Entry created on 1 July 2019 (Revision 1.0) Annotator: Rita Pancsa; Orsolya Kovács
Basic protein information
Accession P45973
Common name HP1α
Gene CBX5
Organism Homo sapiens
Uniprot name Chromobox protein homolog 5
Basic LLPS information
Organelle heterochromatin
Type of experimental evidence
Protein region(s) mediating LLPS
1
-
177
Full length protein without the C-terminal extension
Based on the experimental results of the following publication: 28636604
Molecular features viewer
PDB structures
View on PDBe
Extended LLPS information
Functional description
Constitutive heterochromatin is an important component of eukaryotic genomest hat has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. 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. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension (NTA) or DNA-binding promotes the formation of phase-separated droplets. The LLPs-compatible forms are capable of higher order oligomerisation, while others only form dimers. The phosphorylated residues of NTE in one dimer probably make electrostatic interactions with basic residues in the hinge of another dimer to generate higher-order oligomers. Depending on nuclear context, heterochromatin could exist in a more permissive soluble state or a less permissive phase-separated state. DNA-binding and NTE-phosphorylation could provide qualitatively different means of regulating heterochromatin. Phase-separated HP1α droplets allow the means to physically sequester and compact chromatin while enabling recruitment of repressive factors (PMID:28636604). Solid-state NMR spectroscopy was used to track the conformational dynamics of phosphorylated HP1α during its transformation from the liquid to the gel state. Experiments designed to probe distinct dynamic modes identified regions with varying mobilities within HP1α molecules and show that specific serine residues uniquely contribute to gel formation (PMID:30845353).
Literature supporting the LLPS: 28636604, 30845353, 30471698
Functional class of membraneless organelle: protective storage/reservoir
Binding partners (at biological protein concentrations)
1) DNA (promotes LLPS)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
RNA not required.
Molecular interaction types contributing to LLPS
linear oligomerization/self-association (PMID:28636604) protein-DNA interaction (PMID:28636604) electrostatic (cation-anion) interaction (PMID:28636604)
Determinants of phase separation and droplet properties
1) protein concentration 2) phosphorylation state
Membrane cluster No
Partner-dependent No
RNA-dependent No
PTM required No
Domain-motif interactions No
Discrete oligomerization No
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
Two different phosphorylated versions of HP1α nPhos-HP1α and hPhos-HP1α have been generated in vitro by respectively phosphorylating (protein phosphorylation) HP1α proteins that have their hinge or NTE serine residues mutated to alanine. nPhos-HP1α solution became turbid upon cooling, and revealed liquid droplets (morphology) when investigated by microscopy. The turbid solution became clear upon raising the temperature or upon treatment with alkaline phosphatase (protein dephosphorylation) suggesting reversibility. Wild-type HP1α did not phase-separate upon cooling only in the presence of DNA. nPhos-HP1α forms higher-order oligomers beyond a dimer (physical interaction), so phase separation probably depends on inter-dimer contacts. When mutating a conserved basic patch in the hinge to alanines (residues 89–91, basic patch mutant) phos-HP1α(BPM) was defective for both phosphorylation-driven oligomerization (physical interaction) and phase separation (particle size and count by microscopy). Sequence features of both the hinge and NTE that are specific to HP1α are required for oligomerization. There are several putative inter-HP1α cross-links between the CTE and the hinge, deleting the 14-amino-acid CTE (truncation) in the context of NTE phosphorylation (nPhos-HP1α(ΔCTE) lowers the saturation concentration by approximately tenfold compared to nPhos-HP1α, suggesting that interactions between the CTE and the hinge stabilize the HP1αdimer in a compact auto-inhibited state that cannot make multivalent interactions. Mutating the basic patch in the wild-type HP1α hinge that is proposed to interact with DNA eliminated droplet formation (particle size and count by microscopy). DNA compaction (other change in phenotype/functional readout) appears to be driven largely by electrostatic interactions, as increasing the level of monovalent salts reverses compaction. Results imply that macromolecules that interact with HP1α can remain solvated in the HP1α dominated phase, while others are either excluded or partitioned according to volume. In vivo, in NIH3T3 cells transduced with Cy3-labelled HP1 proteins HP1 formed puncta (PMID:28636604).
Experimental observations supporting the liquid material state of the condensate
temperature-dependence (PMID:28636604) reversibility of formation and dissolution (PMID:28636604)