The Molecular Hallmarks of Epigenetic Control

PERSPECTIVES

EPIGENETICS mainly highlight important mechanistic TIMELINE and conceptual advances. Seminal primary papers are cited, but for in‑depth discussions The molecular hallmarks of epigenetic and additional references the reader is at times referred to the textbook Epigenetics3 control or other timely reviews. Foundation of epigenetics C. David Allis and Thomas Jenuwein Pioneering work carried out between Abstract | Over the past 20 years, breakthrough discoveries of chromatin-modifying 1869 and 1928 by Miescher, Flemming, Kossel and Heitz defined nucleic acids, enzymes and associated mechanisms that alter chromatin in response to chromatin and histone proteins, which physiological or pathological signals have transformed our knowledge of led to the cytological distinction between epigenetics from a collection of curious biological phenomena to a functionally euchromatin and heterochromatin4 (FIG. 1a). dissected research field. Here, we provide a personal perspective on the This was followed by ground-breaking 5 development of epigenetics, from its historical origins to what we define as studies by Muller (in Drosophila melanogaster) and McClintock6 (in maize) ‘the modern era of epigenetic research’. We primarily highlight key molecular on position-effect variegation (PEV) and mechanisms of and conceptual advances in epigenetic control that have changed transposable elements, providing early our understanding of normal and perturbed development. hints of non-Mendelian inheritance. Descriptions of the phenomena of X-chromosome inactivation7 and imprinting8,9 In 1942, Waddington coined the term gained. Most of the known epigenetic subsequently led to the general concept that ‘epigenetics’, which he defined as changes in modifications of chromatin are reversible, identical genetic material can be maintained phenotype without changes in genotype, to offering considerable promise for therapies in different ‘on’ versus ‘off’ states in the same explain aspects of development for which drawing upon the adaptive nature of nucleus, but its underlying mechanisms were there was little mechanistic understanding1,2. epigenetic control. Epigenetics has been poorly understood. Almost three-quarters of a century later, we and will continue to be one of the most know that epigenetic mechanisms transduce innovative research areas in modern biology DNA methylation. Chemical modifications the inheritance of gene expression patterns and medicine. of DNA bases were detected as early as 1948 without altering the underlying DNA Here, we review the development of (REF. 10) and a role for DNA methylation, sequence but by adapting chromatin, which epigenetics from its historical origins to in particular for 5‑methylcytosine is the physiological form of our genetic the ‘modern era of epigenetic research’, (5mC), in gene regulation was proposed information. Epigenetic mechanisms work which we define as the past twenty years in the mid‑1970s by Holliday and Pugh11, in addition to the DNA template to stabilize from 1996 to 2016. We describe seminal among others. By 1980, the functional gene expression programmes and thereby discoveries that culminated in the enzymatic connection between DNA methylation canalize cell-type identities. This importance definition of chromatin states that are and gene repression was established12, of epigenetic control has long been representative of gene activity (euchromatin) as was the existence of CpG islands13. The recognized, but the enzymatic definition and gene repression (heterochromatin), as first ‘epigenetic drug’, 5‑azacytidine (also of distinct chromatin states that stimulate well as mechanistic insights into the role known as 2′-deoxy‑5‑azacytidine and or repress gene activity was lacking. of epigenetics in chromatin stability, gene later called decitabine), which blocks Technological advances, such as regulation, transcriptional silencing and the DNA methylation, was used to alter gene chromatin immunoprecipitation followed reversibility of both histone modifications expression and phenotypes in fibroblast by next-generation sequencing (ChIP– and DNA methylation. We provide cell lines14. Soon thereafter, Feinberg seq) and variations thereof, have enabled an overview of how these mechanistic and Vogelstein15 reported global DNA the analysis of the epigenome at or insights, in turn, have enabled a better hypomethylation in cancer and, a decade near base-pair resolution and allowed understanding of cell-type identities by later, local DNA hypermethy lation of tumour ‘epigenomic profiling’ in both normal and genome-wide chromatin profiling and have suppressor genes was described — findings abnormal cells and tissues. In some cases, opened novel avenues for research into that were collectively reviewed16. These epigenomic profiling has served to better reprogramming, the response of chromatin insights gave a compelling reason to pursue define critical DNA control elements, such to the environment, epigenetic therapy to the ‘enzymology’ of DNA methylation. as gene enhancers and promoters. When improve human health and chromatin The successful purification and cloning of the combined with DNA sequence analyses, inheritance. We describe many — but by no mouse DNA (cytosine‑5)-methyltransferase 1 insights into disease processes have been means all — breakthrough discoveries and (DNMT1) enzyme17,18 and the generation

PERSPECTIVES

a Euchromatin Heterochromatin acetylation is closely linked to gene activity25. Many studies followed, including studies by Grunstein and others on histone-tail mutations in Saccharomyces cerevisiae that perturb gene silencing at telomeres and mating-type loci; this seminal work provided early functional evidence, including the first characterization of silent information regulator proteins26,27. Development of Xi modification- or site-specific antibodies (for example, histone 4 lysine 16 acetylation Gene transcription ON Gene transcription OFF (H4K16ac)) by Turner and others documented non-random patterns of histone acetylation, such as hypoacetylation b Active state Repressed state of the inactive X chromosome in female HAT (p55) KMT (SUV39H1) mammals28 or the silent mating type genes in yeast29, as well as hyperacetylation of Chromo the twofold upregulated X chromosome Bromo 30 Me3 in D. melanogaster males or expressed Ac β-globin genes in chicken red blood cells31. These major discoveries made a compelling argument that histone modifications, in addition to DNA methylation, carry information that can distinguish euchromatin from HDAC (Rpd3) ??? heterochromatin. Powerful genetic screens 32,33,34 35,36 33,37 Figure 1 | Euchromatin and heterochromatin. a | Cytologically visible ground states of active (euchro- in flies , yeast and plants had matic) and repressed (heterochromatic) chromatin. Schematic representationNature of two Reviews interphase | Genetics nuclei identified other key factors for chromatin- from female mouse somatic cells: the left nucleus displays broad and decondensed staining of unique dependent gene regulation, such as DNA sequences and the right nucleus shows the characteristic heterochromatic foci (black dots) that heterochromatin protein 1 (HP1), Suppressor are visualized by DAPI (4′,6‑diamidino‑2‑phenylindole) staining of AT‑rich repeat sequences. In addition, of variegation 3–9 (Su(var)3–9), Enhancer of the densely staining Barr body (an inactive X chromosome (Xi)) at the nuclear periphery is indicated. zeste (E(z)), Polycomb, Trithorax, cryptic loci In the early years, cytologists used various chromatin dyes and DNA-binding fluorochromes to regulator 4 (Clr4) and DECREASED DNA discriminate euchromatic (decondensed and light staining) from heterochromatic (compact and dense METHYLATION 1 (DDM1). However, staining) regions in eukaryotic chromatin. b | Enzymatic definition of chromatin states that stimulate gene activity (histone acetylation by p55 (also known as Gcn5)) or repress gene activity (histone methy the molecular function of these chromatin lation by Su(var)3–9 homologue 1 (SUV39H1)). In 1996, the nuclear histone acetyltransferase (HAT) p55 factors and how chromatin can ‘switch’ from Tetrahymena thermophila was described as a transcriptional co‑activator that acetylates the his- between euchromatic and heterochromatic tone H3 amino-terminal tail. The acetylated (Ac) lysine on H3 (H3K14ac) provides a docking site for states remained unknown. bromodomain (Bromo)-containing accessory proteins that bind to and further stimulate nucleosome accessibility and transcriptional activity. Histone acetylation can be reversed by opposing histone Enzymatic definition of chromatin states deacetylases (HDACs), which often cause transcriptional repression. In 2000, the human histone lysine Gene activity and euchromatin. In 1996, methyltransferase (KMT) SUV39H1 was described as an orthologue of a Drosophila melanogaster using the ciliated protozoan model, Su(var) position-effect variegation factor that methylates the histone H3 N‑terminal tail. The trimethy Tetrahymena thermophila, Allis and lated H3K9 (H3K9me3) provides a docking site for the chromodomain (Chromo)-containing hetero- colleagues38 combined biochemical chromatin protein 1 (HP1), which then impairs nucleosome accessibility and induces gene repression. The reversibility of histone lysine methylation was not known at that time. approaches with an in‑gel assay to purify and clone the first gene encoding a transcription-associated histone and analysis of Dnmt1‑mutant mice19 chromatin subunit model — put forward in acetyltransferase (HAT) from macronuclei proved important advances towards this 1974 (REF. 22) and visualized by the landmark — the active nucleus in this organism. goal. During the same time frame, the first X‑ray crystal structure of the histone Strikingly, this ciliate HAT (p55) was an DNA-methyl-binding protein, MeCP2 octamer–DNA particle in 1997 (REF. 23). As orthologue of the previously described (methyl-CpG-binding protein 2), was was shown, the basic unit of the chromatin transcriptional coactivator Gcn5 from identified20. DNA methylation and 5mC fibre is the nucleosome core particle, which budding yeast, providing a direct link (considered the ‘fifth base’) had been firmly is composed of two copies each of four between histone acetylation and gene established as a crucial epigenetic mechanism histone proteins (a histone octamer) that activity, and the yeast Gcn5 enzyme was in many, but not all, organisms. package 147 bp of DNA. shown to also exhibit HAT activity38 (FIGS 1b,2). Interestingly, the ciliate enzyme The nucleosome. Studies by many groups led Histone modifications. In the mid‑1960s, contained active site residues found in other to the widely accepted model of nucleosomal pioneering work of Allfrey24 on histone acetyltransferases (for example, cytoplasmic organization of chromatin21, which was first modifications, in particular histone Hat1 in yeast)39,40 and a highly conserved articulated in a provocative theory — the acetylation, led to the hypothesis that bromodomain, which was suggested by

PERSPECTIVES

Allis and colleagues38 to direct chromatin This was the first histone-modification- Reuter53, with cloning and characterization recruitment in ways that remained unclear binding domain to be described, suggesting of mammalian orthologues by Jenuwein54. at that time. Conclusive evidence that a novel mechanism (trans effects) for the The SET domain is present in Su(var)3–9, targeted histone acetylation by Gcn5 binding of bromodomain-containing factors E(z) and Trithorax proteins, all of which had leads to gene activation was provided in to acetylated targets in chromatin (FIGS 1b,2). been implicated in epigenetic regulation subsequent studies by Allis and his team41. To date, a multitude of chromatin-binding without evidence of enzymatic activity. Other HATs were identified, including modules have been described, many in Catalytic activity of the SET domain had TATA-box binding protein associated factor atomic resolution with their cognate been predicted by Jenuwein55; however, TFIID subunit 1 (TAF1; also known as modified-histone ligands46,52. refined bioinformatic interrogation was TAF(II)250)42, p300/CBP-associated factor needed to reveal a distant relationship of the (PCAF)43 and CBP/p300 (CREB-binding Gene repression and heterochromatin. SET domain with plant methyltransferases. protein and p300)44,45, thus confirming The discovery of the first histone lysine Together with the exposed modulation of and extending this paradigm to methyltransferase (KMT) combined insights histone H3 phosphorylation by Su(var)3–9 mammalian cells46. from dominant D. melanogaster PEV homologue 1 (SUV39H1)56, this insight Approximately one month after modifier factors containing an evolutionarily suggested a crucial experiment: to test the publication of the p55–Gcn5 HAT conserved SET domain, identified by recombinant SUV39H1 for KMT activity results, Schreiber and colleagues47 reported the purification and cloning of Glossary the first histone deacetylase (HDAC), which they had identified by an affinity Binary switches Polycomb matrix using the HDAC inhibitor The modification of adjacent or nearby histone Originally identified in genetic screens for homeotic residues affecting recognition and binding by reader transformations in Drosophila melanogaster and later (HDACi) trapoxin. Remarkably, the proteins. shown to encode a chromodomain-containing methylated mammalian trapoxin-bound protein histone H3 lysine 27 (H3K27me)-binding factor. was found to be an orthologue of the Cellular reprogramming budding yeast transcriptional co-repressor Conversion of a differentiated cell to an embryonic Position-effect variegation Rpd3 (FIGS 1b,2). This landmark finding state. (PEV). Stochastic and variegated expression of a gene due to juxtaposition to heterochromatic domains. established that histone deacetylation is Charge effects linked to transcriptional repression. Taken The effect of post-translational histone modifications Readers together, the 1996 HAT and HDAC work on altering the electrostatic interaction with DNA. Proteins that recognize and bind chromatin through histone provided a compelling one–two punch modification recognition domains. Enhancer of zeste that histone acetylation and deacetylation (E(z)). Originally identified in genetic screens for SET domain are directly coupled to ‘on’ and ‘off’ states homeotic transformations in Drosophila melanogaster A 120‑amino-acid signature domain for histone lysine of gene regulation, as had been first and later shown to encode a histone H3 lysine 27 (H3K27) methyltransferases (KMTs) that is conserved in Suppressor hypothesized by Allfrey24. methylating enzyme. of variegation 3–9, Enhancer of zeste and Trithorax. The pioneering discoveries of HAT Erasers Silent information regulator proteins and HDAC led to increased interest in Enzymes that remove histone modifications from A complex of trans-acting silencing proteins involved other proteins that might also exhibit these chromatin. in establishing and maintaining heterochromatin in catalytic activities46,48. In 2000, Guarente budding yeast. and colleagues49 demonstrated that a critical Euchromatin Light-staining, decondensed and transcriptionally Suppressor of variegation 3–9 protein required for gene silencing in yeast, accessible regions of the genome. (Su(var)3–9). Originally identified in genetic screens for Sir2, was a NAD-requiring HDAC (FIG. 2). position effect variegation in Drosophila melanogaster Subsequently, seven Sir2‑like enzymes Heterochromatin and later shown to encode a histone H3 lysine 9 (H3K9) were identified in mammalian cells, which Dark-staining, condensed and gene-poor regions of the methylating enzyme. are now known as the Sirtuin protein genome. Topologically associated domains family. Besides having distinct cofactor Histone cassettes (TADs). Large genomic regions promoting regulatory and catalytic requirements to the other Short sequences in histone proteins with clustered interactions by forming higher-order chromatin structures HDACs, the Sir2‑related HDACs triggered histone modifications that direct the biological readout separated by boundary regions. much research interest for their functions in in a combinatorial fashion. Transgenerational inheritance metabolism and ageing, which are still under Imprinting Transmission of epigenetic information that is passed on 50 intense investigation today . A chromatin state defined by whether the gene or to gametes without alteration of the DNA sequence. Despite remarkable progress, how histone genetic locus is inherited from the male or the female acetylation functions to bring about an germ line. Trithorax active chromatin state remained unknown. Originally identified in genetic screens for homeotic Mating-type loci transformations in Drosophila melanogaster and later shown One long-held view was that histone Genetic elements in yeast containing mating-type to encode a histone H3 lysine 4 (H3K4) methylating enzyme. acetylation regulated chromatin structure information (a or α) that is activated by recombination and gene activity by neutralizing the basic from heterochromatic copies of one of the two Writers charge in histones, weakening interactions mating-type alleles. Enzymes that add histone modifications to chromatin.

with DNA (cis effects). In 1999, Zhou and Multivalency X-chromosome inactivation 51 colleagues documented the bromodomain A property in which several histone modifications work A process in which one of the two X chromosomes is from PCAF as an acetyl-lysine binding together to increase the binding of reader proteins or the randomly inactivated in female mammalian cells early module for docking onto acetylated histones. stability of a nucleosomal arrangement. in development.

PERSPECTIVES

(March 1996) Discovery of the first nuclear HAT38. This HAT was purified and cloned from macronuclei in Tetrahymena thermophila 1996 (April 1996) Discovery of the first HDAC47. This HDAC is and is orthologous to the transcriptional coactivator Gcn5 from orthologous to the transcriptional corepressor Rpd3 from Saccharomyces cerevisisae38,41 Saccharomyces cerevisiae47

(July to December 1996) Description of the transcriptional co-activators TAF(II)250, p300/CBP and PCAF as HATs42–45 1998 (May to June 1998) MeCP2 is shown to interact with HDAC and the transcriptional corepressor Sin3A, thereby linking DNA methylation and histone deacetylation in inducing transcriptional repression90,91 1999 (June 1999) The bromodomain from PCAF is reported to dock onto acetylated histones, representing the ﬁrst description of a protein domain that binds histone modiﬁcations51 2000 (February 2000) SIR2 is reported to be an NAD-requiring HDAC49

(August 2000) Discovery of SUV39H1 as the ﬁrst KMT57. This KMT is orthologous to the Drosophila melanogaster PEV modiﬁer factor Su(var)3–9 (REF. 53). SUV39H1 selectively trimethylates histone H3 lysine 9 (H3K9me3)57 2001 (March 2001) The chromodomain of HP1 is shown to bind H3K9 methylated histones58,59, as does the HP1-related protein Swi6 in Schizosaccharomyces pombe60

(July 2001) The SET-domain-containing protein G9a is found as a repressing KMT that regulates H3K9me2 at euchromatin64

(November 2001) The DIM5 KMT is shown to control DNA methylation in Neurospora crassa92, as was shown (March to November 2002) Description of the SET- later for the KRYPTONITE KMT in Arabidopsis thaliana93 domain-containing Trithorax factors (e.g., MLL) as 2002 activating KMTs that regulate H3K4me3 (REFS 69–71) (September 2002) RNAi-type small nuclear RNA (June 2002) The histone variant H3.3 is found to mark active are reported to direct heterochromatin chromatin by transcription-coupled and replication- assembly and transcriptional gene silencing101–104 independent nucleosome assembly118, requiring distinct histone chaperone complexes described later119 (October 2002) Description of the SET-domain-containing Polycomb 2004 factors E(z) in Drosophila melanogaster and EZH2 in human cells as (January 2004) Nucleosome incorporation of the repressing KMT that regulate H3K27me3 (REFS 65–68) histone variant H2A.Z by the SWR1 histone exchanger 121 complex is described . During 1992–1998, pioneering (December 2004) Discovery of the ﬁrst histone lysine work from several laboratories identiﬁed a variety of demethylase: LSD1 (REF. 122). LSD1 is a nuclear amine oxidase distinct chromatin remodelling complexes108–114 2005 that primarily functions as a transcriptional co-repressor

(August 2005 to August 2007) Descriptions for genome-wide profiling of chromatin signatures and their association with regulatory elements in a variety of cell types135–137. These studies represent some of the first examples for the mapping of epigenomes140,141 (February 2006) Discovery of a second group of KDMs that contain catalytic modules known as 123 (April to May 2006) Two studies document the existence of Jumonji domains . Jumonji-domain-containing 2006 KDMs can also erase histone trimethyl marks124–126 developmentally ‘poised’ genes in embryonic stem cells that are marked by both activating and repressive histone modifications138,139. (2006) The first wave of epigenetic drugs (decitabine The simultaneous presence of H3K4me3 and H3K27me3 at regulatory regions in embryonic stem cells has been referred to as ‘bivalent and vorinostat) are approved by the FDA and become 142 available for epigenetic therapy in human cancers chromatin’ (REF. 138) and was biochemically resolved later

(August 2006) Reprogramming of 128,129 somatic cells to iPS cells by a defined (May 2009 to August 2010) Identification of 5hmC and 2009 149 description of a new family of enzymes known as TET1–3 that set of transcription factors is realized convert 5mC to 5hmC129,130. These discoveries demonstrated that DNA methylation can also be enzymatically erased (October 2009) The role of the Polycomb protein EED in 2010 the propagation of repressive H3K27me3 marks is (April 2010) CpG islands provide affinity for described196 and later the structure of PRC2 is resolved197 CFP1, which recruits activating (H3K4me) KMT and prevents DNA methylation98,99 (December 2010) Development of a new class of 2012 (January 2012) First reports of cancer-associated mutations small-molecule inhibitors that block members of the BET family from binding to acetylated histones169-171 in histone genes: that is, ‘oncohistones’ (REFS 212,213)

2015 (February 2015) The NIH Roadmap Epigenomics Consortium publishes 111 human reference epigenomes144

(April 2015) H3K9 methylation is found to be inherited for many mitotic and meiotic cell generations in Schizosaccharomyces pombe193,194

Nature Reviews | Genetics

PERSPECTIVES

on histone substrates57. This experiment extensively in the field63. Histone lysine excitement over insights gained into the revealed robust catalytic activity of the methylation can either be repressive, such fundamental mechanisms that underlie SET domain of recombinant SUV39H1 to as SUV39H1‑mediated H3K9me3 (FIG. 1b), epigenetic control. Chief examples include methylate histone H3 in vitro. Follow‑up or activating, such as H3K4 methylation. conferences specifically dedicated to collaborative studies between the Jenuwein Other groups identified silenced or active epigenetics, by the Cold Spring Harbor and Allis laboratories57 revealed that chromatin states that are developmentally Laboratory, the American Association for SUV39H1 selectively methylates histone H3 controlled (for example, H3K9me2 by G9a64, Cancer Research (AACR), the Gordon on lysine 9 (H3K9me3); this is a rewarding H3K27me3 by Polycomb and Enhancer Research Conferences (GRC) organization, example of epigenetic ‘magic’, as the genetic of zeste homologue 2 (EZH2)65–68, and the Federation of American Societies classification of the Su(var)3–9 gene by H3K4me3 by Trithorax and mixed-lineage for Experimental Biology (FASEB) and Reuter in D. melanogaster apparently leukaemia (MLL)69–73 (FIG. 2)), as well as Keystone Symposia. In addition, several ‘predicted’ the enzymatic substrate- and the repressive chromatin structure of the large consortia were launched, which site-specificity (H3K9 methylation) of inactive X chromosome in mammals74–78. connected many research groups in the encoded enzyme. In 2000, Jenuwein Even non-SET-domain-containing KMTs Europe (The Networks of Excellence published the discovery of SUV39H1 as the have been described that methylate non-tail ‘The Epigenome’ and ‘EpiGeneSys’), the first KMT57 (FIGS 1b,2). sites (for example, DOT1L, which methylates USA (US National Institutes of Health Soon after the SUV39H1 KMT discovery, H3K79 (REF. 79)). In addition to histone lysine (NIH) Roadmap Epigenomics Project and the chromodomain of HP1 was shown methylation, histone arginine methylation ENCODE), Canada, Asia and worldwide to bind methylated H3K9 (REFS 58,59), has been associated with gene regulation, (the International Human Epigenome as did the HP1‑related protein Swi6 in as the co-activator CARM1 (coactivator- Consortium (IHEC)). Schizosaccharomyces pombe60 (FIGS 1b,2). associated arginine methyltransferase 1)80 or Below, we describe major breakthrough Taken together, these findings established a the protein arginine N-methyltransferase 1 discoveries that were brought forward biochemical explanation for the formation (PRMT1)81 can mediate hormone-dependent between 2000 and 2016. These discoveries and propagation of heterochromatin that transcriptional stimulation via H3R17 are chronologically ordered in FIG. 2, had been lacking since 1928 (REF. 4) and (REF. 82) or H4R3 (REF. 81) methylation. although we do not always present them identified the enzyme class for histone lysine Clearly, histone modifications are instrict sequence but rather group them as methylation that had been elusive since the critically important for chromatin- coherent mechanistic and conceptual 1960s (REF. 61). Underscoring its importance, dependent gene regulation. However, advances. the SUV39H1–HP1–H3K9 histone whether histone lysine methylation, like methylation system for gene repression and histone acetylation, was enzymatically The histone code hypothesis and related heterochromatin assembly is now known to reversible (that is, the discovery of lysine theories. An ever-growing number of be more conserved than DNA methylation demethylases) was a remaining key question covalent histone modifications had and is present in unicellular organisms (for that would require more time and insights suggested that the nucleosome carries example, S. pombe), plants and invertebrates from others in the field. epigenetic information84; however, it was (for example, D. melanogaster), as well as unclear whether this information would complex multicellular organisms (mammals The modern era of epigenetic research be imparted by a cis or a trans mechanism. and humans)32,36,37,62. It can be argued that the above studies, The bromodomain–acetyl-lysine binding The SET domain of SUV39H1 together with the development of new discovery by Zhou and co‑workers51 in provided a signature catalytic domain, technologies such as genome-wide 1999 provided the first line of experimental and numerous SET-domain-containing chromatin profiling, ushered in what we evidence that led to the articulation of an proteins were then examined as potential refer to as the ‘modern era of epigenetic influential hypothesis put forward one KMTs. Approximately 50 genes encoding research’, as evidenced by the multitude year later, known as the ‘histone code SET-domain-containing proteins are of publications in this field since the hypothesis’ (REF. 85). This theory proposed found in the mammalian genome, and year 2000 (REF. 83). New meetings and that combinatorial patterns of histone many of these proteins have been studied initiatives supported a growing worldwide modifications specify distinct biological outcomes, in part by the recruitment of downstream effector proteins (called ◀ Figure 2 | Timeline of major discoveries and advances in epigenetic research between 1996 ‘readers’ to match the analogy of ‘writers’ for and 2016. The indicated discoveries and advances are detailed in the text and displayed as primarily histone-modifying enzymes) or complexes ‘activating mechanisms’ (yellow boxes) on the left, or as primarily ‘repressing mechanisms’ (blue boxes) in trans. The histone code hypothesis on the right. Notably, this is not a complete list. Months and years refer to the printed publication dates predicted that readers of other histone as indicated in PubMed (not online publication dates). 5hmC, 5‑hydroxymethylcytosine; 5mC, modifications would be identified. Indeed, 5‑methylcytosine; BET, bromodomain and extraterminal; CBP, CREB-binding protein; CFP1, CXXC- many types of histone modification-binding type zinc finger protein 1; DIM5, defective in methylation 5; EED, embryonic ectoderm development; modules have been recognized (for example, E(z), Enhancer of zeste; EZH2, Enhancer of zeste homologue 2; FDA, US Food and Drug Administration; chromodomains, tudor domains and HAT, histone acetyltransferase; HDAC, histone deacetylase; HP1, heterochromatin protein 1; iPS cells, induced pluripotent stem cells; KDM, histone lysine demethylase; KMT, histone lysine methyltrans- plant homeodomain (PHD) fingers), with ferase; LSD1, lysine-specific histone demethylase 1; MeCP2, methyl-CpG-binding protein 2; MLL, structural insights explaining the binding mixed-lineage leukaemia; NIH, US National Institutes of Health; PCAF, p300/CBP-associated factor; specificity of respective ligands, leading to PEV, position-effect variegation; PRC2, Polycomb repressive complex 2, RNAi, RNA-mediated inter- extensions of the histone code hypothesis, ference; Su(var)3–9, suppressor of variegation 3–9; SUV39H1, Su(var)3–9 homologue 1; TAF(II)250, including its translation into a broader TATA-box binding protein associated factor TFIID subunit 1; TET 1–3, ten-eleven translocation 1–3. ‘epigenetic code’ (REF. 86). Other follow‑up

PERSPECTIVES

extensions of these hypotheses included medium and high levels of CpG DNA accessibility and the exchange of new histone cassettes, binary switches87 and the methylation can also explain whether histones or transcription factors into and multivalency of effector–ligand binding distinct transcription factors gain access to out of chromatin. During 1992–1998, reactions88. Although a healthy debate in their cognate binding sites100. elegant genetic and biochemical studies the scientific community has questioned led to the identification of SWI/SNF108–110, whether the covalent ‘language’ of histone Non-coding RNA and transcriptional gene NURF (nucleosome-remodelling factor)111 modifications fulfils the requirements silencing. Despite remarkable progress on and other ATP-dependent nucleosome for being a true ‘code’ (REF. 89), the larger histone and DNA modifications, little was remodelling complexes112–114, as well as giving point is the unquestioned documentation known as to how these marks were added to early insights into their mechanism of action that trans mechanisms of effector protein specific genomic locations. A partial solution — an area of active epigenetic research to binding, in addition to cis mechanisms to this problem was provided by the discovery this day34,115. This is further stimulated by the (charge effects), play a major part in histone of small RNAs as potential ‘templating’ high-frequency mutations of components of and DNA modification readout. molecules for epigenetic machinery. In 2002, the human BAF (BRG1‑associated factor) four groups, using fission yeast (Grewal101 and remodelling complex in cancer116. Histone modifications and DNA Martienssen102) or T. thermophila (Allis103 Excellent examples of the utility of these methylation. The combinatorial nature and Gorovsky104) models, reported the remodelling complexes are provided with of histone modifications also raised the involvement of small RNAs in interacting the incorporation of an underappreciated question of whether histone modifications with, and presumably directing, chromatin- chromatin component — histone variants. and DNA methylation were functionally modifying activities to genomic targets (FIG. 2). Histone variants, differing by only a small linked. MeCP2 interacts with HDAC and the In contrast to double-stranded RNA (dsRNA) number of amino acids from their major transcriptional co-repressor SIN3A to bring acting to inhibit gene expression by blocking canonical histone counterparts, were about transcriptional repression90,91 (FIG. 2). the translation of messages in the cytoplasm, thought to be too minor and too similar in Selker and colleagues92, using a fungal model, a process known as RNA-mediated amino acid sequences to have important Neurospora crassa, provided compelling interference (RNAi) or post-transcriptional functional consequences117. Focusing on an evidence that histone H3K9 methylation (by gene silencing (PTGS), these small nuclear H3 variant (H3.3) in D. melanogaster tissue the KMT DIM5 (defective in methylation 5)) RNAs operate in a nuclear process known as culture cells, the H3.3 variant was found to is required for DNA methylation (FIG. 2). ‘transcriptional gene silencing’ (TGS) (FIG. 3), be deposited into chromatin independent of Subsequent studies supported these guiding not only heterochromatin assembly DNA replication, and shown to be targeted findings in plants93, in which the H3K9 and gene silencing in S. pombe105 but also preferentially into actively transcribed KMT KRYPTONITE controls DNA directing programmed DNA elimination chromatin118 (FIG. 2). Soon thereafter, methylation. Here, a repressive pathway in T. thermophila106. In both models, small biochemical approaches documented an for the silencing of repeat elements uses RNAs were shown to interact with known H3.3‑selective chaperone (HIRA)119, one that H3K9me as a docking site for the DNMT components of the histone lysine methylation was distinct from that of the major H3 (H3.1 chromomethylase. Multi-domain factors, machinery, leading to provocative suggestions and H3.2) deposition system (chromatin such as ubiquitin-like PHD and RING finger that these systems evolved to protect genomes assembly factor 1; CAF1)120 operating during domain containing protein 1 (UHRF1; also from harmful DNA elements or viruses the S phase. Moreover, the rapid exchange of known as Np95) can bridge between H3K9 that might disrupt genomes if not properly histone variants into and out of chromatin methylation and hemi-methylated DNA silenced. In an extension of this model, brought about by dedicated machinery to stabilize DNMT1 (REF. 63). In a different non-coding RNA (ncRNA) transcription has that recognizes select histone variants121 way, the catalytically inactive DNMT3‑like generally been proposed as a genome-wide adds support to the notion that histone adaptor selectively binds through its ADD surveillance mechanism with roles in RNA variants provide a major mechanism to add (ATRX, DNMT and DNMT3L) domain quality control107. strategic variation to the chromatin fibre115 to unmodified H3K4 but is blocked by Although questions remain regarding the (FIG. 2). Even centromeric chromatin has H3K4me3 chromatin94. The interdependence order of different molecular steps in these its own specialized H3 variant (known between histone modifications and DNA pathways, these findings underscore the in mammals as centromere protein A methylation for developmentally controlled notion that DNA, RNA and histone proteins, (CENP‑A)), with evidence accumulating gene expression95,96 and for Polycomb- along with their modifications, act in a that CENP‑A marks centromeres with their mediated silencing97 has revealed a complex concerted fashion to bring about chromatin own epigenetic identity117. Other non‑H3 relationship. However, whether a distinct states that are important for dictating genomic histone variants have also gained considerable DNA sequence could direct the presence functions. The TGS pathway represents a attention: for example, H2A.X, which, or absence of DNA methylation has sequence-complementary mechanism for when phosphorylated, strongly marks DNA remained unclear. Breakthrough findings RNA-directed heterochromatinization, double-strand breaks in chromatin; H2A.Z, in 2010 identified that CpG islands provide in which RNA signals back to DNA and a variant enriched at transcription start sites affinity for transcription factors, such as establishes a repressed chromatin state that where it is anti-correlated with 5mC; and CXXC-type zinc finger protein 1 (CFP1), can be propagated across many cell divisions. macroH2A, a long H2A isoform originally that recruit activating KMTs and prevent found to be enriched on the inactive DNA methylation98,99 (FIG. 2). Thus, CpG- Nucleosome remodelling and histone X chromosome117 (FIG. 3). Clearly, the puzzle rich DNA can target an active chromatin variants. ATP-dependent chromatin of chromatin-mediated epigenetic regulation structure and protect it from de novo DNA remodelling complexes provided another had many pieces, but histone variants methylation, even in the absence of ongoing important mechanism for altering suggested that even some of the smallest transcription. Differences between low, histone–DNA contacts, promoting DNA pieces were important ones.

PERSPECTIVES

All chromatin marks are reversible. ‘Erasers’ demethylase 1), a nuclear FAD-dependent and KDM research exploded onto a rapidly were only known for histone acetylation amine oxidase homologue (FIG. 2). Still, the growing list of epigenetic regulators with (deacetylases) and phosphorylation inability of LSD1 to demethylate trimethyl- important catalytic and regulatory functions. (phosphatases), two major histone lysine histones raised the intriguing Historically, there was also considerable modifications with well-documented possibility that lysine trimethylation could confusion regarding the fundamental issue turnover properties that provide dynamic be a permanent epigenetic mark. However, of the reversibility of DNA methylation, responses to the transcriptional needs of soon thereafter, Zhang and co‑workers123 in particular with regard to the loss of the cell. By contrast, erasers of histone disproved this notion by introducing the 5mC. Developmental biologists had long methylation were not known, making it field to a second class of lysine demethylases, described waves of genome-wide DNA likely to be a ‘permanent’ histone mark — Fe(II) and α‑ketoglutarate-dependent demethylation that occur in the germline that is, one that might not be enzymatically dioxygenases with catalytic modules and in early embryogenesis127; however, the reversible. This was an attractive concept: known as Jumonji domains (FIG. 2). This process by which DNA methylation is erased if histone methyl marks were stable class of enzymes was capable of removing long remained elusive, leading to studies epigenetic marks, they might be potentially trimethyl-lysine marks in histones124–126, suggesting passive (diluted through DNA inheritable. Shi and colleagues122 shattered lending support to the general view that all replication) versus active (enzymatically this concept when, in 2004, they identified epigenetic marks were probably reversible. driven) processes. A crucial piece of this and characterized the first histone lysine As with other erasers, KDMs exhibited puzzle was provided with the identification demethylase (KDM), LSD1 (lysine-specific remarkable substrate and site specificity, of 5‑hydroxymethylcytosine (5hmC)128,129

All instructive chromatin TFs, TAFs alterations p300/HAT and PCAF Epigenomic TRR KMT RNA polymerase Promoter signatures Enhancer (RNA processing) activity function Transcript quality Enhancer–promoter control communication lncRNA

CTCF Boundaries Trithorax MLL–SET1 3D architecture stimulation family

SWI/SNF Polycomb Remodelling Mod EZH2 and EED BAF silencing PRC1 and PRC2

Me H3.3, Histone variant Bivalent H3K4me3 and H2A.Z exchange and others chromatin H3K27me3

DNA repair DNA methylation DAXX and ATRX H2A.X DNMT and TET Repeat element Inactive X silencing Xist DNA-me Constitutive RNAi Imprinting ESET heterochromatin repression Air SUV39H1 (TGS) HP1 ncRNA

Figure 3 | Key examples of chromatin contribution to epigenome CTCF, CCCTC-binding factor; DNA-me, DNANature methylation; Reviews |DNMT, Genetics DNA function. A chromatin template with four nucleosomes is depicted in the (cytosine‑5)-methyltransferase; EED, embryonic ectoderm development; middle of the figure, together with chief mechanisms, such as histone ESET, ERG-associated protein with SET domain; EZH2, Enhancer of zeste modifications (Mod), DNA methylation (Me), histone variants and remodel- homologue 2; H2A.X, histone H2 variant; H3K4me3, histone H3 lysine 4 tri- ling (yellow nucleosome) and non-coding RNA (ncRNA; wavy blue lines), methylation; HAT, histone acetyltransferase; HP1, heterochromatin pro- that alter chromatin structure and function in an inter-dependent fashion. tein 1; KMT, lysine methyltransferase; lncRNA, long non-coding RNA; Distinct adaptations of this chromatin template have been associated with MLL, mixed-lineage leukaemia; PCAF, p300/CBP-associated factor; various functions of the epigenome (boxed examples). Also shown are some PRC, Polycomb repressive complex; RNAi, RNA-mediated interference; of the major chromatin factors that regulate these chromatin transitions. SUV39H1, Su(var)3–9 homologue 1; TAFs, TATA-box binding protein associ- See text for details. Air, antisense insulin-like growth factor 2 receptor RNA; ated factors; TET, ten-eleven translocation; TFs, transcription factors; ATRX, α-thalassemia/mental retardation syndrome X-linked; BAF, TGS, transcriptional gene silencing; TRR, Trithorax related; Xist, X-inactive BRG1‑associated factor; DAXX, death-domain-associated protein; specific transcript.

PERSPECTIVES

(FIG. 2). Importantly, studies led by Rao129 ‘poised’ genes in ES cells that carry both almost the entire genome is transcribed, and Zhang130 identified a new family of activating and repressive marks. The pattern giving rise to a range of ncRNAs with enzymes known as TET1–3 (ten-eleven of an overlapping presence of H3K4me3 and distinct regulatory functions147, which translocation 1–3) (FIG. 2) with the ability H3K27me3 has been referred to as ‘bivalent probably contribute to epigenetic landscapes to convert 5mC to 5hmC in an oxidation- chromatin’ (REF. 138) (FIGS 2,3). The discovery in important ways that remain under driven reaction that generates other of bivalent signatures of poised genes was active investigation. intermediates (that is, 5‑formylcytosine unexpected and important. It provided the (5fC) and 5‑carboxylcytosine (5caC)). first indication of an ‘intermediate’ state, Development and disease Enzymatic excision of these modified bases wherein bivalently marked genes could be From the many breakthrough discoveries by DNA glycosylases may follow, leading to resolved during development into active or and conceptual advances detailed above, a fully demethylated DNA template131. As inactive states. Bivalent chromatin is not molecular hallmarks of epigenetic control with histone methylation, DNA methylation specific to ES cells; it is well documented in emerged that are important for cell-type is proving to have a rich repertoire of other cell types140,141. identity and cellular reprogramming (FIG. 4). writers, readers and erasers. Clearly, the How bivalent chromatin is established Most importantly, these hallmarks respond complexity of this covalent modification and organized at a nucleosome level to developmental and environmental ‘language’ of DNA62 is increasing as much as remained an important issue. Are opposing changes and are potentially reversible by that of histones. H3K4me3 and H3K27me3 marks on the chemical inhibition of chromatin-modifying same H3 tail, on distinct H3 tails within enzymes and modification reader proteins Bivalent chromatin and epigenomic the same nucleosome or on neighbouring (FIG. 4). Many other aspects of epigenetic signatures. By 2005, histone marks such nucleosomes? Elegant studies spearheaded response — for example, during metabolic as acetylation, phosphorylation and by Reinberg142 showed that the H3K4me3 fluctuations of a varying diet, circadian methylation stood out as a group of intensely and H3K27me3 marks do not exist on the rhythms, ageing and in manifesting studied histone modifications. Sensitive same H3 tail, leading to an asymmetric phenotypic diversification from the same approaches such as mass spectrometry distribution within the nucleosome (FIG. 2). genomic template (such as imprinting continued to reveal a staggering number of This arrangement has implications for and twin studies) — have recently been histone modifications, although many — the establishment and propagation of summarized3 and are beyond the scope of if not most — of these were less abundant bivalent domains. this article. Here, we focus on the role of the than the major marks132. Correspondingly, Not unexpectedly, this complexity molecular hallmarks of epigenetic control in modification-selective antibodies were serves, in part, to fine-tune the marking development (for example, reprogramming) being developed and often used to examine of other genomic cis-regulatory elements and in some key examples of human disease ‘your favourite gene’ using ChIP assays. in chromatin. Cell-type-specific ‘active’ that are being treated or have been shown Several foresighted laboratories took on a enhancers, for example, are often defined to be responsive to epigenetic therapy, such different and powerful approach, pioneering by a subset of epigenetic marks such as as cancer, inflammation and the immune variations of genome-wide ChIP to dissect H3K4me1 and H3K27ac143. Thus, histone response (FIG. 4). epigenetic landscapes more broadly in modifications, when profiled at a genome normal and abnormal settings. Early level, revealed reproducible patterns that An epigenetic barrier to reprogramming. versions of this approach that compared allowed predictions to be made regarding Epigenetic control is crucial for cell-type H3K4me2 (as an ‘on’ mark) with H3K9me2 what genetic elements are functional identity and cellular reprogramming. (as an ‘off’ mark) were both informative (referred to as epigenomic profiling). Pioneering experiments by Weintraub and striking, revealing a conspicuous anti- Chromatin alterations (a collection of and colleagues148 showed that a cis-acting correlation between these marks along large core histone modifications and DNA transcription factor, MyoD (myogenic chromosomal domains133,134. Extending these methylation) were co‑mapped with differentiation) — a factor crucially studies to embryonic stem (ES) cells using nucleosome position and transcription important for muscle differentiation — could ChIP–seq proved especially informative, factor binding sites and integrated with the reprogramme fibroblastic cells. Twenty years in part owing to the ability of ES cells to overall RNA output of the genome (FIG. 3). later, this logic resurfaced when Yamanaka be coaxed into defined differentiation Instructive histone modification patterns and colleagues149 ‘wound back the clock’, pathways135,136. Soon, consistent patterns — such as H3K4me1 and H3K27ac in providing seminal mechanistic insights of histone marks emerged. H3K4me3, enhancer regions, H3K4me3 in promoter into the classic nuclear reprogramming for example, was associated with active regions, H3K36me3 in transcribed regions, experiments of Gurdon, Briggs and promoter elements, whereas H3K27me3 H3K27me3 in Polycomb-mediated repressed others150 (FIG. 2). Their ground-breaking was enriched within developmentally regions and H3K9me3 in heterochromatin studies demonstrated that a small cocktail of controlled repressive chromatin states137 regions (FIG. 3) — have been used by the NIH defined transcription factors (now known (FIGS 2,3). Instructive epigenomic ‘signatures’ Roadmap Epigenomics Consortium and as ‘Yamanaka factors’), when expressed were beginning to emerge, causing an the IHEC to profile reference epigenomes in differentiated adult somatic fibroblasts, interest in genome-wide approaches that and to compare epigenomic signatures in would induce pluripotency, giving rise remains today. normal versus diseased cell states144 (FIG. 2). to induced pluripotent stem cells (iPS Yet, in 2006, this appealingly straight New advances in technology now allow the cells). The potential for reprogramming forward on–off logic for histone marks analysis of single-cell epigenomes with more somatic cells from adult tissues had exciting proved far too simple. Unexpectedly, precision and new insights into cell lineage implications for regenerative medicine, even landmark studies by Lander138 and Fisher139 commitment145,146. Single-cell transcriptomes if the iPS cells process was inefficient and documented the classes of developmentally have extended earlier findings to reveal that not ready for use in humans. To what extent

PERSPECTIVES

does the chromatin state impede the ability of HDACi somatic cells to be reprogrammed? Increased HDACi DNMTi HDACi DNMTi SIRTi KMTi reprogramming efficiency has been brought about by blocking H3K9me3 KMT151,152 • Behaviour • Complex • T cell • Learning human activation or by stimulating Jumonji KDM and TET disorders enzymes with vitamin C153, suggesting • Cancer that heterochromatin stands as a likely barrier contributing, at least in part, to the • Metabolism SAM DNMTi • Immune activation Histone • Circadian acetyl-CoA inefficiency of these reprogramming events VitC • Tumour supression enzymes C rhythms NAD andNADH A n o- (FIG. 4). In support of this notion, ‘pioneer’ N tio fa D la ct hy E or transcription factors have been identified, et s m which are able to bind to repressive r Me M e • Cancer d s e HDACi r • Cancer chromatin regions to recruit co-factors e t • Chromosome o r a α-KG - b • Inﬂammation KDMi r u t o e and chromatin regulators that are capable stability c l h i u t

g r e i t of inducing downstream gene regulatory s s

H cascades that overcome repressive chromatin R

154,155 e

effects . The general view that lineage v m s a e o His r n plasticity (changes in cellular identity) has i d a o • Cancer n e t • Infection Histone l s PARPi t l i an epigenetic underpinning and may have a s e • DNA repair r H • Pathogens mimetics vital role in reprogramming is supported by M Mod o d s alterations in expression levels of chromatin- ifi e n b c on o in at ist ti modifying enzymes and fluctuations of de io H ca rs n ifi • Cancer od co‑factors that change epigenetic machinery, iBET ncRNAs m • Stem cell causing switches in cell fate156,157. • Inflammation reprogramming VitC • Drug-tolerant KMTi • Epigenetic cancer cells HDACi Cancer and epigenetic therapy. Most of inheritance the breakthrough discoveries mentioned Mobile above were not motivated by the need to RNAs have clear disease connections. Cancer Figure 4 | Molecular hallmarks of epigenetic control and examples for their medical relevance, research, for example, traditionally focused Nature Reviews | Genetics together with possible therapeutic modulation. Key mechanisms of epigenetic control and their on genetic alterations (such as mutations, associated co‑factors are displayed in the circle. In contrast to ‘hard’ alterations of the DNA sequence gene rearrangements and copy number (mutations), ‘soft’ adaptations of the chromatin template (modifications) are potentially all reversible. variation) underlying tumorigenesis, leading This distinction represents one of the key hallmarks of epigenetic control, providing a basis for to defined ‘hallmark’ capabilities exhibited ‘epigenetic therapies’. Known examples for the relevance of epigenetic control in development and by most cancers158. Early on, deviant disease are indicated and are described in the text. Pharmacological intervention and possible reversal epigenetic signatures (for example, DNA of dysregulated epigenetic control by small-molecule inhibitors (epigenetic therapy) (for example, methylation) were found to have potential histone deacetylase inhibitors (HDACi) or DNA methylation inhibitors (DNMTi)) or metabolic clinical importance in cancer, providing co‑factors (for example, α-ketoglutarate (α‑KG)) is shown by the small boxes. Although many of these strong motivation to advance epigenetic treatments with epigenetic inhibitors have been shown to ameliorate disease in some clinical settings, some are still at an exploratory stage. Detailed information on the development and use of epigenetic therapies16,48,159. The application of chemical inhibitors48,159,168 and the function of metabolic co-factors50,157 has recently been published. iBET, inhibitors of DNA methylation (DNMTi) by bromodomain and extraterminal inhibitor; KDMi, histone lysine demethylase inhibitor; KMTi, histone 159 Jones and Baylin to reactivate aberrantly lysine methyltransferase inhibitor; ncRNAs, non-coding RNAs; PARPi, poly(ADP-ribose) polymerase silenced tumour suppressor genes, along inhibitor; SAM, S‑adenosylmethionine; SIRTi, sirtuin inhibitor; VitC, vitamin C. Adapted with with the use of HDACi, such as TSA permission from REF. 158, Elsevier. (trichostatin A) and trapoxin developed by Yoshida47, followed by the use of SAHA (suberoylanilide hydroxamic acid; also emerging. Even major pharmaceutical The general concept that cancer cells may known as vorinostat) by Marks160 in the companies were getting into the action have more fragile chromatin and higher clinic, paved the way for this exciting area following the general notion that, unlike ‘epigenetic noise’ (REF. 165) could explain why of epigenetics161 (FIG. 4). genetic alterations, mistakes made in they are more susceptible to selective killing In 2006, these concepts turned into epigenetic signatures would be reversible. by treatment with epigenetic inhibitors reality as the first wave of US Food and Drug Lessons learned from promising clinical combined with, for example, radiation Administration (FDA)-approved epigenetic outcomes with DNMTi and/or HDACi therapy166. Although rapidly emerging drugs (decitabine and vorinostat) became therapies provided a compelling argument literature has provided a wealth of links available for the treatment of human cancers that other classes of writers and erasers between epigenetics and other, non-cancer (FIG. 2). Reversing epigenetic mistakes in might also stand as worthwhile drug disorders167 (FIG. 4), cancer stands as the people with promising clinical outcomes targets162,163 (FIG. 4). Moreover, drug-tolerant most compelling disease that may respond provided one of the most compelling cancer cells respond to combination therapy to epigenetic therapy159,168. In 2012, certain arguments for the importance of epigenetics. of HDACi and depletion of KDM, which types of cancer were even connected to A cottage industry of epigenetics-centred, ablates survival mechanisms and induces ‘driver’ mutations in histones, referred to as reagent-based biotech companies was higher levels of DNA damage164 (FIG. 4). ‘oncohistones’ (BOX 1; FIG. 2).

PERSPECTIVES

Box 1 | Oncohistone mutations histone-modifying enzymes also target many non-histone nuclear and cytoplasmic Histones are encoded in large multi-gene families, so cancer-associated mutations in histone proteins185,186, a notion first described by 212 213 genes were not anticipated. In close succession, however, results from the Jabado and Baker Roeder and colleagues187 for the acetylation laboratories introduced ’oncohistones‘ to the scientific community in 2012. Many, but not all, of the of the tumour suppressor p53, careful use heterozygous mutations were found in histone H3.3, the relatively minor H3 variant, suggesting some kind of ‘dominant-acting’ mechanism. An intriguing clue was provided by the observation and analysis of small-molecule inhibitors that the mutations identified mapped at or near well-known sites of histone modification (for for clinical studies is required. Specifically, example, H3 lysine 27 (H3K27)). Oncohistone mutations act, in part, to inhibit the writers of these the balance between attacking tumour cells marks, such as Enhancer of zeste homologue 2 (EZH2), a subunit of Polycomb repressive complex 2 and not weakening the defending immune (PRC2), in the case of H3K27 methylation214. Other mechanisms are also possible and under active cells through epigenetic therapy needs to be investigation. Moreover, when ‘engineered’ mutations (at select H3 lysine positions to mimic cautiously considered. oncohistone mutations from patient tumours) are added back to human cells in trans, methylation Epigenetic control involving post- at these sites is reduced, which is consistent with the prediction that this form of epigenetic translational modifications of non-histone 214 dysfunction might be found in other cancer types . In support of this, high-frequency histone proteins further extends the modulation of mutations have been identified in other human cancers (for example, H3K36M mutations in 215 functional chromatin output. For example, chondroblastoma) . Although all mutations in histone genes are classified as genetic alterations, 87 the dominant involvement of these mutations in diverting the chromatin structure of cancer distinct modification cassettes in histones , epigenomes makes it likely that additional mutations in histone-encoding genes will be uncovered particularly of the ARKS/T-type, are also at a rapid pace216,217. found in several non-histone proteins and allow post-translational modification and recognition by reader proteins. A short histone mimic within the G9a KMT was Histone-modifying enzymes, both shown for macrophages177. Inflammatory reportedly required to trigger its activity by writers and erasers, have proved to be signals (for example, lipopolysaccharide) automethylation, giving rise to the general attractive drug targets in oncology. Work lead to transcriptional activation of pro- concept of histone ‘mimicry’ (REF. 188). by the Bradner169, Tarakhovskly170 and inflammatory genes (for example, NFKB) The non-structural protein 1 (NS1) of Kouzarides171 groups added readers to that often are already poised by RNA the influenza virus harbours an amino the list of novel epigenetic targets and polymerase II (Pol II) occupancy178, to acid sequence that is very closely related therapies. Here, a select number of enable a rapid response. Stalled RNA Pol II to the histone H3 N-terminus and can bromodomain-containing proteins, such as is blocked for elongation and requires the signal H3K4 methylation. The H3K4‑like members of the BET (bromodomain and PCAF–HAT elongation complex — a feature methylation in NS1 will divert PCAF and extra terminal) family, proved druggable of many genes179. The selective response of attenuate the transcription of antiviral by small molecules (for example, the NFKB to iBET is thought to suppress genes189. Thus, histone mimicry is used inhibitors JQ1 or iBET) (FIG. 2) that bound inflammation by perturbing engagement by pathogen-derived proteins to suppress to the acetyl-lysine binding pocket in ways of PCAF to pro-inflammatory genes170. cellular defences (FIG. 4). These provocative that disrupted critical protein–histone Epigenetic control is also important for findings have been used to formally propose interactions. In keeping with other the activation of immune cells and allows that certain histone peptide mimetics could small-molecule inhibitors of HATs and strengthening of an immune response by be developed into novel epigenetic drugs. HDACs, downstream responses were pharmacological treatment. Unexpectedly, remarkably non-random. For example, one the EZH2 KMT has been shown to Chromatin inheritance (memory). of the better-studied bromodomain proteins transduce T cell activation by methylating A central question in the active debate that is targeted by these small molecules cytoplasmic actin180, providing a clear on epigenetic research has been whether is BRD4, which, in turn, functions in a example that many histone-modifying histones and their modifications are true transcriptional elongation pathway that is enzymes have non-histone substrates. Also, carriers of epigenetic information. Unlike critical for the expression of key tumour- HDACi can maintain T cell activation by DNA methylation, or other modifications promoting oncogenes, such as MYC, preventing activation-induced cell death181 to the nucleic acid template, mechanisms and pro-inflammatory genes, such as (FIG. 4). Pharmacological inhibition of the of histone inheritance have remained NFKB, in haematological cancers (FIG. 4). G9a KMT and the released gene repression unresolved, in part owing to long-standing Other chromatin readers are also being results in the activation of interferon genes debates as to how histones (old versus new) investigated with great interest now that a and leads to increased resistance towards are segregated at the replication fork190. clear precedence has been established with pathogens182 (FIG. 4). DNMTi affects not only Early hints that chromatin transitions may bromodomain inhibitors172,173. tumour suppressor genes but also repetitive be inheritable were provided by Grewal elements that respond to decreasing and Klar191 in S. pombe and Paro and Immune defence. The immune system DNA methylation. Low-dose treatment co‑workers192 in D. melanogaster. Recently, is particularly enriched for chromatin- of several cancer types with DNMTi the Moazed193 and Allshire194 groups mediated gene regulation in the specification activates endogenous retroviruses, leading demonstrated that H3K9 methylation of cell lineages, response to external signals to a dsRNA-mediated immune response can be transiently induced and inherited and induction of cellular memory174. Cells that then targets the tumour cells183,184 for many cell generations in the absence of the haematopoietic lineages integrate (FIG. 4). Breaking immune tolerance and of cis-acting transcription factors or signalling through chromatin alterations strengthening the immune response are DNA sequences used to target it (FIG. 2). that regulate gene activity175,176 and can result proving to be two major mechanisms to Importantly, this chromatin inheritance in ‘memory’ of an activated state, as recently combat cancer cells. Because nearly all requires the depletion of antagonistic factors

PERSPECTIVES

that counteract H3K9 methylation. Similar stress the need to discriminate between 1. Waddington, C. H. The epigenotype. Endeavour 1, 18–20 (1942). 195 findings by Strome and co‑workers have the cause or consequence of epigenomic 2. Waddington, C. H. Canalization of development and revealed Polycomb repressive complex 2 alterations. To this end, we anticipate the inheritance of acquired characters. Nature 150, 563–565 (1942). (PRC2)‑mediated H3K27me3 chromatin the continued use of CRISPR–Cas9 3. Allis, C. D., Caparros, M., Jenuwein, T. & Reinberg, D. inheritance in Caenorhabditis elegans. genome editing technology to allow more (eds) Epigenetics 2nd edn (Cold Spring Harbor Laboratory Press, 2015). Taken together, these studies suggest that, comprehensive dissections of genetic and 4. Heitz, E. Das Heterochromatin der Moose. Jahrb. at least in these models, histone proteins epigenetic control. Chromatin dynamics Wiss. Bot. 69, 762–818 (1928). 5. Muller, H. J. & Altenburg, E. The frequency of can transmit their message, although the can no longer be thought of as a one- or translocations produced by X‑rays in Drosophila. exact molecular details are under active two-dimensional problem, as long-range Genetics 15, 283–311 (1930). 6. McClintock, B. Chromosome organization and genic investigation. To that end, Reinberg, interactions in three-dimensional space — expression. Cold Spring Harb. Symp. Quant. Biol. 16, Gamblin and colleagues196 have shown that giving rise to topologically-associated domains 13–47 (1951). 7. Lyon, M. F. Gene action in the X‑chromosome of the propagation of the repressive histone mark (TADs) and other chromatin territories that mouse (Mus musculus L.). Nature 190, 372–373 H3K27me3 is brought about by positive structure and organize the genome — are (1961). 8. Surani, M. A., Barton, S. C. & Norris, M. L. allosteric regulation of the PRC2 KMT well documented, as are new approaches Development of reconstituted mouse eggs suggests complex through its non-catalytic subunit to define them206–208 (FIG. 3). Furthermore, imprinting of the genome during gametogenesis. Nature 308, 548–550 (1984). EED (embryonic ectoderm development) the central importance of ncRNA in many 9. McGrath, J. & Solter, D. Completion of mouse (FIG. 2). These studies and novel structural aspects of epigenetic control extends well embryogenesis requires both the maternal and paternal genomes. Cell 37, 179–183 (1984). work on PRC2 (REF. 197) are important beyond RNAi-mediated TGS and micro 10. Hotchkiss, R. D. The quantitative separation of as they provide biochemical evidence RNA-dependent PTGS, and reveals an purines, pyrimidines, and nucleosides by paper chromatography. J. Biol. Chem. 175, 315–332 for feed-forward loops in chromatin that ever-growing number of chromatin- (1948). may well contribute to the inheritance of associated RNAs (for example, long ncRNAs, 11. Holliday, R. & Pugh, J. E. DNA modification mechanisms and gene activity during development. histone modifications. enhancer RNAs and repeat RNAs) that Science 187, 226–232 (1975). Can results like these be extended to initiate and stabilize distinct chromatin 12. Razin, A. & Riggs, A. D. DNA methylation and gene function. Science 210, 604–610 (1980). the inheritance of epigenetic factors across states, even among alleles sharing the 13. Bird, A., Taggart, M., Frommer, M., Miller, O. J. & multiple generations, that is, to the general same DNA sequence. In fact, RNA can be Macleod, D. A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA. phenomenon known as transgenerational considered as one of the ‘master molecules’ Cell 40, 91–99 (1985). inheritance? Indeed, new studies in fruitflies of epigenetic control, and important 14. Taylor, S. M. & Jones, P. A. Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with and mice suggest that changes in diet and functions of ncRNA have been detailed in 5‑azacytidine. Cell 17, 771–779 (1979). other environmental factors, in particular recent reviews147,209. 15. Feinberg, A. P. & Vogelstein, B. Hypomethylation distinguishes genes of some human cancers from their the paternal diet, can reprogramme the Exploratory research into more complex normal counterparts. Nature 301, 89–92 (1983). metabolism of offspring in a way that is human disease (for example, metabolic and 16. Feinberg, A. P. & Tycko, B. The history of cancer epigenetics. Nat. Rev. Cancer 4, 143–153 (2004). propagated to future generations, leading neurodegenerative diseases) and habitual 17. Gruenbaum, Y., Cedar, H. & Razin, A. Substrate and to obesity in these generations198,199. functions (such as learning and memory) sequence specificity of a eukaryotic DNA methylase. Nature 295, 620–622 (1982). As with the experiments in S. pombe, is also predicted to reveal responsiveness 18. Bestor, T. H. & Ingram, V. M. Two DNA alterations in histone methylation seem to to combinatorial epigenetic therapies as methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of be involved. As histone marks can influence more precise inhibitors are being developed interaction with DNA. Proc. Natl Acad. Sci. USA 80, enzyme systems responsible for de novo (for example, HDACi, DNMTi, iBET and 5559–5563 (1983). 19. Li, E., Bestor, T. H. & Jaenisch, R. Targeted mutation of DNA methylation as well as the expression sirtuin inhibitors) (FIG. 4). Moreover, novel the DNA methyltransferase gene results in embryonic of ncRNA, other more-conventional nucleic- experimental systems are being used to start lethality. Cell 69, 915–926 (1992). 20. Meehan, R. R., Lewis, J. D., McKay, S., Kleiner, E. L. & acid-templated mechanisms are likely to also analysing the contribution of epigenetics to Bird, A. P. Identification of a mammalian protein that enter into the overall epigenetics inheritance behaviour and phenotypic polymorphism binds specifically to DNA containing methylated CpGs. 210 Cell 58, 499–507 (1989). equation. In plants, the ‘masters of epigenetic in social insects . In particular, for 21. Olins, D. E. & Olins, A. L. Chromatin history: our view control’ — mobile RNA — have been shown metabolic disorders and environment-driven from the bridge. Nat. Rev. Mol. Cell Biol. 4, 809–814 200 (2003). to be carriers of epigenetic information , adaptations, chromatin appears to be the 22. Kornberg, R. D. Chromatin structure: a repeating unit and small RNA sequences have been used physiological template to integrate changing of histones and DNA. Science 184, 868–871 (1974). 201 50,157,211 23. Luger, K., Mader, A. W., Richmond, R. K., to reprogramme fertilized mouse oocytes . inputs . Given the progress made Sargent, D. F. & Richmond, T. J. Crystal structure of ncRNA and tRNA fragments have recently between 1996 and 2016, we anticipate that the nucleosome core particle at 2.8 Å resolution. 202–204 Nature 389, 251–260 (1997). even been detected in sperm , which many more discoveries will continue to 24. Allfrey, V. G., Faulkner, R. & Mirsky, A. E. Acetylation suggests that more than the DNA sequence reveal how chromatin adaptations organize and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl Acad. Sci. (FIG. 4) can be inherited . and expose the information that is stored in USA 51, 786–794 (1964). our genome. 25. Verdin, E. & Ott, M. 50 years of protein acetylation: Outlook from gene regulation to epigenetics, metabolism and C. David Allis is at the Laboratory of Chromatin Biology beyond. Nat. Rev. Mol. Cell Biol. 16, 258–264 The past 20 years have witnessed and Epigenetics, The Rockefeller University, (2015). 26. Kayne, P. S. et al. Extremely conserved histone H4 1230 York Avenue, New York 10065, New York, USA. unanticipated progress in dissecting the N terminus is dispensable for growth but essential for molecular mechanisms of epigenetic control, [email protected] repressing the silent mating loci in yeast. Cell 55, 27–39 (1988). with far-reaching implications for a better Thomas Jenuwein is at the Max Planck Institute of 27. Megee, P. C., Morgan, B. A., Mittman, B. A. & understanding of normal development Immunobiology and Epigenetics, Stübeweg 51, Smith, M. M. Genetic analysis of histone H4: essential Freiburg D-79108, Germany. role of lysines subject to reversible acetylation. Science and the treatment of human disease. Here, 247, 841–845 (1990). [email protected] we advocate for a more refined definition 28. Jeppesen, P. & Turner, B. M. The inactive X chromosome in female mammals is distinguished by of epigenomic signatures, drawing upon doi:10.1038/nrg.2016.59 a lack of histone H4 acetylation, a cytogenetic marker advances in single-cell analyses205,146, but Published online 27 Jun 2016 for gene expression. Cell 74, 281–289 (1993).

PERSPECTIVES

29. Braunstein, M., Sobel, R. E., Allis, C. D., Turner, B. M. 54. Aagaard, L. et al. Functional mammalian homologues 80. Chen, D. et al. Regulation of transcription by a protein & Broach, J. R. Efficient transcriptional silencing in of the Drosophila PEV-modifier Su(var)3‑9 encode methyltransferase. Science 284, 2174–2177 (1999). Saccharomyces cerevisiae requires a heterochromatin centromere-associated proteins which complex with 81. Wang, H. et al. Methylation of histone H4 at arginine histone acetylation pattern. Mol. Cell. Biol. 16, the heterochromatin component M31. EMBO J. 18, 3 facilitating transcriptional activation by nuclear 4349–4356 (1996). 1923–1938 (1999). hormone receptor. Science 293, 853–857 (2001). 30. Bone, J. R. et al. Acetylated histone H4 on the male X 55. Jenuwein, T., Laible, G., Dorn, R. & Reuter, G. SET 82. Bauer, U. M., Daujat, S., Nielsen, S. J., Nightingale, K. chromosome is associated with dosage compensation domain proteins modulate chromatin domains in & Kouzarides, T. Methylation at arginine 17 of histone in Drosophila. Genes Dev. 8, 96–104 (1994). eu- and heterochromatin. Cell. Mol. Life Sci. 54, H3 is linked to gene activation. EMBO Rep. 3, 39–44 31. Hebbes, T. R., Clayton, A. L., Thorne, A. W. & 80–93 (1998). (2002). Crane-Robinson, C. Core histone hyperacetylation 56. Melcher, M. et al. Structure-function analysis of 83. Ushijima, T. Cancer epigenetics: now harvesting fruit co‑maps with generalized DNase I sensitivity in the SUV39H1 reveals a dominant role in heterochromatin and seeding for common diseases. Biochem. Biophys. chicken β-globin chromosomal domain. EMBO J. 13, organization, chromosome segregation, and mitotic Res. Commun. 455, 1–2 (2014). 1823–1830 (1994). progression. Mol. Cell. Biol. 20, 3728–3741 (2000). 84. Turner, B. M. Decoding the nucleosome. Cell 75, 5–8 32. Elgin, S. C. & Reuter, G. Position-effect variegation, 57. Rea, S. et al. Regulation of chromatin structure by (1993). heterochromatin formation, and gene silencing in site-specific histone H3 methyltransferases. Nature 85. Strahl, B. D. & Allis, C. D. The language of covalent Drosophila. Cold Spring Harb. Perspect. Biol. 5, 406, 593–599 (2000). histone modifications. Nature 403, 41–45 (2000). a017780 (2013). 58. Lachner, M., O’Carroll, D., Rea, S., Mechtler, K. & 86. Jenuwein, T. & Allis, C. D. Translating the histone code. 33. Grossniklaus, U. & Paro, R. Transcriptional silencing Jenuwein, T. Methylation of histone H3 lysine 9 Science 293, 1074–1080 (2001). by Polycomb-group proteins. Cold Spring Harb. creates a binding site for HP1 proteins. Nature 410, 87. Fischle, W., Wang, Y. & Allis, C. D. Binary switches and Perspect. Biol. 6, a019331 (2014). 116–120 (2001). modification cassettes in histone biology and beyond. 34. Kingston, R. E. & Tamkun, J. W. Transcriptional 59. Bannister, A. J. et al. Selective recognition of Nature 425, 475–479 (2003). regulation by trithorax-group proteins. Cold Spring methylated lysine 9 on histone H3 by the HP1 88. Ruthenburg, A. J., Li, H., Patel, D. J. & Allis, C. D. Harb. Perspect. Biol. 6, a019349 (2014). chromo domain. Nature 410, 120–124 (2001). Multivalent engagement of chromatin modifications 35. Grunstein, M. & Gasser, S. M. Epigenetics in 60. Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D. & by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, Saccharomyces cerevisiae. Cold Spring Harb. Grewal, S. I. Role of histone H3 lysine 9 methylation 983–994 (2007). Perspect. Biol. 5, a017491 (2013). in epigenetic control of heterochromatin assembly. 89. Schreiber, S. L. & Bernstein, B. E. Signaling network 36. Allshire, R. C. & Ekwall, K. Epigenetic regulation of Science 292, 110–113 (2001). model of chromatin. Cell 111, 771–778 (2002). chromatin states in Schizosaccharomyces pombe. 61. Paik, W. K. & Kim, S. Protein methylation. Science 90. Jones, P. L. et al. Methylated DNA and MeCP2 Cold Spring Harb. Perspect. Biol. 7, a018770 (2015). 174, 114–119 (1971). recruit histone deacetylase to repress transcription. 37. Pikaard, C. S. & Mittelsten Scheid, O. Epigenetic 62. Li, E. & Zhang, Y. DNA methylation in mammals. Nat. Genet. 19, 187–191 (1998). regulation in plants. Cold Spring Harb. Perspect. Biol. Cold Spring Harb. Perspect. Biol. 6, a019133 (2014). 91. Nan, X. et al. Transcriptional repression by the methyl- 6, a019315 (2014). 63. Cheng, X. Structural and functional coordination of CpG-binding protein MeCP2 involves a histone 38. Brownell, J. E. et al. Tetrahymena histone DNA and histone methylation. Cold Spring Harb. deacetylase complex. Nature 393, 386–389 (1998). acetyltransferase A: a homolog to yeast Gcn5p Perspect. Biol. 6, a018747 (2014). 92. Tamaru, H. & Selker, E. U. A histone H3 linking histone acetylation to gene activation. Cell 84, 64. Tachibana, M., Sugimoto, K., Fukushima, T. & methyltransferase controls DNA methylation in 843–851 (1996). Shinkai, Y. SET domain-containing protein, G9a, Neurospora crassa. Nature 414, 277–283 (2001). 39. Kleff, S., Andrulis, E. D., Anderson, C. W. & is a novel lysine-preferring mammalian histone 93. Jackson, J. P., Lindroth, A. M., Cao, X. & Sternglanz, R. Identification of a gene encoding a methyltransferase with hyperactivity and specific Jacobsen, S. E. Control of CpNpG DNA methylation yeast histone H4 acetyltransferase. J. Biol. Chem. selectivity to lysines 9 and 27 of histone H3. by the KRYPTONITE histone H3 methyltransferase. 270, 24674–24677 (1995). J. Biol. Chem. 276, 25309–25317 (2001). Nature 416, 556–560 (2002). 40. Parthun, M. R., Widom, J. & Gottschling, D. E. 65. Czermin, B. et al. Drosophila Enhancer of zeste/ESC 94. Ooi, S. K. et al. DNMT3L connects unmethylated The major cytoplasmic histone acetyltransferase complexes have a histone H3 methyltransferase lysine 4 of histone H3 to de novo methylation of DNA. in yeast: links to chromatin replication and histone activity that marks chromosomal Polycomb sites. Nature 448, 714–717 (2007). metabolism. Cell 87, 85–94 (1996). Cell 111, 185–196 (2002). 95. Bird, A. DNA methylation patterns and epigenetic 41. Kuo, M. H. et al. Transcription-linked acetylation 66. Müller, J. et al. Histone methyltransferase activity memory. Genes Dev. 16, 6–21 (2002). by Gcn5p of histones H3 and H4 at specific lysines. of a Drosophila Polycomb group repressor complex. 96. Cedar, H. & Bergman, Y. Linking DNA methylation Nature 383, 269–272 (1996). Cell 111, 197–208 (2002). and histone modification: patterns and paradigms. 42. Mizzen, C. A. et al. The TAF(II)250 subunit of TFIID 67. Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Nat. Rev. Genet. 10, 295–304 (2009). has histone acetyltransferase activity. Cell 87, Tempst, P. & Reinberg, D. Histone methyltransferase 97. Margueron, R. & Reinberg, D. The Polycomb complex 1261–1270 (1996). activity associated with a human multiprotein complex PRC2 and its mark in life. Nature 469, 343–349 43. Yang, X. J., Ogryzko, V. V., Nishikawa, J., Howard, B. H. containing the Enhancer of zeste protein. Genes Dev. (2011). & Nakatani, Y. A p300/CBP-associated factor that 16, 2893–2905 (2002). 98. Thomson, J. P. et al. CpG islands influence chromatin competes with the adenoviral oncoprotein E1A. 68. Cao, R. et al. Role of histone H3 lysine 27 structure via the CpG-binding protein Cfp1. Nature Nature 382, 319–324 (1996). methylation in Polycomb-group silencing. Science 464, 1082–1086 (2010). 44. Ogryzko, V. V., Schiltz, R. L., Russanova, V., 298, 1039–1043 (2002). 99. Blackledge, N. P. et al. CpG islands recruit a histone H3 Howard, B. H. & Nakatani, Y. The transcriptional 69. Krogan, N. J. et al. COMPASS, a histone H3 (lysine 4) lysine 36 demethylase. Mol. Cell 38, 179–190 (2010). coactivators p300 and CBP are histone methyltransferase required for telomeric silencing of 100. Domcke, S. et al. Competition between DNA acetyltransferases. Cell 87, 953–959 (1996). gene expression. J. Biol. Chem. 277, 10753–10755 methylation and transcription factors determines 45. Bannister, A. J. & Kouzarides, T. The CBP co‑activator (2002). binding of NRF1. Nature 528, 575–579 (2015). is a histone acetyltransferase. Nature 384, 641–643 70. Milne, T. A. et al. MLL targets SET domain 101. Hall, I. M. et al. Establishment and maintenance of a (1996). methyltransferase activity to Hox gene promoters. heterochromatin domain. Science 297, 2232–2237 46. Marmorstein, R. & Zhou, M. M. Writers and readers Mol. Cell 10, 1107–1117 (2002). (2002). of histone acetylation: structure, mechanism, and 71. Nakamura, T. et al. ALL‑1 is a histone 102. Volpe, T. A. et al. Regulation of heterochromatic inhibition. Cold Spring Harb. Perspect. Biol. 6, methyltransferase that assembles a supercomplex silencing and histone H3 lysine 9 methylation by a018762 (2014). of proteins involved in transcriptional regulation. RNAi. Science 297, 1833–1837 (2002). 47. Taunton, J., Hassig, C. A. & Schreiber, S. L. A Mol. Cell 10, 1119–1128 (2002). 103. Taverna, S. D., Coyne, R. S. & Allis, C. D. Methylation mammalian histone deacetylase related to the 72. Yokoyama, A. et al. Leukemia proto-oncoprotein MLL of histone H3 at lysine 9 targets programmed DNA yeast transcriptional regulator Rpd3p. Science 272, forms a SET1‑like histone methyltransferase complex elimination in Tetrahymena. Cell 110, 701–711 (2002). 408–411 (1996). with menin to regulate Hox gene expression. 104. Mochizuki, K., Fine, N. A., Fujisawa, T. & 48. Seto, E. & Yoshida, M. Erasers of histone acetylation: Mol. Cell. Biol. 24, 5639–5649 (2004). Gorovsky, M. A. Analysis of a piwi-related gene the histone deacetylase enzymes. Cold Spring Harb. 73. Dou, Y. et al. Regulation of MLL1 H3K4 implicates small RNAs in genome rearrangement Perspect. Biol. 6, a018713 (2014). methyltransferase activity by its core components. in Tetrahymena. Cell 110, 689–699 (2002). 49. Imai, S., Armstrong, C. M., Kaeberlein, M. & Nat. Struct. Mol. Biol. 13, 713–719 (2006). 105. Martienssen, R. & Moazed, D. RNAi and Guarente, L. Transcriptional silencing and longevity 74. Heard, E. et al. Methylation of histone H3 at Lys‑9 heterochromatin assembly. Cold Spring Harb. protein Sir2 is an NAD-dependent histone is an early mark on the X chromosome during X Perspect. Biol. 7, a019323 (2015). deacetylase. Nature 403, 795–800 (2000). inactivation. Cell 107, 727–738 (2001). 106. Chalker, D. L., Meyer, E. & Mochizuki, K. Epigenetics 50. Berger, S. L. & Sassone-Corsi, P. Metabolic signaling 75. Peters, A. H. et al. Histone H3 lysine 9 methylation is of ciliates. Cold Spring Harb. Perspect. Biol. 5, to chromatin. Cold Spring Harb. Perspect. Biol. an epigenetic imprint of facultative heterochromatin. a017764 (2013). http://dx.doi.org/10.1101/cshperspect.a019463 Nat. Genet. 30, 77–80 (2002). 107. Reyes-Turcu, F. E., Zhang, K., Zofall, M., Chen, E. & (2016). 76. Peters, A. H. et al. Partitioning and plasticity of Grewal, S. I. Defects in RNA quality control factors 51. Dhalluin, C. et al. Structure and ligand of a histone repressive histone methylation states in mammalian reveal RNAi-independent nucleation of acetyltransferase bromodomain. Nature 399, chromatin. Mol. Cell 12, 1577–1589 (2003). heterochromatin. Nat. Struct. Mol. Biol. 18, 491–496 (1999). 77. Rice, J. C. et al. Histone methyltransferases direct 1132–1138 (2011). 52. Patel, D. J. A structural perspective on readout of different degrees of methylation to define distinct 108. Hirschhorn, J. N., Brown, S. A., Clark, C. D. & epigenetic histone and DNA methylation marks. chromatin domains. Mol. Cell 12, 1591–1598 (2003). Winston, F. Evidence that SNF2/SWI2 and SNF5 Cold Spring Harb. Perspect. Biol. 8, a018754 (2015). 78. Plath, K. et al. Role of histone H3 lysine 27 activate transcription in yeast by altering chromatin 53. Tschiersch, B. et al. The protein encoded by the methylation in X inactivation. Science 300, 131–135 structure. Genes Dev. 6, 2288–2298 (1992). Drosophila position-effect variegation suppressor gene (2003). 109. Cote, J., Quinn, J., Workman, J. L. & Peterson, C. L. Su(var)3‑9 combines domains of antagonistic 79. van Leeuwen, F., Gafken, P. R. & Gottschling, D. E. Stimulation of GAL4 derivative binding to nucleosomal regulators of homeotic gene complexes. EMBO J. 13, Dot1p modulates silencing in yeast by methylation DNA by the yeast SWI/SNF complex. Science 265, 3822–3831 (1994). of the nucleosome core. Cell 109, 745–756 (2002). 53–60 (1994).

PERSPECTIVES

110. Kwon, H., Imbalzano, A. N., Khavari, P. A., 136. Heintzman, N. D. et al. Distinct and predictive 167. Zoghbi, H. Y. & Beaudet, A. L. Epigenetics and human Kingston, R. E. & Green, M. R. Nucleosome disruption chromatin signatures of transcriptional promoters disease. Cold Spring Harb. Perspect. Biol. 8, a019497 and enhancement of activator binding by a human and enhancers in the human genome. Nat. Genet. 39, (2015). SW1/SNF complex. Nature 370, 477–481 (1994). 311–318 (2007). 168. Audia, J. E. & Campbell, R. M. Histone modifications 111. Tsukiyama, T. & Wu, C. Purification and properties of 137. Boyer, L. A. et al. Polycomb complexes repress and cancer. Cold Spring Harb. Perspect. Biol. 8, an ATP-dependent nucleosome remodeling factor. developmental regulators in murine embryonic stem a019521 (2015). Cell 83, 1011–1020 (1995). cells. Nature 441, 349–353 (2006). 169. Filippakopoulos, P. et al. Selective inhibition of BET 112. Ito, T., Bulger, M., Pazin, M. J., Kobayashi, R. & 138. Bernstein, B. E. et al. A bivalent chromatin structure bromodomains. Nature 468, 1067–1073 (2010). Kadonaga, J. T. ACF, an ISWI-containing and ATP- marks key developmental genes in embryonic stem 170. Nicodeme, E. et al. Suppression of inflammation by utilizing chromatin assembly and remodeling factor. cells. Cell 125, 315–326 (2006). a synthetic histone mimic. Nature 468, 1119–1123 Cell 90, 145–155 (1997). 139. Azuara, V. et al. Chromatin signatures of pluripotent (2010). 113. Varga-Weisz, P. D. et al. Chromatin-remodelling factor cell lines. Nat. Cell Biol. 8, 532–538 (2006). 171. Dawson, M. A. et al. Inhibition of BET recruitment to CHRAC contains the ATPases ISWI and topoisomerase 140. Mikkelsen, T. S. et al. Genome-wide maps of chromatin chromatin as an effective treatment for MLL-fusion II. Nature 388, 598–602 (1997). state in pluripotent and lineage-committed cells. leukaemia. Nature 478, 529–533 (2011). 114. Lorch, Y., Cairns, B. R., Zhang, M. & Kornberg, R. D. Nature 448, 553–560 (2007). 172. Arrowsmith, C. H., Bountra, C., Fish, P. V., Lee, K. Activated RSC-nucleosome complex and persistently 141. Barski, A. et al. High-resolution profiling of histone & Schapira, M. Epigenetic protein families: a new altered form of the nucleosome. Cell 94, 29–34 methylations in the human genome. Cell 129, frontier for drug discovery. Nat. Rev. Drug Discov. 11, (1998). 823–837 (2007). 384–400 (2012). 115. Becker, P. B. & Workman, J. L. Nucleosome 142. Voigt, P. et al. Asymmetrically modified nucleosomes. 173. Rodriguez, R. & Miller, K. M. Unravelling the genomic remodeling and epigenetics. Cold Spring Harb. Cell 151, 181–193 (2012). targets of small molecules using high-throughput Perspect. Biol. 5, a017905 (2013). 143. Rada-Iglesias, A. et al. A unique chromatin signature sequencing. Nat. Rev. Genet. 15, 783–796 (2014). 116. Kadoch, C. & Crabtree, G. R. Mammalian SWI/SNF uncovers early developmental enhancers in humans. 174. Busslinger, M. & Tarakhovsky, A. Epigenetic control chromatin remodeling complexes and cancer: Nature 470, 279–283 (2011). of immunity. Cold Spring Harb. Perspect. Biol. 6, mechanistic insights gained from human genomics. 144. Roadmap Epigenomics Consortium et al. Integrative a019307 (2014). Sci. Adv. 1, e1500447 (2015). analysis of 111 reference human epigenomes. Nature 175. Foster, S. L., Hargreaves, D. C. & Medzhitov, R. 117. Henikoff, S. & Smith, M. M. Histone variants and 518, 317–330 (2015). Gene-specific control of inflammation by TLR-induced epigenetics. Cold Spring Harb. Perspect. Biol. 7, 145. Grün, D. et al. Single-cell messenger RNA sequencing chromatin modifications. Nature 447, 972–978 a019364 (2015). reveals rare intestinal cell types. Nature 525, 251–255 (2007). 118. Ahmad, K. & Henikoff, S. The histone variant H3.3 (2015). 176. Lara-Astiaso, D. et al. Immunogenetics: chromatin marks active chromatin by replication-independent 146. Battich, N., Stoeger, T. & Pelkmans, L. Control of state dynamics during blood formation. Science 345, nucleosome assembly. Mol. Cell 9, 1191–1200 transcript variability in single mammalian cells. 943–949 (2014). (2002). Cell 163, 1596–1610 (2015). 177. Yoshida, K. et al. The transcription factor ATF7 119. Tagami, H., Ray-Gallet, D., Almouzni, G. & Nakatani, Y. 147. Morris, K. V. & Mattick, J. S. The rise of regulatory mediates lipopolysaccharide-induced epigenetic Histone H3.1 and H3.3 complexes mediate RNA. Nat. Rev. Genet. 15, 423–437 (2014). changes in macrophages involved in innate nucleosome assembly pathways dependent or 148. Davis, R. L., Weintraub, H. & Lassar, A. B. Expression immunological memory. Nat. Immunol. 16, independent of DNA synthesis. Cell 116, 51–61 of a single transfected cDNA converts fibroblasts to 1034–1043 (2015). (2004). myoblasts. Cell 51, 987–1000 (1987). 178. Bhatt, D. M. et al. Transcript dynamics of 120. Smith, S. & Stillman, B. Purification and 149. Takahashi, K. & Yamanaka, S. Induction of pluripotent proinflammatory genes revealed by sequence analysis characterization of CAF‑I, a human cell factor required stem cells from mouse embryonic and adult fibroblast of subcellular RNA fractions. Cell 150, 279–290 for chromatin assembly during DNA replication cultures by defined factors. Cell 126, 663–676 (2006). (2012). in vitro. Cell 58, 15–25 (1989). 150. Hochedlinger, K. & Jaenisch, R. Induced pluripotency 179. Adelman, K. & Lis, J. T. Promoter-proximal pausing 121. Mizuguchi, G. et al. ATP-driven exchange of histone and epigenetic reprogramming. Cold Spring Harb. of RNA polymerase II: emerging roles in metazoans. H2AZ variant catalyzed by SWR1 chromatin Perspect. Biol. 7, a019448 (2015). Nat. Rev. Genet. 13, 720–731 (2012). remodeling complex. Science 303, 343–348 (2004). 151. Chen, J. et al. H3K9 methylation is a barrier during 180. Su, I. H. et al. Polycomb group protein Ezh2 controls 122. Shi, Y. et al. Histone demethylation mediated by the somatic cell reprogramming into iPSCs. Nat. Genet. actin polymerization and cell signaling. Cell 121, nuclear amine oxidase homolog LSD1. Cell 119, 45, 34–42 (2013). 425–436 (2005). 941–953 (2004). 152. Matoba, S. et al. Embryonic development following 181. Cao, K. et al. Histone deacetylase inhibitors prevent 123. Tsukada, Y. et al. Histone demethylation by a family somatic cell nuclear transfer impeded by persisting activation-induced cell death and promote anti-tumor of JmjC domain-containing proteins. Nature 439, histone methylation. Cell 159, 884–895 (2014). immunity. Oncogene 34, 5960–5970 (2015). 811–816 (2006). 153. Blaschke, K. et al. Vitamin C induces Tet-dependent 182. Fang, T. C. et al. Histone H3 lysine 9 di‑methylation 124. Whetstine, J. R. et al. Reversal of histone lysine DNA demethylation and a blastocyst-like state in ES as an epigenetic signature of the interferon response. trimethylation by the JMJD2 family of histone cells. Nature 500, 222–226 (2013). J. Exp. Med. 209, 661–669 (2012). demethylases. Cell 125, 467–481 (2006). 154. Zaret, K. S. & Carroll, J. S. Pioneer transcription 183. Roulois, D. et al. DNA-demethylating agents target 125. Fodor, B. D. et al. Jmjd2b antagonizes H3K9 factors: establishing competence for gene expression. colorectal cancer cells by inducing viral mimicry by trimethylation at pericentric heterochromatin in Genes Dev. 25, 2227–2241 (2011). endogenous transcripts. Cell 162, 961–973 (2015). mammalian cells. Genes Dev. 20, 1557–1562 155. Soufi, A., Donahue, G. & Zaret, K. S. Facilitators and 184. Chiappinelli, K. B. et al. Inhibiting DNA methylation (2006). impediments of the pluripotency reprogramming causes an interferon response in cancer via dsRNA 126. Klose, R. J. et al. The transcriptional repressor factors’ initial engagement with the genome. Cell 151, including endogenous retroviruses. Cell 162, JHDM3A demethylates trimethyl histone H3 lysine 9 994–1004 (2012). 974–986 (2015). and lysine 36. Nature 442, 312–316 (2006). 156. Tee, W. W. & Reinberg, D. Chromatin features and the 185. Glozak, M. A., Sengupta, N., Zhang, X. & Seto, E. 127. Reik, W. & Surani, M. A. Germline and Pluripotent epigenetic regulation of pluripotency states in ESCs. Acetylation and deacetylation of non-histone proteins. Stem Cells. Cold Spring Harb. Perspect. Biol. 7, Development 141, 2376–2390 (2014). Gene 363, 15–23 (2005). a019422 (2015). 157. Gut, P. & Verdin, E. The nexus of chromatin regulation 186. Biggar, K. K. & Li, S. S. Non-histone protein 128. Kriaucionis, S. & Heintz, N. The nuclear DNA base and intermediary metabolism. Nature 502, 489–498 methylation as a regulator of cellular signalling and 5‑hydroxymethylcytosine is present in Purkinje (2013). function. Nat. Rev. Mol. Cell Biol. 16, 5–17 (2015). neurons and the brain. Science 324, 929–930 158. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: 187. Gu, W. & Roeder, R. G. Activation of p53 sequence- (2009). the next generation. Cell 144, 646–674 (2011). specific DNA binding by acetylation of the p53 129. Tahiliani, M. et al. Conversion of 5‑methylcytosine to 159. Jones, P. A. & Baylin, S. B. Epigenetic determinants of C‑terminal domain. Cell 90, 595–606 (1997). 5‑hydroxymethylcytosine in mammalian DNA by MLL cancer. Cold Spring Harb. Perspect. Biol. 6, a019505 188. Sampath, S. C. et al. Methylation of a histone mimic partner TET1. Science 324, 930–935 (2009). (2014). within the histone methyltransferase G9a regulates 130. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC 160. Marks, P. A. Discovery and development of SAHA as an protein complex assembly. Mol. Cell 27, 596–608 conversion, ES‑cell self-renewal and inner cell mass anticancer agent. Oncogene 26, 1351–1356 (2007). (2007). specification. Nature 466, 1129–1133 (2010). 161. Baylin, S. B. & Jones, P. A. A decade of exploring the 189. Marazzi, I. et al. Suppression of the antiviral response 131. He, Y. F. et al. Tet-mediated formation of cancer epigenome — biological and translational by an influenza histone mimic. Nature 483, 428–433 5‑carboxylcytosine and its excision by TDG in implications. Nat. Rev. Cancer 11, 726–734 (2011). (2012). mammalian DNA. Science 333, 1303–1307 (2011). 162. Chi, P., Allis, C. D. & Wang, G. G. Covalent histone 190. Almouzni, G. & Cedar, H. Maintenance of epigenetic 132. Zhao, Y. & Garcia, B. A. Comprehensive catalog of modifications — miswritten, misinterpreted and information. Cold Spring Harb. Perspect. Biol. 8, currently documented histone modifications. miserased in human cancers. Nat. Rev. Cancer 10, a019372 (2015). Cold Spring Harb. Perspect. Biol. 7, a025064 (2015). 457–469 (2010). 191. Grewal, S. I. & Klar, A. J. Chromosomal inheritance 133. Noma, K., Allis, C. D. & Grewal, S. I. Transitions in 163. Dawson, M. A. & Kouzarides, T. Cancer epigenetics: of epigenetic states in fission yeast during mitosis distinct histone H3 methylation patterns at the from mechanism to therapy. Cell 150, 12–27 (2012). and meiosis. Cell 86, 95–101 (1996). heterochromatin domain boundaries. Science 293, 164. Sharma, S. V. et al. A chromatin-mediated reversible 192. Cavalli, G. & Paro, R. The Drosophila Fab‑7 1150–1155 (2001). drug-tolerant state in cancer cell subpopulations. chromosomal element conveys epigenetic inheritance 134. Litt, M. D., Simpson, M., Gaszner, M., Allis, C. D. & Cell 141, 69–80 (2010). during mitosis and meiosis. Cell 93, 505–518 Felsenfeld, G. Correlation between histone lysine 165. Pujadas, E. & Feinberg, A. P. Regulated noise in the (1998). methylation and developmental changes at the epigenetic landscape of development and disease. 193. Ragunathan, K., Jih, G. & Moazed, D. Epigenetic chicken β-globin locus. Science 293, 2453–2455 Cell 148, 1123–1131 (2012). inheritance uncoupled from sequence-specific (2001). 166. Groselj, B., Sharma, N. L., Hamdy, F. C., Kerr, M. recruitment. Science 348, 6230 (2015). 135. Kim, T. H. et al. A high-resolution map of active & Kiltie, A. E. Histone deacetylase inhibitors as 194. Audergon, P. N. et al. Restricted epigenetic promoters in the human genome. Nature 436, radiosensitisers: effects on DNA damage signaling inheritance of H3K9 methylation. Science 348, 876–880 (2005). and repair. Br. J. Cancer 108, 748–754 (2013). 132–135 (2015).

PERSPECTIVES

195. Gaydos, L. J., Wang, W. & Strome, S. Gene repression. 204. Sharma, U. et al. Biogenesis and function of tRNA 214. Lewis, P. W. et al. Inhibition of PRC2 activity by a H3K27me and PRC2 transmit a memory of repression fragments during sperm maturation and fertilization gain‑of‑function H3 mutation found in pediatric across generations and during development. Science in mammals. Science 351, 391–396 (2016). glioblastoma. Science 340, 857–861 (2013). 345, 1515–1518 (2014). 205. Grün, D. & van Oudenaarden, A. Design and analysis 215. Behjati, S. et al. Distinct H3F3A and H3F3B driver 196. Margueron, R. et al. Role of the polycomb protein EED of single-cell sequencing experiments. Cell 163, mutations define chondroblastoma and giant cell in the propagation of repressive histone marks. Nature 799–810 (2015). tumor of bone. Nat. Genet. 45, 1479–1482 (2013). 461, 762–767 (2009). 206. Bickmore, W. A. & van Steensel, B. Genome 216. Roy, D. M., Walsh, L. A. & Chan, T. A. Driver mutations 197. Jiao, L. & Liu, X. Structural basis of histone H3K27 architecture: domain organization of interphase of cancer epigenomes. Protein Cell 5, 265–296 trimethylation by an active Polycomb repressive chromosomes. Cell 152, 1270–1284 (2013). (2014). complex 2. Science 350, 6258 (2015). 207. Dekker, J. & Misteli, T. Long-range chromatin 217. Polak, P. et al. Cell‑of‑origin chromatin organization 198. Ost, A. et al. Paternal diet defines offspring chromatin interactions. Cold Spring Harb. Perspect. Biol. 7, shapes the mutational landscape of cancer. Nature state and intergenerational obesity. Cell 159, a019356 (2015). 518, 360–364 (2015). 1352–1364 (2014). 208. Tang, Z. et al. CTCF-mediated human 3D genome 199. Carone, B. R. et al. Paternally induced architecture reveals chromatin topology for Acknowledgements transgenerational environmental reprogramming of transcription. Cell 163, 1611–1627 (2015). C.D.A. and T.J. are grateful to all of our laboratory members, metabolic gene expression in mammals. Cell 143, 209. Rinn, J. L., & Chang, H. Y. Genome regulation by long past and present, and to our scientific colleagues in the field 1084–1096 (2010). noncoding RNAs. Annu. Rev. Biochem. 81, 145–166 for helping us ‘write’ the history that we review here. Their 200. Baulcombe, D. C. & Dean, C. Epigenetic regulation in (2012). hard work, insights and passion for the field of epigenetics plant responses to the environment. Cold Spring Harb. 210. Yan, H. et al. Eusocial insects as emerging models have made the last 20 years such an enjoyable ride. We Perspect. Biol. 6, a019471 (2014). for behavioural epigenetics. Nat. Rev. Genet. 15, thank P. Jones (Grand Rapids, Michigan, USA) and 201. Rassoulzadegan, M. et al. RNA-mediated non- 677–688 (2014). A. Tarakhovsky (New York, USA) for giving us feedback on mendelian inheritance of an epigenetic change in the 211. Lempradl, A., Pospisilik, J. A. & Penninger, J. M. this manuscript and M. Onishi-Seebacher (Freiburg, mouse. Nature 441, 469–474 (2006). Exploring the emerging complexity in transcriptional Germany) for help with the figure preparations and reference 202. Gapp, K. et al. Implication of sperm RNAs in regulation of energy homeostasis. Nat. Rev. Genet. listings. Our objective in this article is to be more reflective transgenerational inheritance of the effects of early 16, 665–681 (2015). than comprehensive, and admittedly, we have brought for- trauma in mice. Nat. Neurosci. 17, 667–669 212. Schwartzentruber, J. et al. Driver mutations in histone ward our personal views. We ask for understanding from (2014). H3.3 and chromatin remodelling genes in paediatric those colleagues whose important contributions could not be 203. Siklenka, K. et al. Disruption of histone glioblastoma. Nature 482, 226–231 (2012). explicitly mentioned. methylation in developing sperm impairs offspring 213. Wu, G. et al. Somatic histone H3 alterations in pediatric health transgenerationally. Science 350, 6261 diffuse intrinsic pontine gliomas and non-brainstem Competing interests statement (2015). glioblastomas. Nat. Genet. 44, 251–253 (2012). The authors declare no competing interests.