Single-molecule imaging of epigenetic complexes in living cells: insights from studies on Polycomb group proteins

Abstract

Chromatin-associated factors must locate, bind to, and assemble on specific chromatin regions to execute chromatin-templated functions. These dynamic processes are essential for understanding how chromatin achieves regulation, but direct quantification in living mammalian cells remains challenging. Over the last few years, live-cell single-molecule tracking (SMT) has emerged as a new way to observe trajectories of individual chromatin-associated factors in living mammalian cells, providing new perspectives on chromatin-templated activities. Here, we discuss the relative merits of live-cell SMT techniques currently in use. We provide new insights into how Polycomb group (PcG) proteins, master regulators of development and cell differentiation, decipher genetic and epigenetic information to achieve binding stability and highlight that Polycomb condensates facilitate target-search efficiency. We provide perspectives on liquid-liquid phase separation in organizing Polycomb targets. We suggest that epigenetic complexes integrate genetic and epigenetic information for target binding and localization and achieve target-search efficiency through nuclear organization.

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

“…The goal of this protocol is to determine the target-search parameters of an epigenetic protein and evaluate molecular dynamics ( Kent et al., 2020 ; Tatavosian et al., 2018 ; Brown et al., 2021 ). Individual molecules are tracked, and the tracks are linked.…”

“…(Related to measurements of residence times in general) To measure residence times, photobleaching is a major hurdle ( Brown et al., 2021 ). A reduced laser intensity and a long interval time allow measuring binding events with long residence times ( Watanabe and Mitchison, 2002 ; Elf et al., 2007 ).…”

“…A reduced laser intensity and a long interval time allow measuring binding events with long residence times ( Watanabe and Mitchison, 2002 ; Elf et al., 2007 ). Due to the photobleaching, the reported residence times should be considered as “apparent” residence times ( Brown et al., 2021 ). Since the slow-diffusing molecules might be chromatin-bound or confined movement populations ( Garcia et al., 2021b ), when analyzing residence times, the confined fractions should be excluded from the bound ones based on their difference in jump distances or diffusion coefficients ( Garcia et al., 2021b ; Mazza et al., 2012 ; Zhen et al., 2016 ).…”

“…The power-law model, which assumes a continuous binding affinity of proteins to chromatin, fits distributions of dwell times better than the exponential model ( Garcia et al., 2021a , 2021b ). However, it cannot obtain classic kinetic parameters that describe time-related biochemical processes ( Brown et al., 2021 ). Given the continuous distribution of binding affinities, it is challenging to sample, unbiased, all binding events.…”

“…Thus, control experiments such as using Tags (control for transient binding) and histone proteins (control for stable binding) should be performed. Additionally, using mutants with defects in binding is an exquisite control ( Brown et al., 2021 ).…”