Live-cell imaging probes to track chromatin modification dynamics

Abstract

The spatiotemporal organization of chromatin is regulated at different levels in the nucleus. Epigenetic modifications such as DNA methylation and histone modifications are involved in chromatin regulation and play fundamental roles in genome function. While the one-dimensional epigenomic landscape in many cell types has been revealed by chromatin immunoprecipitation and sequencing, the dynamic changes of chromatin modifications and their relevance to chromatin organization and genome function remain elusive. Live-cell probes to visualize chromatin and its modifications have become powerful tools to monitor dynamic chromatin regulation. Bulk chromatin can be visualized both by small fluorescent dyes and fluorescent proteins, and specific endogenous genomic loci have been detected by adapting genome-editing tools. To track chromatin modifications in living cells, various types of probes have been developed. Protein domains that bind to specific modifications weakly, such as chromodomains for histone methylation, can be repeated to create a tighter binding probe that can then be tagged with a fluorescent protein. It has also been demonstrated that antigen-binding fragments and single-chain variable fragments from modification-specific antibodies can serve as binding probes without disturbing cell division, development and, differentiation. These modification-binding modules are used in modification sensors based on fluorescence/Förster resonance energy transfer to measure the intramolecular conformation changes triggered by modifications. Other probes can be created using a bivalent binding system, such as fluorescence complementation, or luciferase chemiluminescence. Live-cell chromatin modification imaging using these probes will address dynamic chromatin regulation and will be useful for assaying and screening effective epigenome drugs in cells and organisms.

Studying Chromatin Epigenetics with Fluorescence Microscopy

“…Along with H3K9ac, specific mintbody to H4K20me1 [ 23 ] and H3K27me3 histone modifications were also published [ 24 ]. To date, only some variants of mintbodies have been invented (for H3K9ac, H3K27me3 and H4K20me1), since using genetically encoded mintbodies is more convenient than Fabs [ 25 ].…”

Single-cell profiling of transcriptome and histone modifications with EpiDamID

“…1A). Such constructs have been previously successfully applied in microscopy, proteomics and ChIP experiments (Sato et al, 2013(Sato et al, , 2016(Sato et al, , 2021Tjalsma et al, 2021;Villaseñor et al, 2020). The different constructs were categorized based on their targets into the following chromatin types: accessible, active, heterochromatin, and Polycomb.…”

Single-cell profiling of transcriptome and histone modifications with EpiDamID

“…In this study, we adapted the DamID method to detect histone PTMs by fusing Dam to one of the following: (1) full-length chromatin proteins, (2) tuples of well-characterized reader domains ( Kungulovski et al., 2016 , 2014 ; Vermeulen et al., 2007 ), or (3) single-chain variable fragments (scFv) also known as mintbodies ( Sato et al., 2016 , 2013 ; Tjalsma et al., 2021 ) ( Figure 1 A, Methods). Similar strategies have been successfully applied in microscopy, proteomics and ChIP experiments ( Sato et al., 2021 , 2016 , 2013 ; Tjalsma et al., 2021 ; Villaseñor et al., 2020 ). Our approach is henceforth referred to as EpiDamID, and the construct fused to Dam as the targeting domain.…”