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KMT5C displays robust retention and liquid-like behavior in phase separated heterochromatin

Hilmar Strickfaden, Kristal Missiaen, Michael J. Hendzel, D. Alan Underhill

Posted on: 30 September 2019 , updated on: 1 October 2019

Preprint posted on 20 September 2019

Another liquid state - heterochromatin protein KMT5C displays liquid behavior only within its functional context at the pericentromere.

Selected by Carmen Adriaens

Cellular compartmentalization goes much beyond the canonical membrane-delineated organelles such as mitochondria, the nucleus and the ER. In fact, we now think of the cell as a liquid demixed environment in which the protein, lipid, and nucleic acid components move freely but coordinately to achieve cellular function [1].

For example, DNA-dense heterochromatic and DNA-sparse euchromatic regions in the nucleus are highly discrete, and many RNA- and DNA-binding as well as structural proteins are involved in this dynamic compartmentalization process through phase separation [1]. For instance, it has been shown that phase separation of HP1, the reader protein for the heterochromatin mark H3K9me2/3, is essential for structural genesis and maintenance of the transcriptionally inactive B-compartment [2,3], and that transcription ‘hubs’ are defined by phase-separated close interactions between RNA pol II, Mediator, and the targeted (super)enhancer/promoter DNA [4,5]. In addition, nucleic-acid rich nuclear bodies such as PML bodies, paraspeckles, nucleoli, and splicing speckles all achieve their partitioning through liquid demixing of (some) of their components [6].

 

In this preprint, Strickfaden et al. [7] describe a novel phase-separated state for the heterochromatin component lysine methyltransferase 5C (KMT5C). Specifically, they find that in contrast to HP1, which rapidly exchanges with the surrounding nucleoplasm, KMT5C only behaves as a liquid within heterochromatic foci. The key experiment to show this is photobleaching of pericentromeres, which, due to their repetitive nature, are tightly compacted into heterochromatic chromocenter bodies. Thus, where whole chromocenter photobleaching causes only low level, slow recovery of KMT5C intensities, partial photobleaching causes a rapid replenishment of the full chromocenter area.

The authors further dissect the molecular basis of these observations through examination of the protein sequence. They find that, unlike HP1a and MeCP2, another protein that displays phase separation and is associated with heterochromatin, KMT5C lacks extended disordered regions, suggesting that another mechanism is at play to drive the liquid-demixing capabilities of this protein. Despite the lack of disordered regions, the Chromocenter Retention Domain (CRD), the amino acid sequence essential for targeting KMT5C to chromocenters, is known and conserved, and was found by the authors to behave similarly (i.e. “locally liquid-like”) in all species studied.

Finally, the authors established that the interaction of KMT5C with chromatin is essential for its dynamic behavior, reminiscent of HP1a needing H3K9me3 and its chromatin binding domain to achieve phase separation in heterochromatic regions of the nucleus. When KMT5C is released from the chromocenters by treatment with a histone deacetylase inhibitor shown to decrease heterochromatin accumulations, a marked spreading of H4K20me3 (KMT5C’s catalytic product) is observed, indicative of the fact that KMT5C’s spatial distribution is key to restrain its enzymatic activity.

 

What is remarkable about these findings is that the authors describe a less mobile, spatially constrained state of the protein, that, importantly, does not acquire gel-like or solid state-like properties. With this, they expand our understanding of the spectrum over which protein physical states can vary. Furthermore, they strengthen the idea that sometimes a given protein won’t be specifically targeted to a sequence motif in the DNA, but rather its partitioning and specificity will depend on the biophysical properties of the environment. This can, for instance, explain early conflicting observations that loss of heterochromatin also leads to an increase of the H4K20me3 throughout the nucleus during aging [8,9].

Thus, the question remains of not only why, but also how sequence specificity is achieved to confer compartmentalization of certain regions of the genome into more tightly packed heterochromatin (see also this preLight on the study by [10]). One can further ask, given the striking differences in nuclear organization between mouse and human nuclei, how much of these processes are conserved, and how they have evolved to make differently shaped nuclei in different species, yet maintaining a similar identity, function, developmental process and gene expression pattern in differentiated tissues. These findings also warrant a more holistic view of the nucleus, and certainly, studying timed heterochromatin formation during dynamic processes such as cell division and differentiation will help us to better understand the 3D architecture of the nucleus and the cell as a whole.

 

References:

[1] Boeynaems et al. (2018) Trends Cell Biol. 28(6):420-435. doi: 10.1016/j.tcb.2018.02.004. Protein Phase Separation: A New Phase in Cell Biology.

[2] Strom et al., (2017) Nature. 13;547(7662):241-245. doi: 10.1038/nature22989. Phase separation drives heterochromatin domain formation.

[3] Larson et al. (2017) Nature. 13;547(7662):236-240. doi: 10.1038/nature22822. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

[4] Cho, Spille et al. (2018) Science. 361(6400):412-415. doi: 10.1126/science.aar4199. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.

[5] Sabari, Dall’Agnese et al. (2018) Science. 361(6400). pii: eaar3958. doi: 10.1126/science.aar3958. Coactivator condensation at super-enhancers links phase separation and gene control.

[6] Sawyer et al. (2019) Semin Cell Dev Biol. 90:94-103. doi: 10.1016/j.semcdb.2018.07.001. Phase separated microenvironments inside the cell nucleus are linked to disease and regulate epigenetic state, transcription and RNA processing.

[7] Strickfaden et al. (2019) bioRxiv. doi: https://doi.org/10.1101/776625.  KMT5C displays robust retention and liquid-like behavior in phase separated heterochromatin

[8] Chicas et al. (2012) Proc Natl Acad Sci U S A. 109:8971–6. H3K4 demethylation by Jarid1a and Jarid1b contributes to retinoblastoma-mediated gene silencing during cellular senescence.

[9] Sarg et al. (2002) J Biol Chem. 277:39195–201. Postsynthetic trimethylation of histone H4 at lysine 20 in mammalian tissues is associated with aging.

[10] Boumendil et al. (2019) Genes Dev. 33(3-4):144-149. doi: 10.1101/gad.321117.118. Nuclear pore density controls heterochromatin reorganization during senescence.

Tags: heterochromatin, liquid behavior, methyl transferase

doi: https://doi.org/10.1242/prelights.14259

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Author's response

prof. Alan D. Underhill shared

The idea that pericentromeric heterochromatin comprises a dynamically exchanging protein population was established in 2003 with studies of heterochromatin protein-1 (1). At the time, it seemed paradoxical that what appeared as a stable structure in the nucleus was maintained by a highly mobile protein. Nearly 15-years later, this behavior was reconciled with the demonstration that HP1 undergoes liquid-liquid demixing (2, 3). At the other extreme, the lysine methyltransferases SUV39H2 and KMT5C appeared immobile within in the same compartment, suggesting they formed a stable scaffold (4, 5). All of these studies used fluorescence recovery after photobleaching (FRAP) to measure protein dynamics in mouse cells where pericentromeres assemble into mesoscale structures called chromocenters. Typically, a circular zone surrounding the entire chromocenter would be selected for photobleaching and fluorescence recovery would be monitored over time. The rate at which proteins recovered was used to infer their mobility (or immobility) and forms our current understanding of chromocenter protein dynamics. FRAP is also a critical tool in assessing liquid-liquid phase separation in cells and has established that rapid exchange of protein constituents is a general property of membraneless organelles (6). Our preprint, however, documents a divergent behavior for KMT5C where it is effectively biocontained within the chromocenter, while nevertheless displaying liquid-like properties (7). As often happens in research, the observation that formed the basis of this study was serendipitous. Our standard photobleaching setup involves using a rectangular area because it simplifies the quantification of diffusion coefficients and residence times (8). In this specific case, the rectangle spanned the width of the nucleus and was sufficiently tall to encompass an entire chromocenter, which inevitably led to partial bleaching of nearby chromocenters. Upon doing so, it was immediately obvious that partially bleached chromocenters recovered fluorescence internally, while fully bleached chromocenters did not, and the story developed from there.

 

  1. T. Cheutin et al., Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 299, 721-725 (2003).
  2. A. G. Larson et al., Liquid droplet formation by HP1alpha suggests a role for phase separation in heterochromatin. Nature 547, 236-240 (2017).
  3. A. R. Strom et al., Phase separation drives heterochromatin domain formation. Nature 547, 241-245 (2017).
  4. M. Hahn et al., Suv4-20h2 mediates chromatin compaction and is important for cohesin recruitment to heterochromatin. Genes Dev 27, 859-872 (2013).
  5. K. Muller-Ott et al., Specificity, propagation, and memory of pericentric heterochromatin. Molecular systems biology 10, 746 (2014).
  6. M. Mir, W. Bickmore, E. E. M. Furlong, G. Narlikar, Chromatin topology, condensates and gene regulation: shifting paradigms or just a phase? Development 146, (2019).
  7. H. Strickfaden, K. Missiaen, M. J. Hendzel, D. A. Underhill, KMT5C displays robust retention and liquid-like behavior in phase separated heterochromatin. bioRxiv, 776625 (2019).
  8. G. Carrero, E. Crawford, M. J. Hendzel, G. de Vries, Characterizing fluorescence recovery curves for nuclear proteins undergoing binding events. Bulletin of mathematical biology 66, 1515-1545 (2004).

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