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.

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities.

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions. While its association with p56lck (Lck), a Src kinase, is important for these functions CD4 also has an Lck-independent role in TCR signaling that is incompletely understood. Here, we identify a conserved GGxxG motif in the CD4 transmembrane domain that is related to the previously described GxxxG motifs of other proteins and predicted to form a flat glycine patch in a transmembrane helix. In other proteins, these patches have been reported to mediate dimerization of transmembrane domains. Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation. However, using Forster’s Resonance Energy Transfer (FRET), we saw no evidence that these mutations decreased CD4 dimerization either in the unliganded state or upon engagement of pMHC concomitantly with the TCR. This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation.

18Mammalian development requires effective mechanisms to repress genes whose expression 19 would generate inappropriately specified cells. The Polycomb Repressive Complex 1 (PRC1) 20 family complexes are central to maintaining this repression 1 . These include a set of canonical 21 PRC1 complexes that each contain four core proteins, including one from the CBX family. These 22 complexes have previously been shown to reside in membraneless organelles called Polycomb 23Here we show that the CBX2 component of canonical PRC1 can phase separate in vitro and 51 generate dynamic puncta in cells. Mutations in CBX2 that impair compaction and proper axial 52 development in mice disrupt phase separation in vitro and formation of puncta in cells. This 53 unites, into a single domain within one component of PRC1, the ability to compact nucleosomes 54 and to phase separate, two functions that might coordinate to generate stable repression. 55We tested various purified PRC1 protein preparations for turbidity, a known 56 characteristic of phase separated solutions 6,8 (Fig. 1b). PRC1 formed a turbid solution in a 57 concentration-dependent manner at near-physiological monovalent salt concentration (100 mM 58 KCl). The CBX2-RING1b heterodimer (heterodimerization is necessary to stabilize full length 59 CBX2) displayed turbidity that was more prominent than other individual PRC1 subunits, 60including RING1b individually. We extended these studies using purified monomeric enhanced 61 GFP (mEGFP) 9 fusions of PRC1 subunits (Extended Data Fig. 1). After centrifugation of 62 purified protein, mEGFP remained distributed throughout the solution, whereas mEGFP-CBX2 63 + RING1b coalesced into a protein-rich pellet (Fig. 1c), indicating that mEGFP-CBX2 + 64RING1b could form a dense phase, separable from bulk solution. Furthermore, fluorescence 65 microscopy revealed the formation of protein-rich foci by purified mEGFP-PRC1 and mEGFP-66 CBX2 + RING1b, while other PRC1 subunits remained diffusely distributed ( Fig. 1d and 67 Extended Data Fig. 2). As seen with other proteins that phase separate 10 , mEGFP-CBX2 + 68RING1b formed spherical droplets that increase in size as a function of concentration (Fig. 1e). 69