Gene silencing by heterochromatin is proposed to occur in part from the ability of HP1 proteins to spread across large regions of the genome, compact the underlying chromatin and recruit repressive activities1–3. 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 other molecules such as the transcription factor TFIIB show no preference. Using single-molecule DNA curtains we find that unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, though 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 based on nuclear context.

Heterochromatin impacts genome function at multiple scales. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus 1,2. It has been proposed that these diverse functions arise in part from compaction of the underlying chromatin. A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates 3-6. Yet how HP1mediated phase separation relates to chromatin compaction remains unclear. Here we demonstrate that chromatin compaction by the S. pombe HP1 protein, Swi6, results in phase-separated liquid condensates. Remarkably, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs chromatin compaction by Swi6 into liquid droplets. Our results indicate that Swi6 couples oligomerization to the phase separation of chromatin by a counter-intuitive mechanism, namely dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

SUMMARY A complete, 52-protein, 2.5 million Dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of crosslinked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders.Protein Prospector addresses these issues through a complementary mass modification strategy in which each peptide is searched and identified separately. We demonstrate this strategy with an analysis of RNA polymerase II. False discovery rates (FDRs) are assessed via comparison of cross-linking data to crystal structure, as well as by using a decoy database strategy. Parameters that are most useful for positive identification of cross-linked spectra are explored. We find that fragmentation spectra generally contain more product ions from one of the two peptides constituting the cross-link. Hence, metrics reflecting the quality of the spectral match to the less confident peptide provide the most discriminatory power between correct and incorrect matches. A support vector machine model was built to further improve classification of cross-linked peptide hits. Furthermore, the frequency with which peptides cross-linked via common acylating reagents fragment to produce diagnostic, cross-linkerspecific ions is assessed.The threshold for successful identification of the cross-linked peptide product depends upon the complexity of the sample under investigation. Protein Prospector, by focusing the reliability assessment on the least confident peptide, is better able to control the FDR for results as larger complexes and databases are ana- Most proteins are organized into stable assemblies that communicate among themselves through transient proteinprotein interaction networks to catalyze cellular phenomena. Chemical cross-linking mass spectrometry directly measures protein-protein interactions by using bifunctional cross-linking reagents to covalently link surfaces of interacting partners (1-3). Following proteolysis, mass spectrometry is used to identify the covalently linked peptides and modified residues. This information, taken together with the geometry of the cross-linking reagent, provides distance constraints that are reflective of the three-dimensional structure of the protein complex. Cross-linking-derived distance constraints provide a powerful means by which to integrate atomic resolution structures of individual protein subunits or subassemblies with low-resolution electron-microscopy-derived structures, as well as to clarify molecular details that are unresolved in electron density maps. For instance, this approach has recently been applied to modeling the RNA Pol II preinitiation complex (4), several chromatin remodeling complexes (5, 6), th...

The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex.DOI: http://dx.doi.org/10.7554/eLife.08719.001