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Chronically persistent viruses are integral components of the organismal ecosystem in humans and animals 1 2 . Many of these viruses replicate and accumulate within the cell nucleus 3 . The nuclear location allows viruses to evade cytoplasmic host viral sensors and promotes viral replication 4 . One of the unexplored and puzzling aspects of the viral nuclear lifecycle involves the virus's ability to deal with the physical constraints of nuclear architecture. To replicate and accumulate within the nucleus in large numbers sufficient for infection spreading, DNA viruses need to overcome the spatial limitations imposed by chromatin and the nuclear matrix. We found that one of the most widespread and potentially lethal human viruses, the JC polyomavirus 5 , interferes with nuclear heterochromatin to create virus-occupied space. The JC virus's impact on heterochromatin is mediated by the viral nonstructural protein, Agnoprotein (Agno). Agno's interference with heterochromatin is governed by structurally diverse mimics of host epigenetic regulators that facilitate virus-induced chromatin reorganization and a dramatic decline in nuclear stiffness in the infected cells. The JCV epigenetic mimicry is critical for the virus infection, as evident from reduced replication of mimic-mutant viruses. Our data suggest that modulation of nuclear mechanical properties is a novel strategy enabling chronicity of the JC and possibly other nuclear virus infections.

ATP-dependent chromatin remodeling enzymes mobilize nucleosomes, but how such mobilization affects chromatin condensation is unclear. Here, we investigate effects of two major remodelers, ACF and RSC using chromatin condensates and single-molecule footprinting. We find that both remodelers inhibit the formation of condensed chromatin. However, the remodelers have distinct effects on pre-formed chromatin condensates. ACF spaces nucleosomes without de-condensing the chromatin, explaining how ACF maintains nucleosome organization in transcriptionally repressed genomic regions. In contrast, RSC catalyzes ATP-dependent de-condensation of chromatin. Surprisingly, RSC also drives micron-scale movements of entire condensates. These newly uncovered activities of RSC explain its central role in transcriptional activation. The biological importance of remodelers may thus reflect both their effects on nucleosome mobilization and the corresponding consequences on chromatin dynamics at the mesoscale., Competing Interests: Competing interests: G.J.N is a co-founder of TippingPoint Biosciences.

The essential architectural protein HMGB1 increases accessibility of nucleosomal DNA and counteracts the effects of linker histone H1. However, HMGB1 is less abundant than H1 and binds nucleosomes more weakly raising the question of how HMGB1 effectively competes with H1. Here, we show that HMGB1 rescues H1's inhibition of nucleosomal DNA accessibility without displacing H1. HMGB1 also increases the dynamics of condensed, H1-bound chromatin. Cryo-EM shows that HMGB1 binds at internal locations on a nucleosome and locally distorts the DNA. These sites, which are away from the binding site of H1, explain how HMGB1 and H1 co-occupy a nucleosome. Our findings lead to a model where HMGB1 counteracts the activity of H1 by distorting nucleosomal DNA and by contacting the H1 C-terminal tail. Compared to direct competition, nucleosome co-occupancy by HMGB1 and H1 allows a greater diversity of dynamic chromatin states and may be generalizable to other chromatin regulators., Competing Interests: Declaration of Interests Y.C. is scientific advisory board member of ShuiMu BioSciences. G.J.N. is a founder and scientific advisory board member of TippingPoint Biosciences.

It is long known that an RNA polymerase transcribing through a nucleosome can generate subnucleosomal particles called hexasomes. These particles lack an H2A-H2B dimer, breaking the symmetry of a nucleosome and revealing new interfaces. Whether hexasomes are simply a consequence of RNA polymerase action or they also have a regulatory impact remains an open question. Recent biochemical and structural studies of RNA polymerases and chromatin remodelers with hexasomes motivated us to revisit this question. Here, we build on previous models to discuss how formation of hexasomes can allow sophisticated regulation of transcription and also significantly impact chromatin folding. We anticipate that further cellular and biochemical analysis of these subnucleosomal particles will uncover additional regulatory roles., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitate collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general., (© 2024. The Author(s).)

Liquid-liquid phase separation (LLPS) is driven by weak multi-valent interactions. Such interactions can result in the formation of puncta in cells and droplets in vitro . The heterochromatin protein HP1α forms droplets with chromatin in vitro and is found in puncta in cells. A common approach to visualize the dynamics of HP1α in cells is to genetically encode fluorescent tags on the protein. HP1α modified with tags such as GFP has been shown to localize to dynamic puncta in vivo . However, whether tagged HP1α retains its intrinsic phase separation properties has not been systematically studied. Here, using different C-terminal tags (AID-sfGFP, mEGFP, and UnaG), we assessed how tag size and linker length affected the phase separation ability of HP1α with DNA in vitro . We found that the AID-sfGFP tag (52 kDa) promoted HP1α phase-separation, possibly driven by the highly disordered AID degron. The mEGFP tag (27 kDa) inhibited phase-separation by HP1α, whereas an UnaG tag (13 kDa) with a 16 amino acid linker showed minimal perturbation. The UnaG tag can thus be used in cellular studies of HP1α to better correlate in vitro and in vivo studies. To test if cellular crowding overcomes the negative effects of large tags in vivo , we used polyethylene glycol (PEG) to mimic crowding in vitro . We found that addition of 10% PEG8000 or PEG4000 enables phase separation by GFP-tagged HP1α at comparable concentrations as untagged HP1α. However, these crowding agents also substantially dampened the differences in phase-separation between wild-type and mutant HP1α proteins. PEG further drove phase-separation of Maltose Binding Protein (MBP), a tag often used to solubilize other proteins. These results suggest that phase-separation of biological macromolecules with PEG should be interpreted with caution as PEG-based crowding agents may change the types of interactions made within the phases.

Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology., (© 2023. The Author(s).)

Unlike other chromatin remodelers, INO80 preferentially mobilizes hexasomes, which can form during transcription. Why INO80 prefers hexasomes over nucleosomes remains unclear. Here, we report structures of Saccharomyces cerevisiae INO80 bound to a hexasome or a nucleosome. INO80 binds the two substrates in substantially different orientations. On a hexasome, INO80 places its ATPase subunit, Ino80, at superhelical location -2 (SHL -2), in contrast to SHL -6 and SHL -7, as previously seen on nucleosomes. Our results suggest that INO80 action on hexasomes resembles action by other remodelers on nucleosomes such that Ino80 is maximally active near SHL -2. The SHL -2 position also plays a critical role for nucleosome remodeling by INO80. Overall, the mechanistic adaptations used by INO80 for preferential hexasome sliding imply that subnucleosomal particles play considerable regulatory roles.

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). To address this issue, we developed graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitated collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general., Competing Interests: Competing Interests The authors declare no competing interests.

Nuclear DNA in eukaryotes is wrapped around histone proteins to form nucleosomes on a chromatin fiber. Dynamic folding of the chromatin fiber into loops and variations in the degree of chromatin compaction regulate essential processes such as transcription, recombination, and mitotic chromosome segregation. Our understanding of the physical properties that allow chromatin to be dynamically remodeled even in highly compacted states is limited. Previously, we reported that chromatin has an intrinsic capacity to phase separate and form dynamic liquid-like condensates, which can be regulated by cellular factors [B. A. Gibson et al. , Cell 179 , 470-484.e421 (2019)]. Recent contradictory reports claim that a specific set of solution conditions is required for fluidity in condensates that would otherwise be solid [J. C. Hansen, K. Maeshima, M. J. Hendzel, Epigenetics Chromatin 14 , 50 (2021); H. Strickfaden et al. , Cell 183 , 1772-1784.e1713 (2020)]. We sought to resolve these discrepancies, as our ability to translate with confidence these biophysical observations to cells requires their precise characterization. Moreover, whether chromatin assemblies are dynamic or static affects how processes such as transcription, loop extrusion, and remodeling will engage them inside cells. Here, we show in diverse conditions and without specific buffering components that chromatin fragments form phase separated fluids in vitro. We also explore how sample preparation and imaging affect the experimental observation of chromatin condensate dynamics. Last, we describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained chromatin movement observed in cells.

ATP-dependent chromatin remodelers are essential enzymes that restructure eukaryotic genomes to enable all DNA-based processes. The diversity and complexity of these processes arethe complexity of the enzymes that carry them out, making remodelers a challenging class of molecular motors to study by conventional methods. Here we use a single molecule biophysical assay to overcome some of these challenges, enabling a detailed mechanistic dissection of a paradigmatic remodeler reaction, that of sliding a nucleosome towards the longer DNA linker. We focus on how two motors of a dimeric remodeler coordinate to accomplish such directional sliding. We find that ATP hydrolysis by both motors promotes coordination, suggesting a role for ATP in resolving the competition for directional commitment. Furthermore, we show an artificially constitutive dimer is no more or less coordinated, but is more processive, suggesting a cell could modulate a remodeler's oligomeric state to modulate local chromatin dynamics., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1ɑ droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin's biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates., (© 2022. The Author(s).)

The critical role of the INO80 chromatin remodeling complex in transcription is commonly attributed to its nucleosome sliding activity. Here, we have found that INO80 prefers to mobilize hexasomes over nucleosomes. INO80's preference for hexasomes reaches up to ∼60 fold when flanking DNA overhangs approach ∼18-bp linkers in yeast gene bodies. Correspondingly, deletion of INO80 significantly affects the positions of hexasome-sized particles within yeast genes in vivo. Our results raise the possibility that INO80 promotes nucleosome sliding by dislodging an H2A-H2B dimer, thereby making a nucleosome transiently resemble a hexasome. We propose that this mechanism allows INO80 to rapidly mobilize nucleosomes at promoters and hexasomes within gene bodies. Rapid repositioning of hexasomes that are generated in the wake of transcription may mitigate spurious transcription. More generally, such versatility may explain how INO80 regulates chromatin architecture during the diverse processes of transcription, replication, and repair., Competing Interests: Declaration of interests Geeta Narlikar is on the Molecular Cell advisory board., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Packaging of the eukaryotic genome into chromatin places fundamental physical constraints on transcription. Clarifying how transcription operates within these constraints is essential to understand how eukaryotic gene expression programs are established and maintained. Here we review what is known about the mechanisms of transcription on chromatin templates. Current models indicate that transcription through chromatin is accomplished by the combination of an inherent nucleosome disrupting activity of RNA polymerase and the action of ATP-dependent chromatin remodeling motors. Collaboration between these two types of molecular motors is proposed to occur at all stages of transcription through diverse mechanisms. Further investigation of how these two motors combine their basic activities is essential to clarify the interdependent relationship between genome structure and transcription., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Biological enzymes significantly speed up chemical reactions in living organisms. The complex environment within cells has long been appreciated as a major regulator of enzymatic activities. Recent advances in the rapidly evolving field of biological condensates, which are spontaneously formed by macromolecules through phase separation, suggest new possibilities for how enzymatic reactions may be modulated within cells. Here, we review the latest studies of enzymatic reactions in biological condensates focusing on basic concepts in enzymology and discussing some context-dependent roles of phase separation in regulating biochemical reactions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Liquid-liquid phase separation (LLPS) has been invoked as an underlying mechanism involved in the formation and function of several cellular membrane-less compartments. Given the explosion of studies in this field in recent years, it has become essential to converge on clear guidelines and methods to rigorously investigate LLPS and advance our understanding of this phenomenon. Here, we describe basic methods to (1) visualize droplets formed by nucleic acid binding proteins and (2) characterize the liquid-like nature of these droplets under controlled in vitro experimental conditions. We discuss the rationale behind these methods, as well as caveats and limitations. Our ultimate goal is to guide scientists interested in learning how to test for LLPS, while appreciating that the field is evolving rapidly and adjusting constantly to the growing knowledge. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Observing phase-separated condensates by microscopy. Support Protocol: Coating of glass-bottom plates. Basic Protocol 2: Assessing condensate reversibility by changing ionic strength. Alternate Protocol 1: Assessing condensate reversibility by dilution. Alternate Protocol 2: Assessing condensate reversibility by altering temperature. Basic Protocol 3: Quantifying phase separation by centrifugation assay. Basic Protocol 4: Quantifying phase separation by turbidity assay., (© 2021 Wiley Periodicals LLC.)

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog's DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale., Competing Interests: MK, DB, LB, RR, HK, CC, BH, SG, GN, SR No competing interests declared, (© 2021, Keenen et al.)

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure., Competing Interests: NA, CM, LH, SK, AK, LE, MK, HG, VR No competing interests declared, JU JGU is an employee of Pacific Biosciences, Inc and holds stock in this company. GN Reviewing editor, eLife, (© 2020, Abdulhay et al.)

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA) n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence-dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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 possibilities 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.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeF x that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn't show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeF x predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation., Competing Interests: JA, NG, SJ, SW, YC No competing interests declared, JL Is affiliated with 3T Biosciences and has no other competing interests to declare, GN Reviewing editor, eLife, (© 2019, Armache et al.)

In eukaryotes, a first step towards the nuclear DNA compaction process is the formation of a nucleosome, which is comprised of negatively charged DNA wrapped around a positively charged histone protein octamer. Often, it is assumed that the complexation of the DNA into the nucleosome completely attenuates the DNA charge and hence the electrostatic field generated by the molecule. In contrast, theoretical and computational studies suggest that the nucleosome retains a strong, negative electrostatic field. Despite their fundamental implications for chromatin organization and function, these opposing views of nucleosome electrostatics have not been experimentally tested. Herein, we directly measure nucleosome electrostatics and find that while nucleosome formation reduces the complex charge by half, the nucleosome nevertheless maintains a strong negative electrostatic field. Our studies highlight the importance of considering the polyelectrolyte nature of the nucleosome and its impact on processes ranging from factor binding to DNA compaction., Competing Interests: MG, SJ, DH No competing interests declared, GN Reviewing editor, eLife, (© 2019, Gebala et al.)

DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity., (Copyright © 2018. Published by Elsevier Inc.)

Heterochromatin is a classic context for studying the mechanisms of chromatin organization. At the core of a highly conserved type of heterochromatin is the complex formed between chromatin methylated on histone H3 lysine 9 and HP1 proteins. This type of heterochromatin plays central roles in gene repression, genome stability, and nuclear mechanics. Systematic studies over the last several decades have provided insight into the biophysical mechanisms by which the HP1-chromatin complex is formed. Here, we discuss these studies together with recent findings indicating a role for phase separation in heterochromatin organization and function. We suggest that the different functions of HP1-mediated heterochromatin may rely on the increasing diversity being uncovered in the biophysical properties of HP1-chromatin complexes., (© 2019 Sanulli et al.; Published by Cold Spring Harbor Laboratory Press.)

In eukaryotic cells, structures called heterochromatin play critical roles in nuclear processes ranging from gene repression to chromosome segregation. Biochemical and in vivo studies over the past several decades have implied that the diverse functions of heterochromatin rely on the ability of these structures to spread across large regions of the genome, to compact the underlying DNA, and to recruit different types of activities. Recent observations have suggested that heterochromatin may possess liquid droplet-like properties. Here, we discuss how these observations provide a new perspective on the mechanisms for the assembly, regulation, and functions of heterochromatin.

ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell., Competing Interests: NG, SJ, MT, AB No competing interests declared, GN Reviewing editor, eLife, (© 2018, Gamarra et al.)

The yeast INO80 chromatin remodeling complex plays essential roles in regulating DNA damage repair, replication, and promoter architecture. INO80's role in these processes is likely related to its ability to slide nucleosomes, but the underlying mechanism is poorly understood. Here we use ensemble and single-molecule enzymology to study INO80-catalyzed nucleosome sliding. We find that the rate of nucleosome sliding by INO80 increases ∼100-fold when the flanking DNA length is increased from 40 to 60 bp. Furthermore, once sliding is initiated, INO80 moves the nucleosome rapidly at least 20 bp without pausing to re-assess flanking DNA length, and it can change the direction of nucleosome sliding without dissociation. Finally, we show that the Nhp10 module of INO80 plays an auto-inhibitory role, tuning INO80's switch-like response to flanking DNA. Our results indicate that INO80 is a highly processive remodeling motor that is tightly regulated by both substrate cues and non-catalytic subunits., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The ability of the heterochromatin protein-1 (HP1) to phase separate into droplets suggests new mechanisms of gene organization in the cell nucleus. An accumulating body of work suggests that other nuclear proteins also display phase separation behaviors in vitro. To understand the mechanistic and biological significance of such droplet formation a rigorous biophysical characterization of this behavior is necessary. Herein we describe procedures for imaging HP1 droplets by brightfield microscopy, and two methods to quantify phase separation., (© 2018 Elsevier Inc. All rights reserved.)

Heterochromatin protein 1 (HP1) family proteins are conserved chromatin binding proteins involved in gene silencing, chromosome packaging, and chromosome segregation. These proteins recognize histone H3 lysine 9 methylated tails via their chromodomain and recruit additional ligand proteins with diverse activities through their dimerization domain, the chromoshadow domain. Species that have HP1 proteins possess multiple paralogs that perform non-overlapping roles in vivo. How different HP1 proteins, which are highly conserved, perform different functions is not well understood. Here, we use the two Schizosaccharomyces pombe HP1 paralogs, Swi6 and Chp2, as model systems to compare and contrast their biophysical properties. We find that Swi6 and Chp2 have similar dimerization and oligomerization equilibria, and that Swi6 binds slightly (~3-fold) more strongly to nucleosomes than Chp2. Furthermore, while Swi6 binding to the H3K9me3 mark is regulated by a previously described auto-inhibition mechanism, the binding of Chp2 to the H3K9me3 mark is not analogously regulated. In the context of chromoshadow domain interactions, we show using a newly identified peptide sequence from the Clr3 histone deacetylase and a previously identified sequence from the protein Shugoshin that the Swi6 chromoshadow domain binds both ligands more strongly than the Chp2. Overall, our findings uncover quantitative differences in how Swi6 and Chp2 interact with nucleosomal and non-nucleosomal ligands and qualitative differences in how their assembly on nucleosomes is regulated. These findings provide a biochemical framework to explain the varied functions of Chp2 and Swi6 in vivo., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.

The hexameric AAA+ ATPases Rvb1 and Rvb2 (Rvbs) are essential for diverse processes ranging from metabolic signaling to chromatin remodeling, but their functions are unknown. While originally thought to act as helicases, recent proposals suggest that Rvbs act as protein assembly chaperones. However, experimental evidence for chaperone-like behavior is lacking. Here, we identify a potent protein activator of the Rvbs, a domain in the Ino80 ATPase subunit of the INO80 chromatin-remodeling complex, termed Ino80INS. Ino80INS stimulates Rvbs' ATPase activity by 16-fold while concomitantly promoting their dodecamerization. Using mass spectrometry, cryo-EM, and integrative modeling, we find that Ino80INS binds asymmetrically along the dodecamerization interface, resulting in a conformationally flexible dodecamer that collapses into hexamers upon ATP addition. Our results demonstrate the chaperone-like potential of Rvb1/Rvb2 and suggest a model where binding of multiple clients such as Ino80 stimulates ATP-driven cycling between hexamers and dodecamers, providing iterative opportunities for correct subunit assembly., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adenosine 5'-triphosphate (ATP)-dependent chromatin remodeling enzymes play essential biological roles by mobilizing nucleosomal DNA. Yet, how DNA is mobilized despite the steric constraints placed by the histone octamer remains unknown. Using methyl transverse relaxation-optimized nuclear magnetic resonance spectroscopy on a 450-kilodalton complex, we show that the chromatin remodeler, SNF2h, distorts the histone octamer. Binding of SNF2h in an activated ATP state changes the dynamics of buried histone residues. Preventing octamer distortion by site-specific disulfide linkages inhibits nucleosome sliding by SNF2h while promoting octamer eviction by the SWI-SNF complex, RSC. Our findings indicate that the histone core of a nucleosome is more plastic than previously imagined and that octamer deformation plays different roles based on the type of chromatin remodeler. Octamer plasticity may contribute to chromatin regulation beyond ATP-dependent remodeling., (Copyright © 2017, American Association for the Advancement of Science.)

Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

ATP-dependent chromatin remodeling complexes carry out diverse transformations of chromatin. Understanding their mechanisms requires assays that can monitor the kinetics or chromatin remodeling. In this chapter, we describe complimentary native gel-based and FRET-based methods for assaying the kinetics of ATP-driven nucleosome sliding. These methods can be readily adapted to investigate other types of nucleosomal transformations carried out by chromatin remodeling ATPases., (© 2016 Elsevier Inc. All rights reserved.)

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

The ATP-dependent chromatin assembly factor (ACF) spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Heterochromatin protein 1 (HP1) proteins were originally identified as critical components in heterochromatin-mediated gene silencing and are now recognized to play essential roles in several other processes including gene activation. Several eukaryotes possess more than one HP1 paralog. Despite high sequence conservation, the HP1 paralogs achieve diverse functions. Further, in many cases, the same HP1 paralog is implicated in multiple functions. Recent biochemical studies have revealed interesting paralog-specific biophysical differences and unanticipated conformational versatility in HP1 proteins that may account for this functional promiscuity. Here we review these findings and describe a molecular framework that aims to link the conformational flexibility of HP1 proteins observed in vitro with their functional promiscuity observed in vivo., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chromatin provides both a means to accommodate a large amount of genetic material in a small space and a means to package the same genetic material in different chromatin states. Transitions between chromatin states are enabled by chromatin-remodeling ATPases, which catalyze a diverse range of structural transformations. Biochemical evidence over the last two decades suggests that chromatin-remodeling activities may have emerged by adaptation of ancient DNA translocases to respond to specific features of chromatin. Here, we discuss such evidence and also relate mechanistic insights to our understanding of how chromatin-remodeling enzymes enable different in vivo processes., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

In Schizosaccharomyces pombe, heterochromatin spread, which is marked by histone 3 lysine 9 methylation (H3K9me), requires the chromodomains (CDs) of the H3K9 methylase Suv39/Clr4 and the HP1/Swi6 protein. It is unclear how the actions of these two H3K9me-recognizing CDs are coordinated. We find that the intrinsic preference of Suv39/Clr4 is to generate dimethylated H3K9 product. The recognition of pre-existing H3K9me marks by the CD of Suv39/Clr4 stimulates overall catalysis, enabling the accumulation of small amounts of trimethylated product in vivo. Coincidentally, the Suv39/Clr4 CD, unlike the HP1/Swi6 CD, has been shown to prefer the trimethyl state over the dimethyl state. We show that this preference enables efficient heterochromatin spread in vivo by reducing competition with HP1 proteins for the more prevalent dimethyl state. Our results reveal a strategy by which "writers" and "readers" of a chromatin mark exploit different methylation states on the same residue in order to facilitate collaboration and avoid competition., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions. Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 methyl mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymerization processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.

Jumonji histone demethylases catalyze removal of methyl marks from lysine residues in histone proteins within nucleosomes. Here, we show that the catalytic domain of demethylase JMJD2A (cJMJD2A) utilizes a distributive mechanism to remove the histone H3 lysine 9 trimethyl mark. By developing a method to assess demethylation of homogeneous, site-specifically methylated nucleosomes, we determined that the kinetic parameters for demethylation of nucleosomes by cJMJD2A are comparable to those of peptide substrates. These findings imply that other domains of the demethylase or its protein partners may contribute to nucleosome recognition in vivo and, in this way, may further regulate demethylation activity and processivity. The quantitative assays of nucleosome demethylation developed in our work provide a platform for future work with complex chromatin substrates and full-length demethylases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)