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Key Points DNA methylation is highly correlated with the histone H3K9me mark in silenced genes from fungi to human. DNA methylation and H3K9me marks crosstalk with each other through certain structural domains that specifically recognize methylated DNA and histones. The fungi Neurospora crassa has a unidirectional regulation pathway from histone to DNA methylation. Recent structural and functional studies have uncovered the complex cross regulatory network between DNA and histone methylation in the model plant Arabidopsis thaliana . In mammals, multiple pathways are involved in both de novo and maintenance DNA methylation linking unmethylated H3K4, H3K9me and H3K36me to various targeting mechanisms. DNA methylation and H3K9 methylation are typically associated with gene silencing. Genetic, genomic, structural and biochemical data reveal functional connections between these two epigenetic marks. They also highlight how specialized protein domains that recognize the marks are essential for their establishment and maintenance at appropriate genomic loci. Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk.

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 Active DNA demethylation in mammals is achieved through TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution of oxidized 5mC or thymine DNA glycosylase (TDG)-mediated excision of 5fC and 5caC coupled with base excision repair. Active DNA demethylation is regulated at various levels, including substrate and cofactor availability, post-transcriptional and post-translational regulation of TET and TDG, and genomic localization of the demethylation machinery. Studies of tissue distribution, genomic distribution and the dynamics of oxidized 5mC provide insights into the mechanism and function of active DNA demethylation as well as the potential roles of oxidized 5mC. Active DNA demethylation and oxidized 5mC are involved in pre-implantation embryo development, primordial germ cell development, pluripotency and differentiation, as well as neuronal functions. In certain biological contexts, such as in pre-implantation embryos, the biological meaning of TET-mediated oxidation is not fully understood. In some other biological contexts, such as neurons, the extent of active DNA demethylation and its function require further study. TET may function in a catalytic-activity-independent manner. Further analysis is needed to distinguish the functions of the TET proteins themselves from the function of active DNA demethylation. Emerging evidence suggests an interplay between TET, active DNA demethylation and genomic instability and the DNA damage response. A key mode of regulating DNA methylation is through active demethylation driven by TET-mediated oxidation of 5-methylcytosine (5mC). This Review discusses our latest understanding of the mechanisms and regulation of active DNA demethylation, and the roles of active demethylation (and the oxidized 5mC intermediates) in gene regulation, genome stability, development and disease. In mammals, DNA methylation in the form of 5-methylcytosine (5mC) can be actively reversed to unmodified cytosine (C) through TET dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution or thymine DNA glycosylase (TDG)-dependent base excision repair. In the past few years, biochemical and structural studies have revealed mechanistic insights into how TET and TDG mediate active DNA demethylation. Additionally, many regulatory mechanisms of this process have been identified. Technological advances in mapping and tracing the oxidized forms of 5mC allow further dissection of their functions. Furthermore, the biological functions of active DNA demethylation in various biological contexts have also been revealed. In this Review, we summarize the recent advances and highlight key unanswered questions.

Heterochromatin is a key architectural feature of eukaryotic chromosomes, which endows particular genomic domains with specific functional properties. The capacity of heterochromatin to restrain the activity of mobile elements, isolate DNA repair in repetitive regions and ensure accurate chromosome segregation is crucial for maintaining genomic stability. Nucleosomes at heterochromatin regions display histone post-translational modifications that contribute to developmental regulation by restricting lineage-specific gene expression. The mechanisms of heterochromatin establishment and of heterochromatin maintenance are separable and involve the ability of sequence-specific factors bound to nascent transcripts to recruit chromatin-modifying enzymes. Heterochromatin can spread along the chromatin from nucleation sites. The propensity of heterochromatin to promote its own spreading and inheritance is counteracted by inhibitory factors. Because of its importance for chromosome function, heterochromatin has key roles in the pathogenesis of various human diseases. In this Review, we discuss conserved principles of heterochromatin formation and function using selected examples from studies of a range of eukaryotes, from yeast to human, with an emphasis on insights obtained from unicellular model organisms.

Heterochromatin is a key characteristic of eukaryotic genomes. Since its cytological description nearly 100 years ago, our understanding of heterochromatin features and functions, including transcription repression and genome stability, have continuously evolved. In this Viewpoint article, experts provide their current opinions on heterochromatin definition, types and functional mechanisms.In this Viewpoint article, experts provide their current opinions on heterochromatin definition, types and functional mechanisms.

Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15 , DYRK2 , FHL2 , MRPS33 , ABCA17P . Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6–8 CpGs in the HOX family of genes altered with age. With HOXD10 , HOXD9 , HOXD8 , HOXA3 , HOXC9 , HOXB1 , HOXB3 , HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3 ) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8 , HOXC9 , HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1 , HOXA3 and HOXC-AS3 . Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

TET2 is shown to associate with OGT, which catalyses O -GlcNAcylation, and the two enzymes are found together at transcription start sites; TET2 facilitates the activity of OGT in O -GlcNAcylation of histone 2B, and epigenetic modifications to both DNA and histones by TET2 and OGT may be important in gene transcription regulation. TET2 facilitates histone modification Enzymes of the TET family catalyse the oxidation of 5-methylcytosine, the 'fifth base' in DNA, into derivatives such as 5-hydroxymethylcytosine, and can influence gene expression. Here, Xiaochun Yu and colleagues show that TET2 associates with O -linked β- N -acetylglucosamine transferase (OGT), an enzyme that catalyses O -GlcNAcylation, and that the two proteins are found concurrently at transcriptional start sites. TET2 facilitates the activity of OGT in O -GlcNAcylation of histone H2B, a histone mark associated with active genes. Thus as well as influencing histone modifications, TET2 can affect DNA modifications of potential importance in transcription regulation Ten eleven translocation (TET) enzymes, including TET1, TET2 and TET3, convert 5-methylcytosine to 5-hydroxymethylcytosine 1 and regulate gene transcription 2 , 3 , 4 , 5 . However, the molecular mechanism by which TET family enzymes regulate gene transcription remains elusive 5 , 6 . Using protein affinity purification, here we search for functional partners of TET proteins, and find that TET2 and TET3 associate with O -linked β- N -acetylglucosamine (O-GlcNAc) transferase (OGT), an enzyme that by itself catalyses the addition of O -GlcNAc onto serine and threonine residues ( O -GlcNAcylation) in vivo 7 , 8 . TET2 directly interacts with OGT, which is important for the chromatin association of OGT in vivo . Although this specific interaction does not regulate the enzymatic activity of TET2, it facilitates OGT-dependent histone O -GlcNAcylation. Moreover, OGT associates with TET2 at transcription start sites. Downregulation of TET2 reduces the amount of histone 2B Ser 112 GlcNAc marks in vivo , which are associated with gene transcription regulation. Taken together, these results reveal a TET2-dependent O -GlcNAcylation of chromatin. The double epigenetic modifications on both DNA and histones by TET2 and OGT coordinate together for the regulation of gene transcription.

The structure of the METTL3–METTL14 complex, which mediates N 6 -adenosine methylation of RNA, suggests that the METTL3 subunit is the catalytic core while METTL14 serves to bind RNA. A window on m 6 A epitranscriptomics The various base modifications now known to occur in messenger RNA and long non-coding RNA are reversible, and are utilized to dynamically modify the function of the RNA. The N 6 -methyladenosine modification is removed by an enzyme complex comprising METTL3 and METTL14. Ping Yin and colleagues have solved structures of the methyltransferase domains of this heterodimeric complex with and without ligand. Surprisingly, the S -adenosyl methionine ligand was found only the METTL3 pocket, not in METTL14. This suggests a model in which there is a single catalytic subunit, with METTL3 functioning as an RNA binding platform. The reported structures provide unprecedented mechanistic insight into m 6 A RNA methylation and suggest new opportunities for the development of therapeutic agents. Chemical modifications of RNA have essential roles in a vast range of cellular processes 1 , 2 , 3 . N 6 -methyladenosine (m 6 A) is an abundant internal modification in messenger RNA and long non-coding RNA that can be dynamically added and removed by RNA methyltransferases (MTases) and demethylases, respectively 2 , 3 , 4 , 5 . An MTase complex comprising methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) efficiently catalyses methyl group transfer 6 , 7 . In contrast to the well-studied DNA MTase 8 , the exact roles of these two RNA MTases in the complex remain to be elucidated. Here we report the crystal structures of the METTL3–METTL14 heterodimer with MTase domains in the ligand-free, S -adenosyl methionine (AdoMet)-bound and S -adenosyl homocysteine (AdoHcy)-bound states, with resolutions of 1.9, 1.71 and 1.61 Å, respectively. Both METTL3 and METTL14 adopt a class I MTase fold and they interact with each other via an extensive hydrogen bonding network, generating a positively charged groove. Notably, AdoMet was observed in only the METTL3 pocket and not in METTL14. Combined with biochemical analysis, these results suggest that in the m 6 A MTase complex, METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform, reminiscent of the target recognition domain of DNA N 6 -adenine MTase 9 , 10 . This structural information provides an important framework for the functional investigation of m 6 A.

Lysine acetylation is a widespread and versatile protein post-translational modification. Lysine acetyltransferases and lysine deacetylases catalyse the addition or removal, respectively, of acetyl groups at both histone and non-histone targets. In this Review, we discuss several features of acetylation and deacetylation, including their diversity of targets, rapid turnover, exquisite sensitivity to the concentrations of the cofactors acetyl-CoA, acyl-CoA and NAD+, and tight interplay with metabolism. Histone acetylation and non-histone protein acetylation influence a myriad of cellular and physiological processes, including transcription, phase separation, autophagy, mitosis, differentiation and neural function. The activity of lysine acetyltransferases and lysine deacetylases can, in turn, be regulated by metabolic states, diet and specific small molecules. Histone acetylation has also recently been shown to mediate cellular memory. These features enable acetylation to integrate the cellular state with transcriptional output and cell-fate decisions.Lysine acetyltransferases and lysine deacetylases regulate gene expression and protein function by controlling acetylation and deacetylation of histones and diverse non-histone proteins. The activity of lysine acetyltransferases and lysine deacetylases is regulated by cellular metabolic states, offering the potential for therapeutic modulation through dietary and pharmacological interventions.