in the The finding that the 10n ? 5 bp spacing between absence of any bound ligand or post-translational nucleosomes promotes phase-separation of chromatin modification, this protein exists in a compact autoin- indicates that the specific molecular arrangement of the hibited dimer form (Larson et al. 2017). Autoinhibition repeating nucleosomal units can regulate phase- 5 Page 4 of 5 Geeta J Narlikar

Figure 2. The schematic shows multiple LLPS droplets across a stretch of the genome and highlights different mechanisms for regulating LLPS in the context of chromatin organization. The droplets are shown in blue and grey. HP1 proteins are shown in green, and other hypothetical chromatin regulators are shown in purple and maroon. Nucleosomes in blue are shown schematically as adopting different shapes to represent different conformations. The H3K9 methyl mark is depicted in red. Thicker arrows represent droplets with higher material strength compared to thinner arrows. separation driven chromatin compaction (Gibson et al. nucleosome spacing could either promote or disrupt 2019). Thus, one can imagine that ATP-dependent chromatin compaction by altering the spacing between chromatin remodeling complexes that rearrange nucleosomes (Zhou et al. 2016). 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