Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation

Erdel, Fabian; Rademacher, Anne; Vlijm, Rifka; Tünnermann, Jana; Frank, Lukas; Weinmann, Robin; Schweigert, Elisabeth; Yserentant, Klaus; Hummert, Johan; Bauer, Caroline; Schumacher, Sabrina; Alwash, Ahmad Al; Normand, Christophe; Herten, Dirk‐Peter; Engelhardt, Johann; Rippe, Karsten

doi:10.1016/j.molcel.2020.02.005

Cited by 254 publications

(301 citation statements)

References 70 publications

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“…It is important to note that a recent study has called into question the biological importance of the ability of HP1-a to phase-separate in vitro on its in vivo function, for example the failure of HP1-a over-expression to increase the size of heterochromatic domains as predicted for simple LLPS systems (Erdel et al, 2020). However, the complexity of feedback within heterochromatin is rife with paradox, for example the requirement for siRNA transcription to drive chromatin compaction and silencing in fission yeast (Yamanaka et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, Swi6 recruits the H3K9me demethylase Epe1 almost exclusively into heterochromatic domains (Zofall and Grewal, 2006), which would be expected to decrease available H3K9me and thereby delocalize H3K9meengaged Swi6 molecules, likely short-circuiting the ability of elevated Swi6 levels to drive heterochromatin spreading. Moreover, the binding of HP1-/Swi6 to H3K9me and its subsequent compaction of heterochromatin involves an integrated set of activities driven by its (1) chromodomain avidity, (2) dimerization, (3) engagement of nucleosome arrays, and (4) its phase separation properties, all of which make it challenging to arrive at mechanistic conclusions without clear separation-of-function mutants or through engineered systems (Erdel et al, 2020). For this reason, the swi6-sm1 allele and others of its kind that disrupt specific functionalities will be essential in future studies.…”

Section: Discussionmentioning

confidence: 99%

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The condensation of HP1-α/Swi6 imparts nuclear stiffness

Williams

Surovtsev

Schreiner

et al. 2020

Preprint

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SummaryLiquid-liquid phase separation (LLPS) has emerged as a major driver of cellular organization. However, it remains unexplored whether the mechanical properties of LLPS domains are functionally important. The heterochromatin protein HP1-α (and the orthologous Swi6 in S. pombe) is capable of LLPS in vitro and promotes formation of LLPS heterochromatin domains in vivo. Here, we demonstrate that LLPS of Swi6 contributes to the emergent mechanical properties of nuclei. Using nuclear fluctuation analysis in live cells and force spectroscopy of isolated nuclei, we find that disrupting histone H3K9 methylation or depleting Swi6 compromises nuclear stiffness, while heterochromatin spreading through loss of the H3K9 demethylase, Epe1, increases nuclear stiffness. Leveraging a separation-of-function allele, we demonstrate that phase separation of Swi6—but not its histone binding or dimerization—is essential for Swi6’s mechanical role. These findings demonstrate that altering chromatin state has mechanical consequences and highlights that phase-separated domains can do mechanical work.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The condensation of HP1-α/Swi6 imparts nuclear stiffness

Williams

Surovtsev

Schreiner

et al. 2020

Preprint

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“…KMT5C, a histone H4 lysine 20 methyltransferase, shows liquid behavior within chromocenters but does not exchange with the nucleoplasm (Strickfaden et al, 2019). In contrast, CBX5, which can phase separate at high concentrations in vitro (Larson et al, 2017;Strom et al, 2017), was found to move rapidly into and out of mouse chromocenters (Erdel et al, 2020;Strom et al, 2017). We therefore performed twochannel FRAP experiments on TAMRA-dUTP labelled cells transfected with fluorescent protein tags on KMT5C or CBX5 ( Figure 2B, C).…”

Section: Replication Labeling Reveals Solid-like Behavior Of Euchromamentioning

confidence: 99%

Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells

Strickfaden

Tolsma

Sharma

et al. 2020

Preprint

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The association of nuclear DNA with histones to form chromatin is essential to the temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of chromatin in vitro and in vivo. Our in vitro studies demonstrate that MgCl2-dependent self-association of native chromatin fragments or reconstituted nucleosomal arrays produced supramolecular condensates whose constituents are physically constrained and solid-like. Liquid chromatin condensates could be generated in vitro, but only using non-physiological conditions. By measuring DNA mobility within heterochromatin and euchromatin in living cells, we show that chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, displayed liquid-like behavior and coalesced around a solid chromatin scaffold. Remarkably, both euchromatin and heterochromatin showed solid-like behavior even when transmission electron microscopy revealed limited interactions between chromatin fibers. Our results therefore argue that chromatin is not liquid but exists in a solid-like material state whose properties are tuned by fiber-fiber interactions. INTRODUCTION:

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“…However further work is needed to decipher the physical nature of these foci. Interestingly, heterochromatin foci in mouse cells were first described as liquid droplets (Strom et al 2017), but a recent study indicates that these foci rather resemble collapsed polymer globules (Erdel et al 2020). Whether specific heterochromatin factors are responsible for this collapse is not known, but HP1, the functional homolog of Sir3 does not seem required.…”

Section: Mechanisms Of Sir3 Mediated Trans-interactionsmentioning

confidence: 99%

The silencing factor Sir3 is a molecular bridge that sticks together distant loci

Ruault

Scolari

Lazar‐Stefanita

et al. 2020

Preprint

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ABSTRACTPhysical contacts between distant loci contribute to regulate genome function. However, the molecular mechanisms responsible for settling and maintaining such interactions remain poorly understood. Here we investigate the well conserved interactions between heterochromatin loci. In budding yeast, the 32 telomeres cluster in 3-5 foci in exponentially growing cells. This clustering is functionally linked to the formation of heterochromatin in subtelomeric regions through the recruitment of the silencing complex SIR composed of Sir2/3/4. Combining microscopy and Hi-C on strains expressing different alleles of SIR3, we show that the binding of Sir3 directly promotes long range contacts between distant regions, including the rDNA, telomeres, and internal Sir3 bound sites. Furthermore, we unveil a new property of Sir3 in promoting rDNA compaction. Finally, using a synthetic approach we demonstrate that Sir3 can bond loci belonging to different chromosomes together, when targeted to these loci, independently of its interaction with its known partners (Rap1, and Sir4), Sir2 activity or chromosome context. Altogether these data suggest that Sir3 represents an uncommon example of protein able to bridge directly distant loci.

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Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation

Cited by 254 publications

References 70 publications

The condensation of HP1-α/Swi6 imparts nuclear stiffness

The condensation of HP1-α/Swi6 imparts nuclear stiffness

Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells

The silencing factor Sir3 is a molecular bridge that sticks together distant loci

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