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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Chemical Dimerization-Induced Protein Condensates on Telomeres

Published: April 12th, 2021

DOI:

10.3791/62173

1Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University, 2Department of Chemistry, School of Arts and Sciences, University of Pennsylvania

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).

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.

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....

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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 .......

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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.......

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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 .......

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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.

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Name Company Catalog Number Comments
0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate Corning MT25053CI
16% Formaldehyde (w/v), Methanol-free Thermo Scientific 28906 Prepare 1% in 1x PBS
6 Well Culture Plate VWR 10861-554
Aluminum Foil Fisher Scientific 01-213-101
Anti-mCherry antibody Abcam Ab183628
Anti-PML antibody Santa Cruz sc966
Anti-SUMO1 antibody Abcam Ab32058
Anti-SUMO2/3 antibody Cytoskeleton Asm23
Blocking Reagent Roche 11096176001
Bovine Serum Albumin (BSA) Fisher Scientific BP9706100
BTX Tube micro 1.5ML  VWR 89511-258
Circle Cover Slips Thermo Scientific 3350
Confocal microscope  Nikon MQS31000
DAPI Fisher Scientific D1306
Dimethyl Sulphoxide  Sigma-Aldrich 472301
DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate Corning MT10017CV
EMCCD Camera iXon Life  897
Ethanol Fisher Scientific 4355221
Fetal Bovine Serum, Qualified, USDA-approved Regions Gibco A4766801
Formamide, Deionized MilliporeSigma 46-101-00ML
Goat anti-Mouse IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647 Invitrogen A28181
Goat anti-Rabbit IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647 Invitrogen A32733
High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps Fisher Scientific 12-000-122
Laser merge module  Nikon NIIMHF47180
Leibovitz's L-15 Medium Gibco 21083027
Lipofectamine 2000 Transfection Reagent Invitrogen 11668027
Figure plotting software, MATLAB The MathWorks
Microscope Slide Box Fisher Scientific 34487
Nail Polish Fisher Scientific 50-949-071
Imaging software, NIS-Elements  Nikon
Opti-MEM Reduced Serum Media Gibco 51985091
Parafilm Bemis 13-374-12
PBS 10x, pH 7.4 Fisher Scientific 70-011-044
Penicillin-Streptomycin Solution,100X Gibco 15140122
Piezo Z-Drive  Physik Instrumente (PI) 91985
Pipet Tips VWR 10017
Plain and Frosted Clipped Corner Microscope Slides Fisher Scientific 22-037-246
Poly-D-Lysine solution Sigma-Aldrich A-003-E
Sodium Azide Fisher Scientific BP922I-500
Spinning disk Yokogawa CSU-X1
Square Cover Slips Thermo Scientific 3305
TBS 10x solution Fisher Scientific BP2471500
TelC-Alexa488 PNA Bio F1004
TMP Synthesized by Chenoweth lab Available upon request
TNH Synthesized by Chenoweth lab Available upon request
Tris Solution Fisher Scientific 92-901-00ML
Triton X-100 10% Solution MilliporeSigma 64-846-350ML Prepare 0.5% in 1x PBS
U2Os cell line From E.V. Makayev lab (Nanyang Technological University, Singapore) HTB-96
VECTASHIELD Antifade Mounting Medium Vector Laboratories NC9524612

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