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Quantifying the global binding and target-search dynamics of epigenetic regulatory factors using live-cell single-molecule tracking

ABSTRACT: Summary This protocol provides instructions to track the global dynamics of single epigenetic regulatory factors in live cells. We describe an approach to generate cell lines that stably express HaloTag-fused proteins. We then use live-cell single-molecule tracking to obtain kinetic populations and residence times. The kinetic parameters obtained can be used to determine important aspects of transcriptional regulation such as target-search time, 3D free diffusion time, and number of non-specific sites sampled before reaching a specific site and compare behaviors across different nuclear environments. For complete details on the use and execution of this protocol, please refer to Kent et al. (2020). Graphical abstract Highlights • Generate cell lines stably expressing HaloTag fusion genes• Quantify global binding and target-search kinetics of epigenetic factors• Map binding dynamics of epigenetic factors within transcriptional condensates This protocol provides instructions to track the global dynamics of single epigenetic regulatory factors in live cells. We describe an approach to generate cell lines that stably express HaloTag-fused proteins. We then use live-cell single-molecule tracking to obtain kinetic populations and residence times. The kinetic parameters obtained can be used to determine important aspects of transcriptional regulation such as target-search time, 3D free diffusion time, and number of non-specific sites sampled before reaching a specific site and compare behaviors across different nuclear environments.

SUBMITTER: Brown K

PROVIDER: S-EPMC8605088 | biostudies-literature |

REPOSITORIES: biostudies-literature

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