Explore the vast range of titles available.

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 Centromeres are defined epigenetically and require the presence of the centromere-specific histone H3 variant centromere protein A (CENP-A). Although DNA sequences are not strictly required for centromere specification, similarities in the organization of centromere DNA suggest that DNA structures contribute to centromere function. CENP-A nucleosomes contain unique sequence and structural features that allow them to stably mark the centromere and be recognized by kinetochore components. CENP-A propagation requires specialized deposition factors and tight regulatory control. The centromere directs the assembly of the kinetochore via the 16-subunit constitutive centromere-associated network (CCAN). Most eukaryotic centromeres are defined epigenetically and require nucleosomes containing the histone H3 variant centromere protein A (CENP-A). We are now gaining insight into the mechanisms that regulate CENP-A deposition and positioning to specify and propagate centromeres during cell division, and into the function of centromeres in recruiting kinetochores to connect chromosomes to spindle microtubules. The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.

Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer.Histone variants differ from canonical histones in their genomic localization, regulation and function. Incorporation of histone variants endows specific genomic regions with unique features to fine-tune gene expression, contributing to animal development and disease pathogenesis.

Key Points Chromatin integrity and functionality is governed by the controlled assembly and disassembly of nucleosomes. An elaborate histone chaperone network governs histone provision, chromatin assembly, histone recycling and histone turnover. Histone chaperone networks operate through histone-dependent co-chaperone interactions and direct chaperone–chaperone contacts. The mode of action of histone chaperones is interpreted from structural and biochemical studies of histone–chaperone complexes. Key molecular functions of histone chaperones include the shielding of functional histone interfaces and trapping histones in non-nucleosomal conformations. The integration of histone chaperone function across DNA metabolic processes acts to maintain genome and epigenome integrity. Histone chaperones safeguard the chromatin template and shield histones from promiscuous interactions to ensure their proper storage, transport, post-translational modification, nucleosome assembly and turnover. The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.

Together with core histones, which make up the nucleosome, the linker histone (H1) is one of the five main histone protein families present in chromatin in eukaryotic cells. H1 binds to the nucleosome to form the next structural unit of metazoan chromatin, the chromatosome, which may help chromatin to fold into higher-order structures. Despite their important roles in regulating the structure and function of chromatin, linker histones have not been studied as extensively as core histones. Nevertheless, substantial progress has been made recently. The first near-atomic resolution crystal structure of a chromatosome core particle and an 11 Å resolution cryo-electron microscopy-derived structure of the 30 nm nucleosome array have been determined, revealing unprecedented details about how linker histones interact with the nucleosome and organize higher-order chromatin structures. Moreover, several new functions of linker histones have been discovered, including their roles in epigenetic regulation and the regulation of DNA replication, DNA repair and genome stability. Studies of the molecular mechanisms of H1 action in these processes suggest a new paradigm for linker histone function beyond its architectural roles in chromatin.

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.

Chromatin state is analyzed for the first time in single cells, revealing new cell subpopulations. Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.

Key Points Histone variants have evolved to endow chromatin with special properties in a locus-specific manner, and they differ from replication-coupled ('canonical') histones in terms of their gene composition, RNA processing, expression and deposition timing, and protein structure. Specific chaperones and chromatin remodellers regulate the chromatin incorporation and removal of histone variants, and they can act in a locus-specific manner. Histone variants are dynamically expressed during early embryonic development and have specialized functions during lineage commitment and during somatic cell reprogramming. In cancer, histone variants and their regulators are frequently deregulated at the level of transcription and in some cases by mutations. The deregulation of histone variants contributes to cancer through multiple mechanisms, including altered transcription, increased epigenetic plasticity and the induction of genomic instability. As an example, lysine residues 27 and 36 of H3.3 (as well as those of the replication-coupled H3.1) can be modified by methylation and are frequently mutated in childhood cancers. These are gain-of-function mutations. The mutant proteins act as inhibitors of specific histone methyltransferases and probably drive cancer by perturbing epigenetic regulation in a restricted developmental time window. Core histone proteins are deposited on chromatin during DNA replication, whereas their replication-independent variants are deposited throughout the cell cycle by specific chaperones and chromatin remodellers. This dynamic deposition of histone variants has important roles in cell fate specification and has been implicated in development and tumorigenesis. Histone variants endow chromatin with unique properties and show a specific genomic distribution that is regulated by specific deposition and removal machineries. These variants — in particular, H2A.Z, macroH2A and H3.3 — have important roles in early embryonic development, and they regulate the lineage commitment of stem cells, as well as the converse process of somatic cell reprogramming to pluripotency. Recent progress has also shed light on how mutations, transcriptional deregulation and changes in the deposition machineries of histone variants affect the process of tumorigenesis. These alterations promote or even drive cancer development through mechanisms that involve changes in epigenetic plasticity, genomic stability and senescence, and by activating and sustaining cancer-promoting gene expression programmes.

Key Points Histone variants replace canonical histones to carry out diverse roles in replication, transcription and heterochromatin formation, all of which are mediated by the activity of chaperones, chromatin remodellers and histone-modifying enzymes. Some chaperones have evolved to distinguish between histone variants and canonical histones and direct them into specialized assembly pathways, whereas other chaperones process variants and canonical histones similarly. The MCM2 subunit of the replication helicase does not distinguish between canonical H3 and its variants, and may pass different H3 variants as well as post-translationally modified H3 from the front to the back of the replication fork. By contrast, new nucleosomes comprising canonical histones are deposited behind the fork by the chaperone chromatin assembly factor 1 (CAF1), which excludes H3 variants. H2A.Z has a conserved role in transcription initiation, which nevertheless varies between organisms and contexts. H2A.Z is found flanking promoters and in some enhancers and can recruit RNA polymerase II, but is then evicted by the transcription machinery. Other H2A variants — H2A.B and macroH2A — can occupy specific promoters in specific cell types. H2A.B, which wraps only ∼120 bp of DNA, appears to facilitate transcription, whereas macroH2a may reinforce active or repressed expression states. H3.3 has high turnover rates at regulatory elements such as enhancers. HIRA deposits H3.3 in gene bodies to replace nucleosomes evicted during transcription, whereas ATRX–DAXX (alpha thalassemia mental retardation syndrome X-linked–death domain associated protein) complex deposits H3.3 into heterochromatin, where it is necessary for maintaining H3 Lys9 trimethylation and preventing transcription of silenced repetitive elements. H2A.Z is necessary for the maintenance of heterochromatin in animals, possibly because chaperones for canonical H2A are not active in heterochromatin outside of S phase. In plants, H2A.W, which wraps 162 bp of DNA, is necessary for heterochromatin condensation. Histone variants are typically incorporated into chromatin independently of DNA replication and modify chromatin properties. Recent studies have elucidated how particular histone variants are substrates of histone chaperones, chromatin remodellers and histone-modifying enzymes, thereby modifying DNA replication and repair, transcription and chromatin packaging. Most histones are assembled into nucleosomes behind the replication fork to package newly synthesized DNA. By contrast, histone variants, which are encoded by separate genes, are typically incorporated throughout the cell cycle. Histone variants can profoundly change chromatin properties, which in turn affect DNA replication and repair, transcription, and chromosome packaging and segregation. Recent advances in the study of histone replacement have elucidated the dynamic processes by which particular histone variants become substrates of histone chaperones, ATP-dependent chromatin remodellers and histone-modifying enzymes. Here, we review histone variant dynamics and the effects of replacing DNA synthesis-coupled histones with their replication-independent variants on the chromatin landscape.

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.