Review Article | Published:

Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells

Nature Reviews Genetics volume 18, pages 457472 (2017) | Download Citation

Abstract

The establishment and maintenance of chromatin states involves multiscale dynamic processes integrating transcription factor and multiprotein effector dynamics, cycles of chemical chromatin modifications, and chromatin structural organization. Recent developments in genomic technologies are emerging that are enabling a view beyond ensemble- and time-averaged properties and are revealing the importance of dynamic chromatin states for cell fate decisions, differentiation and reprogramming at the single-cell level. Concurrently, biochemical and single-molecule methodologies are providing key insights into the underlying molecular mechanisms. Combining results from defined in vitro and single-molecule studies with single-cell genomic approaches thus holds great promise for understanding chromatin-based transcriptional memory and cell fate. In this Review, we discuss recent developments in biochemical, single-molecule biophysical and single-cell genomic technologies and review how the findings from these approaches can be integrated to paint a comprehensive picture of dynamic chromatin states.

Key points

  • Chromatin is a highly dynamic complex, and chromatin dynamics operate over several orders of magnitude of space and time.

  • Local chromatin dynamics are involved in dictating access for transcription factors (TFs), the gene expression machinery and other chromatin effectors. Local chromatin dynamics can be probed in vitro and in vivo using a range of experimental approaches, including single-molecule force spectroscopy and single-molecule tracking methods.

  • The long-range establishment, maintenance and remodelling of chromatin states in the 3D space of the nucleus are involved in the regulation of transcriptional programmes and cell differentiation.

  • Genome-wide studies based on time-resolved chromatin immunoprecipitation and single-cell omics enable the interrogation of dynamic processes on the genomic scale and the single-cell scale.

  • For a complete picture of chromatin function, short-range and rapid dynamics need to be integrated with slower dynamics on the genomic scale; such integration will require new experimental and theoretical approaches.

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Acknowledgements

Dedicated to the memory of Jörg Langowski. B.F. gratefully acknowledges the Sandoz Family Foundation, the Swiss National Science Foundation (grant 31003A_149789), the NCCR Chemical Biology, Systems X (51PHP0_163580) and EPFL for financial support. O.C. acknowledges CNRS/INSB and the grant support of Fondation pour la Recherche Médicale (Equipe FRM DEQ20160334940).

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Affiliations

  1. Laboratoire de Biologie Moléculaire des Eucaryotes (LBME), Center for Integrative Biology (CBI), Universite Paul Sabatier Toulouse III, CNRS, Bâtiment IBCG, 31062 Toulouse, France.

    • Olivier Cuvier
  2. Laboratory of Biophysical Chemistry of Macromolecules (LCBM), Institute of Chemical Sciences and Engineering (ISIC), École Polytechnique Fédérale de Lausanne, CH B3 485, Station 6, CH-1015 Lausanne, Switzerland.

    • Beat Fierz

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Beat Fierz.

Glossary

Transcriptional programmes

A subset of genes actively transcribed in a subpopulation of cells, a cell type or at a developmental stage. Transcriptional programmes depend on transcription factors, chromatin modifications and dynamics to regulate transcription initiation, elongation and termination.

Local feedback loops

Networks of effector activity that result in the amplification (positive feedback) or the dampening (negative feedback) of a local perturbation in the system.

Chromatin effector proteins

Proteins or protein complexes that interact with and alter the local function of chromatin; for example, by modulating chromatin structure, chemically modifying DNA or histones, or by recruiting further biochemical activity to this site.

Emergent properties

Properties of a complex molecular system that are not observable in the individual molecules but that emerge from their interactions.

Multivalent interactions

The simultaneous engagement of multiple binding sites between two binding partners; for example, the simultaneous binding of multiple histone post-translational modifications by an effector protein that contains several specific reader domains, resulting in an increase in binding affinity.

Chromatin-remodelling complexes

Protein complexes that use ATP to move nucleosomes, to exchange histone variants, or to disrupt and remove nucleosomes, thereby contributing to the regulation of chromatin structure, composition and gene regulation.

Fluorescence correlation spectroscopy

(FCS). A spectroscopic method that allows the determination of dynamic parameters (such as molecular diffusion and structural dynamics) from the time-correlation of intensity fluctuations of the sample fluorescence emission.

Fluorescence recovery after photobleaching

(FRAP). A microscopic method to measure the diffusion and interaction kinetics of protein factors in cells. Fluorescently labelled proteins are bleached in the confocal microscope at a defined site, followed by measurement of the local recovery of fluorescence by back-diffusion of fluorescent molecules.

Single-molecule tracking

(SMT). A method to track single, fluorescently labelled (or otherwise tagged) molecules, particularly in living cells, using microscopy techniques. Computational analysis of the individual tracks yields local residence times, diffusion coefficients and diffusion behaviour, depending on the cellular environment.

Chromatin immunoprecipitation

(ChIP). Affinity enrichment of formaldehyde-fixed, fragmented (usually by sonication) chromatin using an antibody against an epitope of interest, such as a histone post-translational modification or a bound protein, followed by DNA identification using PCR amplification or sequencing.

Pioneer TFs

Transcription factors (TFs) that initially bind to the regulatory regions of silenced genes, resulting in opening of the chromatin structure, the facilitation of the assembly of further factors, and thus leading to gene activation.

Fluorescence resonance energy transfer

(FRET). A non-radiative, distance-dependent energy transfer process between a donor and an acceptor fluorophore. It enables the measurement of the physical distance between the dye pairs as a function of transfer efficiency. When dyes are at defined sites within macromolecules, structural transitions can be measured in real time.

Force spectroscopy

Methods to study interaction forces between molecules by manipulating the interaction partners using mechanical means, magnetism or light.

Optical tweezers

A force spectroscopy method in which microscopic beads (to which the molecules under investigation are attached) are trapped within the focal spot of a laser. Modulating the strength or the position of the optical trap then exerts defined forces onto the target molecules.

Nucleosome dyad

The central axis of the nucleosome, dividing the particle into two halves related by pseudo two-fold symmetry.

Total internal reflection fluorescence

(TIRF). A microscopic method in which the sample is illuminated by an evanescent field that decays within a few hundred nanometres distance from the coverslip. This allows the imaging of molecules close to the surface with very low background, making this method highly useful for single-molecule imaging.

Confocal single-molecule detection

A microscopy technique that focuses the excitation light onto a diffraction-limited spot and removes out-of-focus emission light by the addition of a pinhole at the confocal plane of the imaging lens. Only molecules contained in the confocal volume (in the order of femtolitres) are observed, making this method useful for single-molecule observation.

Cryo-electron-microscopy

(Cryo-EM). A transmission electron microscopy technique of unstained samples in a vitrified water layer at cryogenic temperature. Particle averaging of homogenous sample preparation allows 3D reconstruction of the molecular structure, making cryo-EM a highly powerful tool for structural biology.

Micrococcal nuclease

(MNase). A highly active endo-exonuclease that rapidly digests all DNA that is not protected by bound proteins, thus it is often used for protein footprinting applications.

Enhanceosome

A set of factors forming a higher-order protein complex that can recognize enhancer DNA, followed by transcription activation through interactions with core transcriptional factors.

Paired-end sequencing

A DNA sequencing method in which each individual DNA fragment is sequenced from both the 5′ and the 3′ ends, thus increasing sequencing fidelity and facilitating the detection of genomic rearrangements and mapping of repetitive regions.

Affinity enrichment

Enrichment of a molecule from a complex mixture based on its affinity to a specific binder, for example, an antibody.

Chromosome conformation capture (3C) techniques

Analysis methods of genome organization by crosslinking DNA and chromatin-interacting proteins in situ within the nucleus, followed by DNA fragmentation, extraction of the crosslinked products and DNA re-ligation. Analysis of the DNA ligation products produces maps indicating regions of contact within the nucleus, thus allowing a reconstruction of the native chromatin structure. Several 3C-derived techniques exist: 3C investigates interactions between a pair of loci, 4C interrogates interactions between a single locus and the whole genome, 5C captures all interactions within a defined genomic locus of limited size, and Hi-C probes the interactions between all genomic loci.

Epimutation accumulation approach

Monitors the stability of existing epigenomic signatures and the emergence of alternative patterns, including DNA methylation, histone modifications and gene expression traits, in clonal cell populations over time.

Bisulfite sequencing

DNA methylation profiling through DNA sequencing upon bisulfite treatment, which converts cytosine into uracil but leaves 5-methylcytosine (5mC) unaffected. Comparison to the original sequence reveals 5mC sites.

Unique molecular identifiers

(UMIs). Barcoding of DNA molecules by ligation of unique sequence identifiers for the absolute counting of independent molecules in a sequencing library. This allows DNA molecules obtained independently (distinct UMIs) to be distinguished from sequences that originate from the same fragment (identical UMI), for example, due to PCR-mediated pre-amplification steps in library preparation.

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DOI

https://doi.org/10.1038/nrg.2017.28

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