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Expression and phase separation potential of heterochromatin proteins during early mouse development.

ABSTRACT: In most eukaryotes, constitutive heterochromatin is associated with H3K9me3 and HP1?. The latter has been shown to play a role in heterochromatin formation through liquid-liquid phase separation. However, many other proteins are known to regulate and/or interact with constitutive heterochromatic regions in several species. We postulate that some of these heterochromatic proteins may play a role in the regulation of heterochromatin formation by liquid-liquid phase separation. Indeed, an analysis of the constitutive heterochromatin proteome shows that proteins associated with constitutive heterochromatin are significantly more disordered than a random set or a full nucleome set of proteins. Interestingly, their expression begins low and increases during preimplantation development. These observations suggest that the preimplantation embryo is a useful model to address the potential role for phase separation in heterochromatin formation, anticipating exciting research in the years to come.

SUBMITTER: Guthmann M

PROVIDER: S-EPMC6893284 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Expression and phase separation potential of heterochromatin proteins during early mouse development.

Guthmann Manuel M Burton Adam A Torres-Padilla Maria-Elena ME

EMBO reports 20191107 12

In most eukaryotes, constitutive heterochromatin is associated with H3K9me3 and HP1α. The latter has been shown to play a role in heterochromatin formation through liquid-liquid phase separation. However, many other proteins are known to regulate and/or interact with constitutive heterochromatic regions in several species. We postulate that some of these heterochromatic proteins may play a role in the regulation of heterochromatin formation by liquid-liquid phase separation. Indeed, an analysis ...[more]

PMID: 31701657

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Project description:Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments. We show that Drosophila HP1a protein undergoes liquid-liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. We conclude that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. We propose that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.

Project description:The octamer motif is a common cis-acting regulatory element that functions in the transcriptional control regions of diverse genes and in viral origins of replication. The ability of a consensus octamer motif to stimulate transcription of a histone H2B promoter in frog oocytes suggests that oocytes contain a transcriptionally active octamer-binding protein(s). We show here that frog oocytes and developing embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer binding-proteins were isolated from Xenopus cDNA libraries by virtue of their homology with the DNA binding (POU) domain of Oct-1. The predicted POU domains of these proteins were most highly related to mammalian Oct-3 (also termed Oct-4), a germ line-specific gene required for mouse early development. Transcripts from these amphibian POU-domain genes were most abundant during early embryogenesis and absent from most adult somatic tissues. One of the genes, termed Oct-60, was primarily expressed as a maternal transcript localized in the animal hemisphere in mature oocytes. The protein encoded by this gene was present in oocytes and early embryos until the gastrula stage of development. Transcripts from a second POU-domain gene, Oct-25, were present at low levels in oocytes and early embryos and were dramatically upregulated during early gastrulation. In contrast to the Oct-60 mRNA, translation of Oct-25 mRNA appeared to be developmentally regulated, since the corresponding protein was detected in embryos during gastrulation but not in oocytes or rapidly cleaving embryos. Transcripts from the third POU protein gene, Oct-91, were induced after the midblastula transition and reached their highest levels of accumulation during late gastrulation. The expression of all three genes decreased during late gastrulation and early neurulation. By analogy with other members of the POU-domain gene family, the products of these genes may play critical roles in the determination of cell fate and the regulation of cell proliferation.

Project description:Elastin-like proteins (ELPs) are known to undergo liquid-liquid phase separation reversibly above a concentration-dependent transition temperature. Previous studies suggested that, as temperature increases, ELPs experience an increased propensity for type II β-turns. However, how the ELPs behave below the phase transition temperature itself is still elusive. Here, we investigate the importance of β-turn formation during the early stages of ELP self-association. We examined the behavior of two ELPs, a 150-repeat construct that had been investigated previously (ELP[V5G3A2-150] as well as a new 40-repeat construct (ELP40) suitable for nuclear magnetic resonance measurements. Structural analysis of ELP40 reveals a disordered conformation, and chemical shifts throughout the sequence are insensitive to changes in temperature over 20°C. However, a low population of β-turn conformation cannot be ruled out based on chemical shifts alone. To examine the structural consequences of β-turns in ELPs, a series of structural ensembles of ELP[V5G3A2-150] were generated, incorporating differing amounts of β-turn bias throughout the chain. To mimic the early stages of the phase change, two monomers were paired, assuming preferential interaction at β-turn regions. This approach was justified by the observation that buried hydrophobic turns are commonly observed to interact in the Protein Data Bank. After dimerization, the ensemble-averaged hydrodynamic properties were calculated for each degree of β-turn bias, and the results were compared with analytical ultracentrifugation experiments at various temperatures. We find that the temperature dependence of the sedimentation coefficient (s20,wo) can be reproduced by increasing the β-turn content in the structural ensemble. This analysis allows us to estimate the presence of β-turns and weak associations under experimental conditions. Because disordered proteins frequently exhibit weak biases in secondary structure propensity, these experimentally-driven ensemble calculations may complement existing methods for modeling disordered proteins generally.

Dataset Information

Expression and phase separation potential of heterochromatin proteins during early mouse development.

Publications

Expression and phase separation potential of heterochromatin proteins during early mouse development.

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