Name: chemical dimerization-induced protein condensates on telomeres
Uploaded: 2021-04-12T13:00:00
Description: Watch this Scientific Journal Video about Chemical Dimerization-Induced Protein Condensates on Telomeres at JoVE.com

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.

1. Production of transient cell lines
<ol>
	<li>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.</li>
	<li>Replace growth medium with 1 mL transfection medium (growth medium without Penicillin-Streptomycin solution) prior to transfection.</li>
	<li>For ...

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+droplet+formation+by+HP1&#945;+suggests+a+role+for+phase+separation+in+heterochromatin.">Liquid droplet formation by HP1&#945; suggests a role for phase separation in heterochromatin.</a> Nature. 547 (7662), 236-240 (2017).</li><li>Boija, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+activate+genes+through+the+phase-separation+capacity+of+their+activation+domains.">Transcription factors activate genes through the phase-separation capacity of their activation domains.</a> Cell. 175 (7), 1842-1855 (2018).</li><li>Hur, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CDK-Regulated+phase+separation+seeded+by+histone+genes+ensures+precise+growth+and+function+of+histone+locus+bodies.">CDK-Regulated phase separation seeded by histone genes ensures precise growth and function of histone locus bodies.</a> Developmental Cell. 54 (3), 379-394 (2020).</li><li>McSwiggen, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+lengthening+of+telomeres:+DNA+repair+pathways+converge.">Alternative lengthening of telomeres: DNA repair pathways converge.</a> Trends in Genetics. 33 (12), 921-932 (2017).</li><li>Draskovic, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+PML+body+function+in+ALT+cells+reveals+spatiotemporal+requirements+for+telomere+recombination.">Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination.</a> Proceedings of the National Academy of Sciences. 106 (37), 15726 (2009).</li><li>Zhang, J. 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R., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SMC5/6+complex+maintains+telomere+length+in+ALT+cancer+cells+through+SUMOylation+of+telomere-binding+proteins.">The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins.</a> Nature Structural and Molecular Biology. 14 (7), 581-590 (2007).</li><li>Chung, I., Leonhardt, H., Rippe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+assembly+of+a+PML+nuclear+subcompartment+occurs+through+multiple+pathways+and+induces+telomere+elongation.">De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation.</a> Journal of Cell Science. 124 (21), 3603-3618 (2011).</li><li>Shen, T. H., Lin, H. K., Scaglioni, P. P., Yung, T. M., Pandolfi, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitination+and+SUMOylation+in+telomere+maintenance+and+dysfunction.">Ubiquitination and SUMOylation in telomere maintenance and dysfunction.</a> Frontiers in Genetics. 8, 1-15 (2017).</li><li>Sarangi, P., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-mediated+regulation+of+DNA+damage+repair+and+responses.">SUMO-mediated regulation of DNA damage repair and responses.</a> Trends in Biochemical Sciences. 40 (4), 233-242 (2015).</li><li>Banani, S. 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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.&#160;
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 ...

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 transcription6, &#65279; LLPS of nascent mRNAs and multi-sex combs protein generates histone locus bodies to regulate the transcription and processing of histone mRNAs7. &#160;However, despite many examples of chromatin-associated condensates being discovered, the underlying mechanisms of condensate formation, regulation and function remain poorly understood. In particular, not all chromatin-associated condensates are formed through LLPS and careful evaluations of condensate formation in live cells are still needed8,9. For example, &#65279; HP1 protein in mouse is shown to have only a weak capacity to form liquid droplets in live cells and &#65279;heterochromatin foci behave as collapsed polymer globules10. Therefore, tools to induce de novo condensates on chromatin in living cells are desirable, particularly those that allow the use of live imaging and biochemical assays to monitor the kinetics of condensate formation, the physical and chemical properties of the resulting condensates, and the cellular consequences of condensate formation.
This protocol reports a chemical dimerization system to induce protein condensates on chromatin11 (Figure 1A). The dimerizer consists of two linked protein-interacting ligands: trimethoprim (TMP) and Halo ligand and can dimerize proteins fused to the cognate receptors: Escherichia coli dihydrofolate reductase (eDHFR) and a bacterial alkyldehalogenase enzyme (Halo enzyme), respectively12. The interaction between Halo ligand and Halo enzyme is covalent, allowing &#160;Halo enzyme to be used as an anchor by fusing it to a chromatin-binding protein to recruit a phase-separating protein fused to eDHFR to chromatin. After the initial recruitment, increased local concentration of the phase separating protein passes the critical concentration needed for phase separation and thus nucleates a condensate at the anchor (Figure 1B). By fusing fluorescent proteins (e.g. mCherry and eGFP) to eDHFR and Halo, nucleation and growth of condensates can be visualized in real time with fluorescence microscopy. Because the interaction between eDHFR and TMP is non-covalent, excess free TMP can be added to compete with the dimerizer for eDHFR binding. This will then release the phase separation protein from the anchor and dissolve the chromatin-associated condensate.
We used this tool to induce de novo promyelocytic leukemia (PML) nuclear body formation on telomeres in telomerase-negative cancer cells that use an alternative lengthening of telomeres&#160;(ALT) pathway for telomere maintenance13,14. &#160;PML nuclear bodies are membrane-less compartments involved in many nuclear processes15,16 and are uniquely localized to ALT telomeres to form APBs, for ALT telomere-associated PML bodies17,18. Telomeres cluster within APBs, presumably to provide repair templates for homology-directed telomere DNA synthesis in ALT19. Indeed, telomere DNA synthesis has been detected in APBs and APBs play essential roles in enriching DNA repair factors on telomeres20,21. However, the mechanisms underlying APB assembly and telomere clustering within APBs were unknown. Since telomere proteins in ALT cells are uniquely modified by small ubiquitin-like modifier (SUMOs)22, many APB components contain sumoylation sites 22,23,24,25 and/or SUMO-interacting motifs (SIMs)26,27 and SUMO-SIM interactions drive phase separation28, we hypothesized that sumoylation on telomeres leads to enrichment of SUMO/SIM containing proteins and SUMO-SIM interactions between those proteins lead to phase separation. &#160;PML protein, which has three sumoylation sites and one SIM site, can be recruited to sumoylated telomeres to form APBs and coalescence of liquid APBs leads to telomeres clustering. To test this hypothesis, we used the chemical dimerization system to mimic sumoylation-induced APB formation by recruiting SIM to telomeres (Figure 2A)11. GFP is fused to Haloenzyme for visualization and to the telomere-binding protein TRF1 to anchor the dimerizer to telomeres. SIM is fused to eDHFR and mCherry. Kinetics of condensate formation and droplet fusion-induced telomere clustering are followed with live cell imaging. &#160;Phase separation is reversed by adding excess free TMP to compete with eDHFR binding. Immunofluorescence (IF) and fluorescence in situ hybridization (FISH) are used to determine condensate composition and telomeric association. Recruiting SIM enriches SUMO on telomeres and the induced condensates contain PML and therefore are APBs. Recruiting a SIM mutant that cannot interact with SUMO does not enrich SUMO on telomeres or induce phase separation, indicating that the fundamental driving force for APB condensation is SUMO-SIM interaction. Agreeing with this observation, polySUMO-polySIM polymers that fused to a TRF2 binding factor RAP1 can also induce APB formation29. Compared to the polySUMO-polySIM fusion system where phase separation occurs as long as enough proteins are produced, the chemical dimerization approach presented here induces phase separation on demand and thus offers better temporal resolution to monitor the kinetics of phase separation and telomere clustering process. In addition, this chemical dimerization system permits the recruitment of other proteins to assess their ability in inducing phase separation and telomere clustering. For example, a disordered protein recruited to telomeres can also form droplets and cluster telomeres without inducing APB formation, suggesting telomere clustering is independent of APB chemistry and only relies on APB liquid property11.&#160;

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			<td >0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate</td>
			<td >Corning</td>
			<td >MT25053CI</td>
			<td ></td>
		</tr>
		<tr >
			<td >16% Formaldehyde (w/v), Methanol-free</td>
			<td >Thermo Scientific</td>
			<td >28906</td>
			<td >Prepare 1% in 1x PBS</td>
		</tr>
		<tr >
			<td >6 Well Culture Plate</td>
			<td >VWR</td>
			<td >10861-554</td>
			<td ></td>
		</tr>
		<tr >
			<td >Aluminum Foil</td>
			<td >Fisher Scientific</td>
			<td >01-213-101</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-mCherry antibody</td>
			<td >Abcam</td>
			<td >Ab183628</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-PML antibody</td>
			<td >Santa Cruz</td>
			<td >sc966</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-SUMO1 antibody</td>
			<td >Abcam</td>
			<td >Ab32058</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-SUMO2/3 antibody</td>
			<td >Cytoskeleton</td>
			<td >Asm23</td>
			<td ></td>
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			<td >Blocking Reagent</td>
			<td >Roche</td>
			<td >11096176001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumin (BSA)</td>
			<td >Fisher Scientific</td>
			<td >BP9706100</td>
			<td ></td>
		</tr>
		<tr >
			<td >BTX Tube micro 1.5ML&#160;</td>
			<td >VWR</td>
			<td >89511-258</td>
			<td ></td>
		</tr>
		<tr >
			<td >Circle Cover Slips</td>
			<td >Thermo Scientific</td>
			<td >3350</td>
			<td ></td>
		</tr>
		<tr >
			<td >Confocal microscope&#160;</td>
			<td >Nikon</td>
			<td >MQS31000</td>
			<td ></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Fisher Scientific</td>
			<td >D1306</td>
			<td></td>
		</tr>
		<tr >
			<td >Dimethyl Sulphoxide&#160;</td>
			<td >Sigma-Aldrich</td>
			<td >472301</td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td >Corning</td>
			<td >MT10017CV</td>
			<td ></td>
		</tr>
		<tr >
			<td >EMCCD Camera</td>
			<td >iXon Life&#160;</td>
			<td >897</td>
			<td ></td>
		</tr>
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			<td >Ethanol</td>
			<td >Fisher Scientific</td>
			<td >4355221</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum, Qualified, USDA-approved Regions</td>
			<td >Gibco</td>
			<td >A4766801</td>
			<td ></td>
		</tr>
		<tr >
			<td >Formamide, Deionized</td>
			<td >MilliporeSigma</td>
			<td >46-101-00ML</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-Mouse IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647</td>
			<td >Invitrogen</td>
			<td >A28181</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-Rabbit IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647</td>
			<td >Invitrogen</td>
			<td >A32733</td>
			<td ></td>
		</tr>
		<tr >
			<td >High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps</td>
			<td >Fisher Scientific</td>
			<td >12-000-122</td>
			<td ></td>
		</tr>
		<tr >
			<td >Laser merge module&#160;</td>
			<td >Nikon</td>
			<td >NIIMHF47180</td>
			<td ></td>
		</tr>
		<tr >
			<td >Leibovitz&#39;s L-15 Medium</td>
			<td >Gibco</td>
			<td >21083027</td>
			<td ></td>
		</tr>
		<tr >
			<td >Lipofectamine 2000 Transfection Reagent</td>
			<td >Invitrogen</td>
			<td >11668027</td>
			<td ></td>
		</tr>
		<tr >
			<td >Figure plotting software, MATLAB</td>
			<td >The MathWorks</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Microscope Slide Box</td>
			<td >Fisher Scientific</td>
			<td >34487</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nail Polish</td>
			<td >Fisher Scientific</td>
			<td >50-949-071</td>
			<td ></td>
		</tr>
		<tr >
			<td >Imaging software, NIS-Elements&#160;</td>
			<td >Nikon</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Opti-MEM Reduced Serum Media</td>
			<td >Gibco</td>
			<td >51985091</td>
			<td ></td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td >Bemis</td>
			<td >13-374-12</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS 10x, pH 7.4</td>
			<td >Fisher Scientific</td>
			<td >70-011-044</td>
			<td ></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution,100X</td>
			<td >Gibco</td>
			<td >15140122</td>
			<td ></td>
		</tr>
		<tr >
			<td >Piezo Z-Drive&#160;</td>
			<td >Physik Instrumente (PI)</td>
			<td >91985</td>
			<td ></td>
		</tr>
		<tr >
			<td >Pipet Tips</td>
			<td >VWR</td>
			<td >10017</td>
			<td ></td>
		</tr>
		<tr >
			<td >Plain and Frosted Clipped Corner Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >22-037-246</td>
			<td ></td>
		</tr>
		<tr >
			<td >Poly-D-Lysine solution</td>
			<td >Sigma-Aldrich</td>
			<td >A-003-E</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Azide</td>
			<td >Fisher Scientific</td>
			<td >BP922I-500</td>
			<td ></td>
		</tr>
		<tr >
			<td >Spinning disk</td>
			<td >Yokogawa</td>
			<td >CSU-X1</td>
			<td ></td>
		</tr>
		<tr >
			<td >Square Cover Slips</td>
			<td >Thermo Scientific</td>
			<td >3305</td>
			<td ></td>
		</tr>
		<tr >
			<td >TBS 10x solution</td>
			<td >Fisher Scientific</td>
			<td >BP2471500</td>
			<td ></td>
		</tr>
		<tr >
			<td >TelC-Alexa488</td>
			<td >PNA Bio</td>
			<td >F1004</td>
			<td ></td>
		</tr>
		<tr >
			<td >TMP</td>
			<td >Synthesized by Chenoweth lab</td>
			<td ></td>
			<td >Available upon request</td>
		</tr>
		<tr >
			<td >TNH</td>
			<td >Synthesized by Chenoweth lab</td>
			<td ></td>
			<td >Available upon request</td>
		</tr>
		<tr >
			<td >Tris Solution</td>
			<td >Fisher Scientific</td>
			<td >92-901-00ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Triton X-100 10% Solution</td>
			<td >MilliporeSigma</td>
			<td >64-846-350ML</td>
			<td >Prepare 0.5% in 1x PBS</td>
		</tr>
		<tr >
			<td >U2Os cell line</td>
			<td >From E.V. Makayev lab (Nanyang Technological University, Singapore)</td>
			<td >HTB-96</td>
			<td ></td>
		</tr>
		<tr >
			<td >VECTASHIELD Antifade Mounting Medium</td>
			<td >Vector Laboratories</td>
			<td >NC9524612</td>
			<td ></td>
		</tr>
	</tbody>
</table>

chemical dimerization-induced protein condensates on telomeres

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.

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...

Watch this Scientific Journal Video about Chemical Dimerization-Induced Protein Condensates on Telomeres at JoVE.com

Chemical Dimerization-Induced Protein Condensates on Telomeres

This protocol illustrates a chemically induced protein dimerization system to create condensates on chromatin.&#160; 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 ...

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