Chemical Dimerization-Induced Protein Condensates on Telomeres.
J Vis Exp
; (170)2021 04 12.
Article
en En
| MEDLINE
| ID: mdl-33900288
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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Tetrahidrofolato Deshidrogenasa
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Trimetoprim
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Telómero
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Proteínas de Escherichia coli
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Escherichia coli
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Multimerización de Proteína
Límite:
Humans
Idioma:
En
Año:
2021
Tipo del documento:
Article