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

The X-ray crystal structure of the Head module, one-third of the Mediator of transcriptional regulation, has been determined as a complex with the C-terminal domain (CTD) of RNA polymerase II. The structure reveals multiple points of interaction with an extended conformation of the CTD; it suggests a basis for regulation by phosphorylation of the CTD. Biochemical studies show a requirement for Mediator-CTD interaction for transcription.transcriptional initiation | X-ray crystallography | cross-linking | yeast M ediator, a megaDalton multiprotein complex, enables the regulation of transcription (reviewed in refs 1 and 2); it bridges between gene activator proteins at enhancers and RNA polymerase II (pol II) at promoters. Mediator makes multiple contacts with pol II, including with the carboxyl-terminal domain (CTD) of the Rpb1 subunit and with the Rpb3 subunit (3). The importance of contact with the CTD is suggested by the consequences of CTD truncation. The CTD comprises 27 repeats of a heptapeptide in yeast and 52 repeats of the same sequence in human cells. Truncation of the yeast CTD to 10-12 repeats causes conditional growth phenotypes, and truncation to fewer than 10 repeats results in loss of viability (4). CTD truncation also correlates with a dampened transcriptional response, and CTD lengthening rescues activator protein mutations (5-7). Suppressors of CTD truncation phenotypes identified the first genes for Mediator subunits (8), later shown to be components of the purified Mediator complex (9).Phosphorylation of the CTD plays multiple roles in transcription. Pol II, with an unmodified CTD, enters a preinitiation complex and is phosphorylated on Ser-5 of the heptapeptide repeat by TFIIH (10-12). Phosphorylation disrupts a Mediatorpol II complex in vitro (13), and phosphorylated pol II is devoid of Mediator in vivo (14). Phosphorylation of Ser-5 creates a binding site for the mRNA capping apparatus (15), which acts on nascent transcripts about 25 residues in length, whereupon the transition-to-transcription elongation takes place. Phosphorylation may be obligatory for the transition to elongation, as inhibition of phosphorylation increases stalling of pol II at promoters in vitro (16) and decreases the occupancy of elongating pol II on yeast ORFs in vivo (17). Elongating pol II is dephosphorylated at Ser-5 and rephosphorylated at Ser-2, leading to the recruitment of RNA splicing and cleavage/polyadenylation factors. Recently, the number of physiologically significant CTD modifications has been expanded with Thr-4 phosphorylation, found to function in histone mRNA 3′ processing (18) and mammalian RNA pol II elongation, and Tyr-1 phosphorylation, which appears to affect the association of the CTD with elongation and termination factors (19).Electron microscopy has suggested a division of Mediator in three modules, termed Head, Middle, and Tail (20). Evidence for discrete modules comes from the separate isolation of Head and Middle modules, and from the creation of Tail-less Mediator by deletion of genes f...

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Summary Paragraph During cell division, remodeling of the nuclear envelope (NE) enables chromosome segregation by the mitotic spindle 1 . The reformation of sealed nuclei requires Endosomal Sorting Complexes Required for Transport (ESCRTs) and LEM2, a transmembrane ESCRT adapter 2 – 4 . Here, we show how LEM2’s ability to condense on microtubules governs ESCRT activation and coordinated spindle disassembly. The LEM motif of LEM2 binds barrier-to-autointegration factor (BAF), conferring affinity for chromatin 5 , 6 , while an adjacent low complexity domain (LCD) confers the ability to phase separate. A proline-arginine-rich sequence within the LCD binds microtubules, targeting LEM2 condensation to spindle microtubules traversing the nascent NE. Furthermore, LEM2’s winged-helix (WH) domain activates the ESCRT-II/ESCRT-III hybrid protein, CHMP7, to form co-oligomeric rings. Disrupting these events in cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to nuclear integrity defects and DNA damage. We propose that during nuclear reassembly, LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin, and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins 7 , 8 , suggest that phase separation may contribute to other critical envelope functions, including interphase repair 8 – 13 and chromatin organization 14 – 17 .