Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 13;33(2):108248.
doi: 10.1016/j.celrep.2020.108248.

Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging

Samantha Kent  1 , Kyle Brown  1 , Chou-Hsun Yang  1 , Njood Alsaihati  1 , Christina Tian  1 , Haobin Wang  1 , Xiaojun Ren  2
Affiliations

Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging

Samantha Kent et al. Cell Rep. .

Abstract

Compartmentalization by liquid-liquid phase separation is implicated in transcription. It remains unclear whether and how transcriptional condensates accelerate the search of transcriptional regulatory factors for their target sites. Furthermore, the molecular mechanisms by which regulatory factors nucleate on chromatin to assemble transcriptional condensates remain incompletely understood. The CBX-PRC1 complexes compartmentalize key developmental regulators for repression through phase-separated condensates driven by the chromobox 2 (CBX2) protein. Here, by using live-cell single-molecule imaging, we show that CBX2 nucleates on chromatin independently of H3K27me3 and CBX-PRC1. The interactions between CBX2 and DNA are essential for nucleating CBX-PRC1 on chromatin to assemble condensates. The assembled condensates shorten 3D diffusion time and reduce trials for finding specific sites through revisiting the same or adjacent sites repetitively, thereby accelerating CBX2 in searching for target sites. Overall, our data suggest a generic mechanism by which transcriptional regulatory factors nucleate to assemble condensates that accelerate their target-search process.

Keywords: CBX2; PRC1; PcG; chromatin; compartmentalization; epigenetics; liquid-liquid phase separation; nucleation; single-molecule imaging; target-search kinetics.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. CBX2 Drives the LLPS of CBX-PRC1
(A) CBX-PRC1 complexes in mESCs. (B) Example fluorescence images for CBX-PRC1 proteins fused with HaloTag in mESCs. Scale bar, 5.0 μm. (C) Numbers of condensates of HaloTag-PRC1 fusion proteins quantified from (B). Error bars represent SD. (D) Measuring residence times by live-cell single-molecule tracking. The red arrowhead indicates molecules that bind stably to chromatin (τsb), and the green arrowhead represents molecules that bind transiently to chromatin (τtb). Scale bar, 2.0 μm. (E–G) Survival probability distribution of the dwell times. HT-NLS, HaloTag fused with nuclear localization sequence. The numbers of cells and trajectories used are listed in Table S1. (H–J) Specific residence times (τsb) quantified from (E)–(G). Non-specific residence times (τtb) are shown in Figure S1. Error bars represent standard error for the derived parameter.
Figure 2.
Figure 2.. The Binding Stability of CBX2 Is Independent of PRC1 and PRC2
(A) Schematic representation of CBX2 and its variants. CD denotes chromodomain; AT, AT-hook; SRR, serine-rich region; ATL, AT-hook-like; HPCR, highly positively charged region; and Cbox, Chromobox. (B) Sketch of the CBX2-PRC1 complex. The Cbox motif of CBX2 interacts with RING1B. (C and F) Survival probability distribution of the dwell times. The numbers of cells and trajectories used are listed in Table S1. (E) The hypothesis tests whether H3K27me3 affects the binding stability of CBX2 through interaction with the CD motif of CBX2. (D and G) Specific residence times (τsb) quantified from (C) and (F). Non-specific residence times (τtb) are shown in Figure S2. Error bars represent standard error for the derived parameter.
Figure 3.
Figure 3.. Effects of Mutation and Deletion on the Condensate Formation and Binding Stability of CBX2
(A) Schematic representation of CBX2 variants used in this study. The underlined residues highlighted in red were mutated to Ala (P2A, positively charged residues to Ala; S2A, Ser to Ala; N2A, negatively charged residues to Ala) or Glu (S2E, Ser to Glu). (B) Survival probability distribution of the dwell times for CBX2 and variants, respectively. The numbers of cells and trajectories used are listed in Table S1. (C) Specific residence times (τsb) for CBX2 and its variants quantified from (B). Non-specific residence times (τsb) are shown in Figure S3. Error bars represent standard error for the derived parameter. (D) Schematic representation of the elements of CBX2 that mediate the interactions with chromatin. The AT motif stabilizes CBX2 on chromatin. The ATL motif also contributes to the stabilization of CBX2 on chromatin but to a lesser extent compared with the AT motif. The HPCR motif antagonizes the binding stability of CBX2 on chromatin.
Figure 4.
Figure 4.. CBX2 Binds DNA, which Promotes LLPS In Vitro and In Vivo
(A) Determination of the binding of CBX2 and CBX2AT-P2A to DNA by EMSA. (B) Quantification of EMSA gel from (A) to estimate the dissociation constant of CBX2 to DNA. (C) Example DIC images of CBX2 condensates on the surface of coverslip in the absence or presence of PEG, DNA, or both. Scale bar, 5.0 μm. (D) Number of condensates quantified from (C). Error bars represent SD. (E) Example live-cell epifluorescence images of CBX2 and its variants with impaired DNA-binding capacity. Scale bars, 2.0 μm.
Figure 5.
Figure 5.. The Target-Search Process of CBX2 and Its Variants
(A) Schematic representation of the quantification of the target-search process. See STAR Methods for details. (B–E) F1sb (B), Ntrial (C), τ3D (D), and τsearch (E) for CBX2 and its variants in wild-type mESCs and for CBX2 in PcG-knockout mESCs as well as for the control HT-NLS in wild-type mESCs. Displacement histograms are in Figure S5. The numbers of cells and displacements used are listed in Table S1. Error bars represent SD.
Figure 6.
Figure 6.. LLPS Speeds up the Target-Search Process of CBX2
(A) Example live-cell epifluorescence images for CBX2 and its variants in wild-type mESCs as well as for CBX2 in PcG-knockout mESCs. Scale bar, 5.0 μm. (B) CLLPS for CBX2 and its variants in wild-type mESCs as well as for CBX2 in PcG-knockout mESCs. Error bars represent SD. (C–E) Dependence of τtb, τsb, Ntrial, τ3D, τsearch, and F1sb on CLLPS for CBX2 and its variants.
Figure 7.
Figure 7.. LLPS Alters the Target-Search Pathway
(A) Representative overlay images of SMT trajectories on epifluorescence images of CBX2. The images are represented as total trajectories (left), trajectories not bound to chromatin (middle), and trajectories bound to chromatin (right). The black circles indicate the start position of trajectories. The colors of trajectories are randomly assigned for each image. Scale bar, 5.0 μm. (B) Percentile of chromatin-bound CBX2 molecules that are inside and outside of condensates. Error bars represent SD. (C) Examples of the angular distribution between consecutive steps of single-molecule tracking traces. (D) Representative angular distribution for diffusive CBX2 inside and outside of condensates as well as for CBX2AT-P2A, CBX289–532, and HT-NLS in whole cells. The major ticks of radial scale are 0.6%, 1.2%, and 1.8%. (E) Quantification of the relative probability of moving backward compared with moving forward ([180° ± 30°]/[0° ± 30°]) for diffusive CBX2 inside and outside of condensates as well as for CBX2AT-P2A, CBX289–532, and HT-NLS in whole cells. Error bars represent SD. (F) A proposed mechanism underpinning that CBX2 undergoes LLPS to form condensates, which then speeds up the target-search kinetics of CBX2, thereby enhancing its genomic occupancy. Our data indicate that phase-separated condensates shorten the target-search process through reducing the 3D free diffusion time and the number of non-specific trials.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alberti S, Gladfelter A, and Mittag T (2019). Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell 176, 419–434. - PMC - PubMed
    1. Annunziata O, Asherie N, Lomakin A, Pande J, Ogun O, and Benedek GB (2002). Effect of polyethylene glycol on the liquid-liquid phase transition in aqueous protein solutions. Proc. Natl. Acad. Sci. U S A 99, 14165–14170. - PMC - PubMed
    1. Aranda S, Mas G, and Di Croce L (2015). Regulation of gene transcription by Polycomb proteins. Sci. Adv 1, e1500737. - PMC - PubMed
    1. Banani SF, Rice AM, Peeples WB, Lin Y, Jain S, Parker R, and Rosen MK (2016). Compositional control of phase-separated cellular bodies. Cell 166, 651–663. - PMC - PubMed
    1. Banani SF, Lee HO, Hyman AA, and Rosen MK (2017). Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol 18, 285–298. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources