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Sophisticated gene-regulatory mechanisms probably evolved in prokaryotes billions of years before the emergence of modern eukaryotes, which inherited the same basic enzymatic machineries. However, the epigenomic landscapes of eukaryotes are dominated by nucleosomes, which have acquired roles in genome packaging, mitotic condensation and silencing parasitic genomic elements. Although the molecular mechanisms by which nucleosomes are displaced and modified have been described, just how transcription factors, histone variants and modifications and chromatin regulators act on nucleosomes to regulate transcription is the subject of considerable ongoing study. We explore the extent to which these transcriptional regulatory components function in the context of the evolutionarily ancient role of chromatin as a barrier to processes acting on DNA and how chromatin proteins have diversified to carry out evolutionarily recent functions that accompanied the emergence of differentiation and development in multicellular eukaryotes.Eukaryotes differ substantially from bacteria and archaea owing to their nucleosome-based packaging of DNA. In this Review, Talbert, Meers and Henikoff place gene regulation in an evolutionary context by discussing how the emergence and diversification of eukaryotic chromatin provided both challenges and opportunities for intricate mechanisms of gene regulation in eukaryotes.

Physical access to DNA is a highly dynamic property of chromatin that plays an essential role in establishing and maintaining cellular identity. The organization of accessible chromatin across the genome reflects a network of permissible physical interactions through which enhancers, promoters, insulators and chromatin-binding factors cooperatively regulate gene expression. This landscape of accessibility changes dynamically in response to both external stimuli and developmental cues, and emerging evidence suggests that homeostatic maintenance of accessibility is itself dynamically regulated through a competitive interplay between chromatin-binding factors and nucleosomes. In this Review, we examine how the accessible genome is measured and explore the role of transcription factors in initiating accessibility remodelling; our goal is to illustrate how chromatin accessibility defines regulatory elements within the genome and how these epigenetic features are dynamically established to control gene expression.Chromatin accessibility comprises the positions, compaction and dynamics of nucleosomes, as well as the occupancy of DNA by other proteins such as transcription factors. In this Review, the authors discuss diverse methods for characterizing chromatin accessibility, how accessibility is determined and remodelled in cells and the regulatory roles of accessibility in gene expression and development.

Isw1 and Chd1 are ATP-dependent nucleosome-spacing enzymes required to establish regular arrays of phased nucleosomes near transcription start sites of yeast genes. Cells lacking both Isw1 and Chd1 have extremely disrupted chromatin, with weak phasing, irregular spacing and a propensity to form close-packed dinucleosomes. The Isw1 ATPase subunit occurs in two different remodeling complexes: ISW1a (composed of Isw1 and Ioc3) and ISW1b (composed of Isw1, Ioc2 and Ioc4). The Ioc4 subunit of ISW1b binds preferentially to the H3-K36me3 mark. Here we show that ISW1b is primarily responsible for setting nucleosome spacing and resolving close-packed dinucleosomes, whereas ISW1a plays only a minor role. ISW1b and Chd1 make additive contributions to dinucleosome resolution, such that neither enzyme is capable of resolving all dinucleosomes on its own. Loss of the Set2 H3-K36 methyltransferase partly phenocopies loss of Ioc4, resulting in increased dinucleosome levels with only a weak effect on nucleosome spacing, suggesting that Set2-mediated H3-K36 trimethylation contributes to ISW1b-mediated dinucleosome separation. The H4 tail domain is required for normal nucleosome spacing but not for dinucleosome resolution. We conclude that the nucleosome spacing and dinucleosome resolving activities of ISW1b and Chd1 are critical for normal global chromatin organisation.

The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (γH2AX), which act as seeds for the formation of DNA-damage response foci 1 . It is unclear how these foci are rapidly assembled to establish a ‘repair-prone’ environment within the nucleus. Topologically associating domains are a key feature of 3D genome organization that compartmentalize transcription and replication, but little is known about their contribution to DNA repair processes 2 , 3 . Here we show that topologically associating domains are functional units of the DNA damage response, and are instrumental for the correct establishment of γH2AX–53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model in which H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the importance of chromosome conformation in the maintenance of genome integrity and demonstrates the establishment of a chromatin modification by loop extrusion. During the repair of double-stranded DNA breaks, cohesin mediates the extrusion of loops of DNA along which phosphorylated H2AX spreads to establish a repair zone.

Key Points Histone exchange involves the partial or complete exchange of nucleosomes for newer or altered components. This process occurs sequentially through the removal and the replacement of the H2A–H2B dimers followed by the H3–H4 tetramer. Several factors that affect the stability of the nucleosome influence the process of histone exchange. These include chromatin modifiers, chromatin remodellers and histone chaperones. Destabilization of the nucleosome allows histone exchange to proceed, often resulting in the replacement of canonical histones with variants that carry out specialized cellular functions. Histone exchange features prominently during the process of transcription initiation and elongation. A combination of variant exchange and turnover of histone subunits drives RNA polymerase II (Pol II)-mediated transcription. Resetting of chromatin is a crucial process used by the cell to reassemble the nucleosomes that are lost during the transcription process. The co-transcriptional histone H3 lysine 36 methylation mark uses a multipronged approach to prevent histone exchange over coding regions. Limiting unobstructed histone exchange over coding regions of genes is necessary to prevent aberrant initiation of transcription. Given the importance of non-coding RNA in the development of diseases, understanding how they are produced has immense value. Access of RNA polymerase II to DNA is regulated by the ordered disassembly of nucleosomes and by histone exchange. Chromatin modifications, chromatin remodellers, histone chaperones and histone variants control nucleosomal dynamics, and dysregulation of these components results in aberrant transcription. The packaging of DNA into strings of nucleosomes is one of the features that allows eukaryotic cells to tightly regulate gene expression. The ordered disassembly of nucleosomes permits RNA polymerase II (Pol II) to access the DNA, whereas nucleosomal reassembly impedes access, thus preventing transcription and mRNA synthesis. Chromatin modifications, chromatin remodellers, histone chaperones and histone variants regulate nucleosomal dynamics during transcription. Disregulation of nucleosome dynamics results in aberrant transcription initiation, producing non-coding RNAs. Ongoing research is elucidating the molecular mechanisms that regulate chromatin structure during transcription by preventing histone exchange, thereby limiting non-coding RNA expression.

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity. The order of DNA methylation and histone modifications during transcription remained unclear. Here the authors show that HMGA2 induces DNA nicks at TGFB1-responsive genes, promoting nucleosome incorporation containing γ-H2AX, which is required for repair-mediated DNA demethylation and transcription.

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. A full understanding of chromatin in diverse cellular processes requires the consideration of its dynamics, but most standard chromatin assays provide only a static snapshot. This Review describes various emerging methods for probing chromatin dynamics across a wide range of temporal and spatial scales, and discusses the resulting biological insights. 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.

Understanding how the genome is structurally organized as chromatin is essential for understanding its function. Here, we review recent developments that allowed the readdressing of old questions regarding the primary level of chromatin structure, the arrangement of nucleosomes along the DNA and the folding of the nucleosome fiber in nuclear space. In contrast to earlier views of nucleosome arrays as uniformly regular and folded, recent findings reveal heterogeneous array organization and diverse modes of folding. Local structure variations reflect a continuum of functional states characterized by differences in post-translational histone modifications, associated chromatin-interacting proteins and nucleosome-remodeling enzymes.Technological advances have led to new insights into genome-wide arrangements of nucleosomes along the DNA and the folding of the chromosome fiber in nuclear space, revealing unexpected diversity.

Over the past few decades, epigenetics has evolved from a collection of curious biological phenomena to a functionally dissected research field. In this article, the authors provide a personal perspective on the advances of research into epigenetics — from its historical origins to its modern era — with a focus on molecular breakthroughs. Over the past 20 years, breakthrough discoveries of chromatin-modifying enzymes and associated mechanisms that alter chromatin in response to physiological or pathological signals have transformed our knowledge of epigenetics from a collection of curious biological phenomena to a functionally dissected research field. Here, we provide a personal perspective on the development of epigenetics, from its historical origins to what we define as 'the modern era of epigenetic research'. We primarily highlight key molecular mechanisms of and conceptual advances in epigenetic control that have changed our understanding of normal and perturbed development.

Key Points The dynamic regulation of chromatin involves four subfamilies of ATP-dependent nucleosome-remodelling complexes: imitation switch (ISWI), chromodomain helicase DNA-binding (CHD), switch/sucrose non-fermentable (SWI/SNF) and INO80. Each subfamily is specialized to preferentially achieve particular chromatin outcomes: assembly, access or editing. Diversity in the protein composition of remodellers enables their specific interaction with particular transcription activators, repressors and histone modifications, which together specify targeting. Although diverse in protein composition, all remodellers have a similar ATPase 'motor' that translocates DNA from a common location within the nucleosome, which breaks histone–DNA contacts. The diverse specialized proteins and domains in each remodeller subfamily are also involved in detecting nucleosome epitopes, which differentially regulate the conserved ATPase–translocase motor to achieve the various chromatin-remodelling outcomes. We propose an 'hourglass' model of chromatin remodelling that involves convergence on a DNA translocation mechanism, which is preceded and followed by remodeller diversity, in terms of differential remodeller targeting and remodelling outcomes, respectively. Remodellers are emerging as 'smart' machines that are informed about whether or how to utilize DNA translocation to conduct chromatin remodelling. Nucleosome-remodelling complexes can slide or eject histones, or incorporate histone variants, but they share an ATPase–translocase 'motor' and a common DNA translocation mechanism. In a unifying 'hourglass' model of remodeller function, the different remodeller subfamilies use different modules for targeting to nucleosomes but converge on a DNA translocation mechanism and then diverge again to achieve various outcomes. Cells utilize diverse ATP-dependent nucleosome-remodelling complexes to carry out histone sliding, ejection or the incorporation of histone variants, suggesting that different mechanisms of action are used by the various chromatin-remodelling complex subfamilies. However, all chromatin-remodelling complex subfamilies contain an ATPase–translocase 'motor' that translocates DNA from a common location within the nucleosome. In this Review, we discuss (and illustrate with animations) an alternative, unifying mechanism of chromatin remodelling, which is based on the regulation of DNA translocation. We propose the 'hourglass' model of remodeller function, in which each remodeller subfamily utilizes diverse specialized proteins and protein domains to assist in nucleosome targeting or to differentially detect nucleosome epitopes. These modules converge to regulate a common DNA translocation mechanism, to inform the conserved ATPase 'motor' on whether and how to apply DNA translocation, which together achieve the various outcomes of chromatin remodelling: nucleosome assembly, chromatin access and nucleosome editing.