Chemical Dimerization-Induced Protein Condensates on Telomeres

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Chromatin-associated condensates are crucial for nuclear processes, but their formation mechanisms are not fully understood.
• Elucidating the dynamic behavior and composition of these condensates is essential for understanding their function.

Purpose of the Study:

• To present a novel protocol for chemically inducing and reversing protein condensates at specific genomic locations, such as telomeres.
• To enable the real-time investigation of chromatin condensate dynamics, including formation, dissolution, localization, and composition.

Main Methods:

• A chemically induced protein dimerization system using Halo ligand and trimethoprim (TMP) was employed.
• Halo-enzyme fused to telomere proteins anchored the system, while TMP-eDHFR recruited phase-separating proteins to induce condensate formation.
• Condensate reversal was achieved by competing TMP-eDHFR binding with excess free TMP.

Main Results:

• The protocol successfully induced and reversed condensates on telomeres, demonstrated by forming promyelocytic leukemia (PML) nuclear bodies.
• The method allowed for the determination of condensate growth, dissolution, localization, and composition.
• Adaptability to other genomic locations using Halo-fusions or dCas9 targeting was shown.

Conclusions:

• This chemically inducible system provides temporal control over condensate formation and dissolution at specific genomic loci.
• The method is suitable for both single-cell live imaging and population-based biochemical assays, facilitating comprehensive studies of chromatin condensate biology.