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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin.  The formation of promyelocytic leukemia (PML) nuclear body on telomeres with chemical dimerizers is demonstrated. Droplet growth, dissolution, localization and composition are monitored with live cell imaging, immunofluorescence (IF) and fluorescence in situ hybridization (FISH).

Abstract

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

Introduction

Many proteins and nucleic acids undergo liquid-liquid phase separation (LLPS) and self-assemble into biomolecular condensates to organize biochemistry in cells1,2. LLPS of chromatin-binding proteins leads to the formation of condensates that are associated with specific genomic loci and are implicated in various local chromatin functions3. For example, LLPS of HP1 protein underlies the formation of heterochromatin domains to organize the genome4,5, LLPS of transcription factors forms transcription centers to regulate transcripti....

Protocol

1. Production of transient cell lines

  1. Culture U2OS acceptor cells on 22 x 22 mm glass coverslips (for live imaging) or 12 mm diameter circular coverslips (for IF or FISH) coated with poly-D-lysine in 6-well plate with growth medium (10% fetal bovine serum and 1% Penicillin-Streptomycin solution in DMEM) until they reach 60-70% confluency.
  2. Replace growth medium with 1 mL transfection medium (growth medium without Penicillin-Streptomycin solution) prior to transfection.
  3. For .......

Representative Results

Representative images of telomeric localization of SUMO identified by telomere DNA FISH and SUMO protein IF are shown in Figure 2. Cells with SIM recruitment enriched SUMO1 and SUMO 2/3 on telomeres compared to cells with SIM mutant recruitment. This indicates that SIM dimerization-induced SUMO enrichment on telomeres depends on SUMO-SIM interactions.

A representative time lapse movie of TRF1 and SIM after dimerization is shown in Video 1. Snapsho.......

Discussion

This protocol demonstrated the formation and dissolution of condensates on telomeres with a chemical dimerization system. Kinetics of phase separation and droplet-fusion-induced telomere clustering are monitored with live imaging. Condensate localization and composition are determined with DNA FISH and protein IF. 

There are two critical steps in this protocol. The first is to determine protein and dimerizer concentration. The success in inducing local phase separation at a genomic locus .......

Acknowledgements

This work was supported by US National Institutes of Health (1K22CA23763201 to H.Z., GM118510 to D.M.C.) and Charles E. Kaufman foundation to H.Z. The authors would like to thank Jason Tones for proofreading the manuscript.

....

Materials

NameCompanyCatalog NumberComments
0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium BicarbonateCorningMT25053CI
16% Formaldehyde (w/v), Methanol-freeThermo Scientific28906Prepare 1% in 1x PBS
6 Well Culture PlateVWR10861-554
Aluminum FoilFisher Scientific01-213-101
Anti-mCherry antibodyAbcamAb183628
Anti-PML antibodySanta Cruzsc966
Anti-SUMO1 antibodyAbcamAb32058
Anti-SUMO2/3 antibodyCytoskeletonAsm23
Blocking ReagentRoche11096176001
Bovine Serum Albumin (BSA)Fisher ScientificBP9706100
BTX Tube micro 1.5ML VWR89511-258
Circle Cover SlipsThermo Scientific3350
Confocal microscope NikonMQS31000
DAPIFisher ScientificD1306
Dimethyl Sulphoxide Sigma-Aldrich472301
DMEM with L-Glutamine, 4.5g/L Glucose and Sodium PyruvateCorningMT10017CV
EMCCD CameraiXon Life 897
EthanolFisher Scientific4355221
Fetal Bovine Serum, Qualified, USDA-approved RegionsGibcoA4766801
Formamide, DeionizedMilliporeSigma46-101-00ML
Goat anti-Mouse IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647InvitrogenA28181
Goat anti-Rabbit IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647InvitrogenA32733
High Precision Straight Tapered Ultra Fine Point Tweezers/ForcepsFisher Scientific12-000-122
Laser merge module NikonNIIMHF47180
Leibovitz's L-15 MediumGibco21083027
Lipofectamine 2000 Transfection ReagentInvitrogen11668027
Figure plotting software, MATLABThe MathWorks
Microscope Slide BoxFisher Scientific34487
Nail PolishFisher Scientific50-949-071
Imaging software, NIS-Elements Nikon
Opti-MEM Reduced Serum MediaGibco51985091
ParafilmBemis13-374-12
PBS 10x, pH 7.4Fisher Scientific70-011-044
Penicillin-Streptomycin Solution,100XGibco15140122
Piezo Z-Drive Physik Instrumente (PI)91985
Pipet TipsVWR10017
Plain and Frosted Clipped Corner Microscope SlidesFisher Scientific22-037-246
Poly-D-Lysine solutionSigma-AldrichA-003-E
Sodium AzideFisher ScientificBP922I-500
Spinning diskYokogawaCSU-X1
Square Cover SlipsThermo Scientific3305
TBS 10x solutionFisher ScientificBP2471500
TelC-Alexa488PNA BioF1004
TMPSynthesized by Chenoweth labAvailable upon request
TNHSynthesized by Chenoweth labAvailable upon request
Tris SolutionFisher Scientific92-901-00ML
Triton X-100 10% SolutionMilliporeSigma64-846-350MLPrepare 0.5% in 1x PBS
U2Os cell lineFrom E.V. Makayev lab (Nanyang Technological University, Singapore)HTB-96
VECTASHIELD Antifade Mounting MediumVector LaboratoriesNC9524612

References

  1. Shin, Y., Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science. 357 (6357), (2017).
  2. Banani, S. F., Lee, H. O., Hyman, A. A., Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry.

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