Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

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. 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 molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although 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 on the basis of nuclear context

HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Keenen, M. M., Brown, D., Brennan, L. D., Renger, R., Khoo, H., Carlson, C. R., Huang, B., Grill, S. W., Narlikar, G. J., & Redding, S. HP1 proteins compact DNA into mechanically and positionally stable phase separated domains. Elife, 10, (2021): e64563, https://doi.org/10.7554/eLife.64563.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.MMK was supported by the Discovery Fellows Program at UCSF and NCI grants F31CA243360 and F99CA245719. RR was support from the NOMIS foundation, Rostock, Germany. BH acknowledges support though NIH R21 GM129652, R01 CA231300 and R01 GM131641. BH is also a Chan Zuckerberg Biohub Investigator. SWG was supported by the DFG (SPP 1782, GSC 97, GR 3271/2, GR 3271/3, GR 3271/4) and the European Research Council (grant 742712). GJN acknowledges support from NIH grant R35 GM127020 and NSF grant 1921794. Support to SR through the UCSF Program for Breakthrough Biomedical Research (PBBR), Sandler Foundation, and Whitman Foundation at the Marine Biological Laboratories

Chapter Three Visualization and Quantitation of Phase-Separated Droplet Formation by Human HP1α

The ability of the heterochromatin protein-1 (HP1) to phase separate into droplets suggests new mechanisms of gene organization in the cell nucleus. An accumulating body of work suggests that other nuclear proteins also display phase separation behaviors in vitro. To understand the mechanistic and biological significance of such droplet formation a rigorous biophysical characterization of this behavior is necessary. Herein we describe procedures for imaging HP1 droplets by brightfield microscopy, and two methods to quantify phase separation

A Transmembrane Domain GGxxG Motif in CD4 Contributes to Its Lck-Independent Function but Does Not Mediate CD4 Dimerization.

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

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

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. 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 molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although 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 on the basis of nuclear context

Mutating the CD4 TMD GGxxG motif does not impair dimerization upon TCR engagement.

<p>(A) Mobility of lipid bilayers was assessed by measuring recovery of streptavidin-PE molecules into a bleached region of the lipid bilayer and normalized to a reference region that was not bleached. (B) FRET_E values for CD28^GFP/Ch, PD1^GFP/Ch and CD4T^GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-E^k). Data are representative of those obtained with two independently generated cell lines. (C) FRET_E values for CD4WT^GFP/Ch vs. CD4T^GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-E^k). Data is concatenated from two independently generated cell lines as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132333#pone.0132333.g004" target="_blank">Fig 4D</a>. (D) FRET_E values for CD4T^GFP/Ch vs. CD4T^TMD-GFP/Ch cells imaged by TIRFM on mobile bilayers containing agonist pMHC (MCC-E^k). Data are representative of those obtained with two independently generated cell lines. Analysis was performed as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132333#pone.0132333.g004" target="_blank">Fig 4</a>. Dots represent single cells and green bars represent median values (*p<0.05, **p<0.001; ***p<0.0001; Mann-Whitney).</p

Mutating the CD4 transmembrane domain GGxxG motif impairs T cell activation.

<p>(A) Alignment of the wild-type CD4 TMD with the CD4 TMD mutant (G403V/G406L). Mutated residues are highlighted in red. Bulky side chains were introduced to disrupt the glycine patch. (B) 58α^-β^- T cell hybridomas were retrovirally transduced with the 5c.c7 TCR and either CD4T, CD4T^TMD or CD4T^Δbind, which is mutated in the region known to bind MHC class II. Surface expression of TCR and CD4 were assessed by flow cytometry as labeled. (C) IL-2 secretion from 58α^-β^- T cell hybridomas after 16 hours of co-culture with M12 cells expressing agonist (MCC), weak agonist (T102S), or null (HB) peptide tethered to I-E^k. Data are representative of four independent experiments with independently generated cell lines. (D) IL-2 secretion from four independently generated CD4T^TMD 58α^-β^- T cell hybridomas after 16 hours of co-culture with MCC:I-E^k+ M12 cells normalized to matched CD4T controls within the same experiment to determine relative IL-2 concentration. (E) IL-2 secretion from 58α^-β^- T cell hybridomas after 16 hours of co-culture with Chinese hamster ovary (CHO) cells ectopically expressing I-E^k (CHO E^k) pulsed with MCC peptide at the indicated concentrations. Data are representative of three independent experiments with independently generated cell lines. (*p<0.05; Mann-Whitney).</p