Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells

Abstract

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid−liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair

“…To gain further insights into the nuclear dynamics of optoSLX4 condensates, we examined fluorescence recovery after photobleaching. Two minutes after SLX4 foci photobeaching, fluorescence recovery was approximately 50%, suggesting that SLX4 foci contain a high mobility and a low mobility population of SLX4 molecules (Figure 1H, I), consistent with the dynamics of proteins within condensates formed on chromatin [61][62][63][64] . To investigate the contribution of the subcellular environment to the formation of optogenetic SLX4 condensates, we deleted a predicted bipartite nuclear localization signal (NLS) in SLX4 (Figure 1J).…”

Cell‐Permeable Ubiquitin and Histone Tools for Studying Post‐translational Modifications

“…Fierz et al. used click chemistry to develop modular cell‐permeable multivalent heterochromatin probes, comprising a scaffold bearing multiple modified histone peptides, cell penetrating peptides and fluorescent dyes [37] (Figure 4B). These probes proved capable of entering cells and targeting HP1 proteins revealing the condensed state of heterochromatin in living cells.…”

“…The hPTMs probes described above can be used to profile cell lysates, but the activity of some proteins may be lost during cell lysis, limiting the amount of interaction information that can be obtained. Fierz et al used click chemistry to develop modular cell-permeable multivalent heterochromatin probes, comprising a scaffold bearing multiple modified histone peptides, cell penetrating peptides and fluorescent dyes [37] (Figure 4B). These probes proved capable of entering cells and targeting HP1 proteins revealing the condensed state of heterochromatin in living cells.…”

Virtual Special Issue: Epigenetics 2022

“…We also include several research articles that develop chemical probes to study chromatin, , study an emerging glycation on histones, and investigate how the local chemical environment may perturb the histone acetylation reaction . With chemical changes on DNA and histones constituting the molecular basis for epigenetic regulation, chemical biologists will continue to play vital roles in this broad area of biology that affects almost every life process in high eukaryotes.…”

“…We have included reviews that introduce chemical biology approaches to inhibit histone methyltransferase 1 and chromatin interacting proteins, 2 visualize ensembles of the nucleosome, 3 summarize recent progresses on histone modification reader proteins, 4 and propose new concepts on dynamic regulation through reversible histone modifications. 5 We also include several research articles that develop chemical probes to study chromatin, 6,7 study an emerging glycation on histones, 8 and investigate how the local chemical environment may perturb the histone acetylation reaction. 9 With chemical changes on DNA and histones constituting the molecular basis for epigenetic regulation, chemical biologists will continue to play vital roles in this broad area of biology that affects almost every life process in high eukaryotes.…”