REVIEWS Author addresses 'Structural Genomics Consortium.Nuffield Department of Clinical Medici e to herapeutics.0Cambridge Park Drive.Cambridge. of H4K5 bly and de poNdgbndgss toteinsthatareinoivedih feenec on among cor ature in a diverse set of proteins unit 1).For example,the recent modor the multi- e to th also fpecihcprotcinstoge omic loci that contain For ex mple, of the enzym dino nitrogens)or i terminal guanidino nitr ns).As with acetylation Dost-ra read sone mark methylation is dynamic. SAM ark stream xoglutarate-depe s or the I nce ofd states n as KDMIA)and LSD2 (also onal s are often ade the of the marks.The e the charged ysine or arginine residu loes no as br of proteins. hat compa omes together or bring ins to ee t on n i n 1964 (REF.12),it has b ed tha ignal that is recogniz y highly evolved methyl-lysine of en nd HD r.HAT 1).Thus,trir thylated Lys ofhistone3 each interac rudes from the nucleo ple,H3K4me3 is a hallmark of transcriptionally active atin ad pts a more relaxe genes,wherea K9me3 and H3 27me3 (RE 23.2 se the effects of HATs and reverse the ace tylation of nine methylation is abundant and has been known for es to rest their positive charge and stabiliz NATURE REVIEWSIDRUG DISCOVERY VOLUME 11 MAY 2012 385Epigenetics Heritable changes in gene expression or phenotype that are stable between cell divisions, and sometimes between generations, but do not involve changes in the underlying DNA sequence of the organism. Differentiation The process by which a stem cell, or other precursor cell, commits towards a more specialized cell type with a specific function, and represents an exit from self-renewal. Differentiation is controlled by cell signalling pathways and maintained through epigenetic mechanisms. Post-translational modification A chemical modification of proteins that acts as a signal to other proteins that recognize the modification. In the context of epigenetic signalling, post-translational modifications are often called ‘marks’. Epigenome The combination of histone and DNA post-translational modifications and related interacting proteins that together package the genome and help define the transcriptional programme in a given cell. Heterochromatin A tightly packed form of DNA associated with transcriptionally silent or repressed genes. It is highly correlated with di- and trimethlyated H3K9 (Lys9 of histone 3) marks. Euchromatin A more loosely packed form of DNA that is associated with transcriptionally active genes. studied, and have a large number of druggable proteins that mediate their dynamic activity. A feature of epi￾genetic regulation that is mediated by histone marks is the collaboration among combinations of marks to affect specific cellular outcomes — often referred to as the his￾tone code hypothesis7–10 (FIG. 1). For example, the recent mapping of nine acetyl and methyl histone marks across the genomes of nine different cell types showed that com￾binations of marks defined 15 chromatin states related to the transcriptional activity of surrounding genes11. Individual marks and combinations of marks are recognized by several classes of conserved protein domains, usually within the context of larger multipro￾tein complexes. Thus, histone marks and the multi￾protein complexes that bind to them contribute to the physical make-up of chromatin and to the recruitment of specific proteins to genomic loci that contain specific histone marks. For example, most of the enzymes that are ‘writers’ of methyl or acetyl histone marks are large proteins that, in addition to their catalytic domain, con￾tain other domains or regions that ‘read’ histone marks and/or interact with DNA or other proteins. Together, these proteins form complexes that integrate upstream cellular and environmental signals to establish and main￾tain cellular identity and contribute to the genesis and/ or maintenance of disease states10. Owing to remarkable progress over the past decade, we now know the basic complement of regulatory proteins that ‘read’, ‘write’ and ‘erase’ the major histone marks. These are summarized in TABLE 1, and further delineated in FIG. 2 as phylogenetic trees of structurally and evolutionarily related families of proteins. Histone acetylation. Since the first description of histone acetylation in 1964 (REF. 12), it has been established that this is a highly dynamic process that is regulated by two families of enzymes — histone acetyltransferases (HATs) and HDACs — that operate in an opposing manner. HATs use acetyl-CoA as a cofactor and catalyse the transfer of an acetyl group to the ε-amino group of lysine side chains on the histone protein. This neutralizes the positive charge on lysine, thus reducing the affinity of the histone tail that protrudes from the nucleosome core of DNA. As a result, chromatin adopts a more relaxed structure, enabling the recruitment of the transcriptional machinery. HDACs oppose the effects of HATs and reverse the acetylation of lysine residues to restore their positive charge and stabilize the local chromatin architecture. Among the various sites of histone lysine acetylation, Lys16 of histone 4 (H4K16) appears to be crucial in the regulation of chromatin folding and in the switch from heterochromatin to euchromatin13. In addition to the acety￾lation of histone tails, there are several lysine substrates within the globular core of the histone proteins (such as H3K56), which suggests that acetylation can also directly affect the interaction between histones and DNA14. There is evidence that histone acetylation, particularly of H4K5 and H4K12, is important for the recognition of chaper￾ones during histone assembly and deposition into DNA. Histone acetylation also promotes transcription by providing binding sites for proteins that are involved in gene activation. In particular, the bromodomain-containing family of proteins recognize (that is, ‘read’) modified lysine residues within histone proteins. Bromodomains are a common feature in a diverse set of proteins united by their importance in transcriptional co-activation, and the ability of bromodomains to identify and bind to acetylated lysine residues within histone proteins is key to their activity15,16. Histone methylation The significance of and the associated mechanisms of histone methylation have been gradually elucidated over the past decade. Lysine residues on histones can be monomethylated, dimethylated or trimethylated. Arginine residues are also subject to monomethylation and dimethylation. Dimethylation of arginine residues can occur in a symmetric manner (via monomethyla￾tion of both terminal guanidino nitrogens) or in an asymmetric manner (via dimethylation of one of the terminal guanidino nitrogens). As with acetylation, methylation is dynamic. Methyl marks are written by S-adenosylmethionine (SAM)-dependent methyl￾transferases and erased by either the Jumonji family of 2-oxoglutarate-dependent demethylases17 or the flavin￾dependent enzymes lysine-specific histone demethyl￾ase 1 (LSD1; also known as KDM1A) and LSD2 (also known as KDM1B)18. Because methylation does not change the charged state of a lysine or arginine residue, it does not appear to effect chromatin structure directly. Instead, the vari￾ous methyl marks act as binding sites for other proteins that compact nucleosomes together19,20 or bring addi￾tional regulatory proteins to chromatin sites marked by methylation21,22. Each type of mark constitutes a specific signal that is recognized by highly evolved methyl-lysine￾binding domains that recognize the level of methyla￾tion and, in many cases, the surrounding amino acid sequence (TABLE 1). Thus, trimethylated Lys4 of histone 3 (H3K4me3), H3K9me3 and H4K20me2 each interact with a distinct set of reader domains. Histone lysine methylation can be associated with either transcriptional activation or repression. For exam￾ple, H3K4me3 is a hallmark of transcriptionally active genes, whereas H3K9me3 and H3K27me3 (REFS 23,24) are associated with silenced genes. Although protein argi￾nine methylation is abundant and has been known for a long time, histone arginine methylation has only recently become recognized as an important transcriptional Author addresses 4 Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7LD, UK. 5 Worldwide Medicinal Chemistry, Pfizer, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK. 6 Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK. 7 Department of Pharmacology & Toxicology, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada. *Present address: Pfizer Biotherapeutics, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140, USA. REVIEWS NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | MAY 2012 | 385 © 2012 Macmillan Publishers Limited. All rights reserved