Chemical Dimerization-Induced Protein Condensates on Telomeres

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 cells^1,2. 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 genome^4,5, 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 .......

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 .......

Materials

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