Histones were discovered in 1884 by  Albrecht Kossel . [ 116 ]  The word "histone" dates from the late 19th century and is derived from the German word  "Histon" , a word itself of uncertain origin, perhaps from Ancient Greek  ἵστημι  (hístēmi, “make stand”) or  ἱστός  (histós, “loom”).


In the early 1960s, before the types of histones were known and before histones were known to be highly conserved across taxonomically diverse organisms,  James F. Bonner  and his collaborators began a study of these proteins that were known to be tightly associated with the DNA in the nucleus of higher organisms. [ 117 ]  Bonner and his postdoctoral fellow  Ru Chih C. Huang  showed that isolated chromatin would not support RNA transcription in the test tube, but if the histones were extracted from the chromatin, RNA could be transcribed from the remaining DNA. [ 118 ]  Their paper became a citation classic. [ 119 ]   Paul T'so and James Bonner had called together a World Congress on Histone Chemistry and Biology in 1964, in which it became clear that there was no consensus on the number of kinds of histone and that no one knew how they would compare when isolated from different organisms. [ 120 ] [ 117 ]   Bonner and his collaborators then developed methods to separate each type of histone, purified individual histones, compared amino acid compositions in the same histone from different organisms, and compared amino acid sequences  of the same histone from different organisms in collaboration with  Emil Smith  from UCLA. [ 121 ]  For example, they found Histone IV sequence to be highly conserved between peas and calf thymus. [ 121 ]   However, their work on the biochemical characteristics of individual histones did not reveal how the histones interacted with each other or with DNA to which they were tightly bound. [ 120 ]


Also in the 1960s, Vincent Allfrey and  Alfred Mirsky  had suggested, based on their analyses of histones, that acetylation and methylation of histones could provide a transcriptional control mechanism, but did not have available the kind of detailed analysis that later investigators were able to conduct to show how such regulation could be gene-specific. [ 122 ]   Until the early 1990s, histones were dismissed by most as inert packing material for eukaryotic nuclear DNA, a view based in part on the models of  Mark Ptashne  and others, who believed that transcription was activated by protein-DNA and protein-protein interactions on largely naked DNA templates, as is the case in bacteria.


During the 1980s, Yahli Lorch and  Roger Kornberg [ 123 ]  showed that a nucleosome on a core promoter prevents the initiation of transcription in vitro, and  Michael Grunstein [ 124 ]  demonstrated that histones repress transcription in vivo, leading to the idea of the nucleosome as a general gene repressor.  Relief from repression is believed to involve both histone modification and the action of chromatin-remodeling complexes.  Vincent Allfrey and Alfred Mirsky had earlier proposed a role of histone modification in transcriptional activation, [ 125 ]  regarded as a molecular manifestation of epigenetics. Michael Grunstein [ 126 ]  and David Allis [ 127 ]  found support for this proposal, in the importance of histone acetylation for transcription in yeast and the activity of the transcriptional activator Gcn5 as a histone acetyltransferase.


The discovery of the H5 histone appears to date back to the 1970s, [ 128 ]  and it is now considered an  isoform  of  Histone H1 . [ 2 ] [ 4 ] [ 5 ] [ 6 ]


Histone acetyltransferases  ( HATs ) are  enzymes  that  acetylate  conserved  lysine   amino acids  on  histone  proteins by transferring an  acetyl group  from  acetyl-CoA  to form  ε- N -acetyllysine . DNA is wrapped around histones, and, by transferring an acetyl group to the histones, genes can be turned on and off. In general, histone acetylation increases gene expression.


In general, histone acetylation is linked to  transcriptional  activation and associated with  euchromatin .  Euchromatin, which is less densely compact, allows transcription factors to bind more easily to regulatory sites on DNA, causing transcriptional activation.  When it was first discovered, it was thought that  acetylation  of lysine neutralizes the positive  charge  normally present, thus reducing affinity between histone and (negatively charged) DNA, which renders DNA more accessible to  transcription factors .  Research has emerged, since, to show that lysine acetylation and other  posttranslational modifications  of histones generate binding sites for specific protein–protein interaction domains, such as the acetyllysine-binding  bromodomain [ citation needed ] . Histone acetyltransferases can also acetylate non-histone proteins, such as nuclear receptors and other transcription factors to facilitate gene expression.


HATs are traditionally divided into two different classes based on their subcellular localization. [ 2 ]  Type A HATs are located in the  nucleus  and are involved in the regulation of gene expression through  acetylation of nucleosomal histones  in the context of chromatin. [ 3 ]   They contain a  bromodomain , which helps them recognize and bind to acetylated lysine residues on histone substrates. Gcn5,  p300/CBP , and  TAF II 250  are some examples of type A HATs that cooperate with activators to enhance transcription. Type B HATs are located in the  cytoplasm  and are responsible for acetylating newly synthesized histones prior to their assembly into  nucleosomes . These HATs lack a bromodomain, as their targets are unacetylated. The acetyl groups added by type B HATs to the histones are removed by  HDACs  once they enter the nucleus and are incorporated into  chromatin .  Hat1  is one of the few known examples of a type B HAT. [ 4 ]   Despite this historical classification of HATs, some HAT proteins function in multiple complexes or locations and would thus not easily fit into a particular class. [ 5 ]


HATs can be grouped into several different families based on sequence homology as well as shared structural features and functional roles. The Gcn5-related  N -acetyltransferase (GNAT) family includes Gcn5,  PCAF , Hat1,  Elp3 , Hpa2, Hpa3,  ATF-2 , and Nut1. These HATs are generally characterized by the presence of a bromodomain, and they are found to acetylate lysine residues on histones  H2B ,  H3 , [ 6 ]  and  H4 . [ 2 ]   All members of the GNAT family are characterized by up to four conserved motifs (A-D) found within the catalytic HAT domain. This includes the most highly conserved motif A, which contains an Arg/Gln-X-X-Gly-X-Gly/Ala sequence that is important for  acetyl-CoA  recognition and binding. [ 4 ]   The C motif is found in most GNATs, but it is not present in the majority of other known HATs. [ 5 ]   The yeast Gcn5 (general control nonderepressible-5) HAT is one of the best-characterized members of this family. It has four functional domains, including an N-terminal domain, a highly conserved catalytic (HAT) domain, an Ada2 interaction domain, and a C-terminal bromodomain. PCAF (p300/CBP-associated factor) and GCN5 are mammalian GNATs that share a high degree of homology throughout their sequences. These proteins have a 400-residue N-terminal region that is absent in yeast Gcn5, but their HAT functions are evolutionarily conserved with respect to the latter. Hat1 was the first HAT protein to be identified. It is responsible for most of the cytoplasmic HAT activity in yeast, and it binds strongly to histone H4 by virtue of its association with an additional subunit, Hat2. Elp3 is an example of a type A HAT found in yeast. It is part of the  RNA polymerase II holoenzyme  and plays a role in  transcriptional elongation .


The MYST family of HATs is named after its four founding members  MOZ , Ybf2 (Sas3), Sas2, and  Tip60 . [ 2 ]   Other important members include  Esa1 ,  MOF ,  MORF , and  HBO1 . These HATs are typically characterized by the presence of  zinc fingers  and  chromodomains , and they are found to acetylate lysine residues on histones  H2A , H3, and H4. Several MYST family proteins contain zinc fingers as well as the highly conserved motif A found among GNATs that facilitates acetyl-CoA binding. [ 4 ]   A cysteine-rich region located in the N terminus of the HAT domain of MYST proteins is involved in zinc binding, which is essential for HAT activity. [ 7 ]   Tip60 (Tat-interactive protein, 60 kDa) was the first human MYST family member to exhibit HAT activity. Sas3 found in yeast is a homolog of MOZ (monocytic leukemia zinc finger protein), which is an  oncogene  found in humans. Esa1 was the first essential HAT to be found in yeast, and MOF is its homolog in fruit flies. The HAT activity of the latter is required for the twofold increased transcription of the male X chromosome ( dosage compensation ) in flies. Human HBO1 (HAT bound to ORC1) was the first HAT shown to associate with components of the  origin of replication complex . MORF (MOZ-related factor) exhibits very close homology to MOZ throughout its entire length. [ 5 ]   It contains an N-terminal repression region that decreases its HAT activity  in vitro  as well as a C-terminal activation domain that is functional in the absence of the HAT domain.


In addition to those that are members of the GNAT and MYST families, there are several other proteins found typically in higher eukaryotes that exhibit HAT activity. These include p300/CBP, nuclear receptor coactivators (e.g., ACTR/SRC-1), TAF II 250, TFIIIC, Rtt109, and  CLOCK . p300/CBP are  metazoan -specific [ 8 ]  and contain several zinc finger regions, a bromodomain, a catalytic (HAT) domain, and regions that interact with other transcription factors. [ 4 ]   Importantly, the HAT domain shows no sequence homology to other known HATs, [ 9 ]   and it is required for p300/CBP to function in transcriptional activation. [ 4 ]   In addition, these proteins contain several HAT domain motifs (A, B, and D) that are similar to those of the GNATs. They also possess a novel motif E that is homologous to sequences in the HAT domains of GNATs. TFIIIC is one of the general transcription factors involved in  RNA polymerase III -mediated transcription. Three components in the human protein have been shown to possess independent HAT activity ( hTFIIIC220 ,  hTFIIIC110 , and  hTFIIIC90 ). [ 10 ]   Rtt109 is a  fungal -specific HAT that requires association with histone chaperone proteins for activity. [ 8 ]   The HAT activities of the human TAF II 250 and CLOCK coactivators have not been studied as extensively. TAF II 250 is one of the TBP-associated factor subunits of  TFIID , and it shares a Gly-X-Gly pattern with Gcn5 that is important for HAT activity. [ 5 ]   CLOCK is a circadian rhythm master regulator that functions with  BMAL1  to carry out its HAT activity. [ 11 ]


Three important nuclear receptor coactivators that display HAT activity are  SRC-1 ,  ACTR , and  TIF-2 . Human SRC-1 (steroid receptor coactivator-1) is known to interact with p300/CBP and PCAF, and its HAT domain is located in its C-terminal region. ACTR (also known as RAC3, AIB1, and TRAM-1 in humans) shares significant sequence homology with SRC-1, in particular in the N-terminal and C-terminal (HAT) regions as well as in the receptor and coactivator interaction domains. [ 5 ]   ACTR also interacts with p300/CBP and PCAF. The former can prevent ACTR from binding to and activating its receptor by acetylating it in its receptor interaction domain. TIF-2 (transcriptional intermediary factor 2; also known as GRIP1) is another nuclear receptor coactivator with HAT activity, and it also interacts with p300/CBP.


A table summarizing the different families of HATs along with their associated members, parent organisms, multisubunit complexes, histone substrates, and structural features is presented below. [ 2 ] [ 5 ] [ 8 ] [ 10 ] [ 12 ] [ 13 ] [ 14 ] [ 15 ] [ 16 ] [ 17 ]


(nuclear receptor coactivators)


In general, HATs are characterized by a structurally conserved core region made up of a three-stranded  β-sheet  followed by a long  α-helix  parallel to and spanning one side of it. [ 7 ] [ 8 ]   The core region, which corresponds to motifs A, B, and D of the GNAT proteins, [ 4 ]   is flanked on opposite sides by N- and C-terminal α/β segments that are structurally unique for a given HAT family. [ 7 ] [ 8 ]   The central core and the flanking segments together form a cleft over the former, which is where histone substrates can bind prior to catalysis. [ 8 ]   While the central core domain (motif A in GNATs) is involved in acetyl-CoA binding and catalysis, the N- and C-terminal segments assist in binding histone substrates. [ 7 ]   Unique features related to the sequence and/or structure of the N- and C-terminal regions for different HAT families may help to explain some observed differences among HATs in histone substrate specificity. CoA binding has been observed to widen the histone binding groove in the central core by moving the C-terminal segment of Gcn5 outward. In addition, since contacts between CoA and protein facilitate the formation of favorable histone-protein contacts, it is likely that CoA binding precedes histone binding  in vivo .


HATs in the GNAT family are most notably characterized by an approximately 160-residue HAT domain and a C-terminal bromodomain, which binds to acetylated lysine residues. [ 7 ]   Those in the MYST family have HAT domains that are about 250 residues in length. Many MYST proteins also contain a cysteine-rich, zinc-binding domain within the HAT region in addition to an N-terminal chromodomain, which binds to  methylated lysine residues .


On a broader scale, the structures of the catalytic domains of GNAT proteins (Gcn5, PCAF) exhibit a mixed α/β globular fold with a total of five α-helices and six β-strands. [ 4 ]   The overall topology resembles a  vise , with the central core of the protein at the base and the N- and C-terminal segments on the sides.


The p300/CBP HATs have larger HAT domains (about 500 residues) than those present in the GNAT and MYST families. [ 7 ]   They also contain a bromodomain as well as three cysteine/histidine-rich domains that are thought to mediate interactions with other proteins. The structure of p300/CBP is characterized by an elongated globular domain, which contains a seven-stranded β-sheet in the center that is surrounded by nine α-helices and several loops. [ 9 ]   The structure of the central core region associated with acetyl-CoA binding is conserved with respect to GNAT and MYST HATs, but there are many structural differences in the regions flanking this central core. Overall, the structural data is consistent with the fact that p300/CBP HATs are more promiscuous than GNAT and MYST HATs with respect to substrate binding.


The structure of Rtt109 is very similar to that of p300, despite there only being 7% sequence identity between the two proteins. [ 9 ]  There is a seven-stranded β-sheet that is surrounded by α-helices as well as a loop that is involved in acetyl-CoA substrate binding. Despite the conserved structure, Rtt109 and p300/CBP are functionally unique. For instance, the substrate binding site of the former is more similar to that of the GNAT and MYST HATs. In addition, the residues in the active site of each enzyme are distinct, which suggests that they employ different catalytic mechanisms for acetyl group transfer.


The basic mechanism catalyzed by HATs involves the transfer of an acetyl group from acetyl-CoA to the ε-amino group of a target lysine side-chain within a histone. [ 8 ]   Different families of HATs employ unique strategies in order to effect such a transformation.


Members of the GNAT family have a conserved glutamate residue that acts as a general base for catalyzing the nucleophilic attack of the lysine amine on the acetyl-CoA thioester bond. [ 8 ]   These HATs use an ordered sequential bi-bi mechanism wherein both substrates (acetyl-CoA and histone) must bind to form a  ternary complex  with the enzyme before catalysis can occur. Acetyl-CoA binds first, followed by the histone substrate. A conserved glutamate residue (Glu173 in yeast Gcn5) activates a water molecule for removal of a proton from the amine group on lysine, which activates it for direct nucleophilic attack on the carbonyl carbon of enzyme-bound acetyl-CoA. After the reaction, the acetylated histone is released first followed by CoA. [ 4 ] [ 8 ]


Studies of yeast Esa1 from the MYST family of HATs have revealed a  ping-pong mechanism  involving conserved glutamate and cysteine residues. [ 18 ]  The first part of the reaction involves the formation of a covalent intermediate in which a cysteine residue becomes acetylated following nucleophilic attack of this residue on the carbonyl carbon of acetyl-CoA. Then, a glutamate residue acts as a general base to facilitate transfer of the acetyl group from the cysteine to the histone substrate in a manner analogous to the mechanism used by GNATs. When Esa1 is assembled in the piccolo  NuA4  complex, it loses its dependence on the cysteine residue for catalysis, which suggests that the reaction may proceed via a ternary bi-bi mechanism when the enzyme is part of a physiologically relevant multiprotein complex.


In human p300, Tyr1467 acts as a general acid and Trp1436 helps orient the target lysine residue of the histone substrate into the active site. [ 8 ]   These two residues are highly conserved within the p300/CBP HAT family and, unlike enzymes in the GNAT and MYST families, p300 does not employ a general base for catalysis. Rather, it is likely that members of the p300/CBP family use a Theorell-Chance (i.e., “hit-and-run”) acetyl transfer mechanism.


Rtt109 is likely to employ a mechanism that is different from that of the other HATs. [ 9 ]   The yeast enzyme has very low catalytic activity in the absence of the histone chaperone proteins  Asf1  and Vps75, which may be involved in delivering histone substrates to the enzyme for acetylation. [ 8 ]   Moreover, a general acid or base have not yet been identified for this HAT.


The structures of several HAT domains bound to acetyl-CoA and histone substrate peptides reveal that the latter bind across a groove on the protein that is formed by the central core region at the base and is flanked on opposite sides by the variable N- and C-terminal segments that mediate the majority of the interactions with the substrate peptide. [ 8 ]   It is likely that these variable regions are at least in part responsible for the observed specificity of different HATs for various histone substrates.


Members of the GNAT and MYST families as well as Rtt109 exhibit greater substrate selectivity than p300/CBP, which is rather promiscuous with regard to substrate binding. [ 8 ]   Whereas it appears that only three to five residues on either side of the lysine to be acetylated are necessary for effective substrate binding and catalysis by members of the GNAT and p300/CBP families, more distal regions of the substrate may be important for efficient acetylation by MYST family HATs. [ 19 ]


Different HATs, usually in the context of multisubunit complexes, have been shown to acetylate specific lysine residues in histones.


Gcn5 cannot acetylate nucleosomal histones in the absence of other protein factors. [ 5 ]   In the context of complexes like SAGA and ADA, however, Gcn5 is able to acetylate H3K14 among other sites within histones H2B, H3, and H4 (e.g., H3K9, H3K36, H4K8, H4K16). [ 3 ] [ 4 ] [ 7 ] [ 19 ]   Both Gcn5 and PCAF have the strongest site preference for H3K14, either as a free histone or within a nucleosome. [ 4 ] [ 7 ]   Hat1 acetylates H4K5 and H4K12, and Hpa2 acetylates H3K14  in vitro . [ 4 ] [ 5 ]


In flies, acetylation of H4K16 on the male X chromosome by MOF in the context of the MSL complex is correlated with transcriptional upregulation as a mechanism for dosage compensation in these organisms. [ 2 ]   In humans, the MSL complex carries out the majority of genome-wide H4K16 acetylation. In the context of their cognate complexes, Sas2 (SAS) and Esa1 (NuA4) also carry out acetylation of H4K16, in particular in the  telomere  regions of chromosomes. Sas2 is also observed to acetylate H3K14  in vitro  on free histones. [ 12 ]   Esa1 can also acetylate H3K14  in vitro  on free histones as well as H2AK5, H4K5, H4K8, and H4K12 either  in vitro  or  in vivo  on nucleosomal histones. H2AK7 and H2BK16 are also observed to be acetylated by Esa1  in vivo . Notably, neither Sas2 nor Esa1 can acetylate nucleosomal histones  in vitro  as a free enzyme. This happens to be the case as well for Sas3, which is observed to acetylate H3K9 and H3K14  in vivo  as well as lysine residues on H2A and H4. MOZ can also acetylate H3K14. [ 19 ]


p300/CBP acetylate all four nucleosomal core histones equally well. [ 4 ]   In vitro , they have been observed to acetylate H2AK5, H2BK12, H2BK15, H3K14, H3K18, H4K5, and H4K8. [ 5 ]   SRC-1 acetylates H3K9 and H3K14, TAF II 230 (Drosophila homolog of human TAF II 250) acetylates H3K14, and Rtt109 acetylates H3K9, H3K23, [ 19 ]   and H3K56 in the presence of either Asf1 or Vps75. [ 9 ]


In addition to the core histones, certain HATs acetylate a number of other cellular proteins including  transcriptional activators ,  basal transcription factors , structural proteins,  polyamines , and proteins involved in nuclear import. [ 4 ]   Acetylation of these proteins can alter their ability to interact with their cognate DNA and/or protein substrates. The idea that acetylation can affect protein function in this manner has led to inquiry regarding the role of acetyltransferases in signal transduction pathways and whether an appropriate analogy to  kinases  and phosphorylation events can be made in this respect.


PCAF and p300/CBP are the main HATs that have been observed to acetylate a number of non-histone proteins. For PCAF, these include the non-histone chromatin ( high-mobility group (HMG) ) proteins  HMG-N2/HMG17  and  HMG-I(Y) , the transcriptional activators  p53 ,  MyoD ,  E2F(1-3) , and  HIV Tat , and the general transcription factors  TFIIE  and  TFIIF . [ 5 ]   Other proteins include  CIITA , Brm (chromatin remodeler),  NF-κB  (p65),  TAL1/SCL ,  Beta2/NeuroD ,  C/EBPβ ,  IRF2 ,  IRF7 ,  YY1 ,  KLF13 ,  EVI1 , AME,  ER81 , and the  androgen receptor (AR) . [ 20 ]   PCAF has also been observed to acetylate  c-MYC ,  GATA-2 ,  retinoblastoma (Rb) ,  Ku70 , and  E1A  adenovirus protein. [ 21 ]   It can also autoacetylate, which facilitates intramolecular interactions with its bromodomain that may be involved in the regulation of its HAT activity. [ 4 ]


p300/CBP have many non-histone substrates, including the non-histone chromatin proteins  HMG1 ,  HMG-N1/HMG14 , and HMG-I(Y), the transcriptional activators p53,  c-Myb ,  GATA-1 ,  EKLF ,  TCF , and HIV Tat, the nuclear receptor coactivators ACTR, SRC-1, and TIF-2, and the general transcription factors TFIIE and TFIIF. [ 5 ]   Other substrates include the transcription factors Sp1,  KLF5 ,  FOXO1 ,  MEF2C ,  SRY ,  GATA-4 , and  HNF-6 , [ 12 ]   HMG-B2 , [ 21 ]   STAT3 , the androgen and  estrogen (α)  receptors, GATA-2,  GATA-3 , MyoD, E2F(1-3),  p73 α, retinoblastoma (Rb), NF-κB (p50, p65),  Smad7 ,  importin-α , Ku70,  YAP1 , [ 22 ]  E1A adenovirus protein, and S-HDAg ( hepatitis delta virus  small delta antigen). [ 21 ]   p300/CBP have also been observed to acetylate  β-catenin ,  RIP140 ,  PCNA , the DNA metabolic enzymes  flap endonuclease-1 ,  thymine DNA glycosylase , and  Werner syndrome DNA helicase ,  STAT6 ,  Runx1 (AML1) , UBF, Beta2/NeuroD,  CREB ,  c-Jun , C/EBPβ,  NF-E2 ,  SREBP , IRF2,  Sp3 , YY1, KLF13, EVI1,  BCL6 ,  HNF-4 , ER81 and  FOXO4 (AFX) . [ 20 ]


The formation of multisubunit complexes has been observed to modulate the substrate specificity of HATs. [ 12 ]   In general, while recombinant HATs are able to acetylate free histones, HATs can acetylate nucleosomal histones only when they are in their respective  in vivo  HAT complexes. [ 5 ]   Some of the proteins that associate with HATs in these complexes function by targeting the HAT complex to nucleosomes at specific regions in the  genome . [ 2 ] [ 12 ]   For instance, it has been observed that HAT complexes (e.g. SAGA, NuA3) often use  methylated histones  as docking sites so that the catalytic HAT subunit can carry out histone acetylation more effectively. [ 2 ]


In addition, the formation of multisubunit HAT complexes influences the lysine specificity of HATs. [ 12 ]   The specific lysine residues that a given HAT acetylates may become either broader or more restricted in scope upon association with its respective complex. For example, the lysine specificity of MYST family HATs toward their histone substrates becomes more restricted when they associate with their complexes. In contrast, Gcn5 acquires the ability to acetylate multiple sites in both histones H2B and H3 when it joins other subunits to form the SAGA and ADA complexes. [ 4 ]   Moreover, the acetylation site specificity of Rtt109 is dictated by its association with either Vps75 or Asf1. [ 19 ]   When in complex with the former, Rtt109 acetylates H3K9 and H3K27, but, when in complex with the latter, it preferentially acetylates H3K56. [ 8 ]


The catalytic activity of HATs is regulated by two types of mechanisms: (1) interaction with regulatory protein subunits and (2) autoacetylation. [ 8 ]   A given HAT may be regulated in multiple ways, and the same effector may actually lead to different outcomes under different conditions. [ 4 ]   Although it is clear that the association of HATs with multiprotein complexes provides a mechanism for the regulation of both HAT activity and substrate specificity  in vivo , the molecular basis for how this actually occurs is still largely unknown. [ 8 ]   However, data suggests that associated subunits may contribute to catalysis at least in part by facilitating productive binding of the HAT complex to its native histone substrates.


The MYST family of HATs, p300/CBP, and Rtt109 have all been shown to be regulated by autoacetylation. [ 8 ]   Human MOF as well as yeast Esa1 and Sas2 are autoacetylated at a conserved active site lysine residue, and this modification is required for their function  in vivo . Human p300 contains a highly basic loop embedded in the middle of its HAT domain that is hyperacetylated in the active form of the enzyme. [ 8 ] [ 9 ]   It has been proposed that, upon autoacetylation, this loop is released from the electronegative substrate binding site where it sits in the inactive HAT. [ 23 ]   Acetylation of yeast Rtt109 at Lys290 is also required for it to exhibit full catalytic activity. [ 24 ]   Some HATs are also inhibited by acetylation. For example, the HAT activity of the nuclear receptor coactivator ACTR is inhibited upon acetylation by p300/CBP. [ 4 ]


Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are recruited to their target promoters through physical interactions with sequence-specific transcription factors. They usually function within a multisubunit complex in which the other subunits are necessary for them to modify histone residues around the binding site. These enzymes can also modify non-histone proteins.


Histone acetyltransferases serve many biological roles inside the cell.  Chromatin  is a combination of proteins and  DNA  found in the  nucleus , and it undergoes many structural changes as different cellular events such as  DNA replication ,  DNA repair , and  transcription  occur. [ 25 ]   Chromatin in the cell can be found in two states: condensed and uncondensed. The latter, known as  euchromatin , is transcriptionally active, whereas the former, known as  heterochromatin , is transcriptionally inactive. [ 25 ] [ 26 ]   Histones comprise the protein portion of chromatin. There are five different  histone  proteins: H1, H2A, H2B, H3, and H4. A core histone is formed when two of each histone subtype, excluding H1, form a quaternary complex. This octameric complex, in association with the 147 base pairs of DNA coiled around it, forms the  nucleosome . [ 4 ]   Histone H1 locks the nucleosome complex together, and it is the last protein to bind in the complex.


Histones tend to be positively charged proteins with N-terminal tails that stem from the core. The phosphodiester backbone of DNA is negative, which allows for strong ionic interactions between histone proteins and DNA. Histone acetyltransferases transfer an  acetyl  group to specific  lysine  residues on histones, which neutralizes their positive charge and thus reduces the strong interactions between the histone and DNA. [ 25 ]   Acetylation is also thought to perturb interactions between individual nucleosomes and act as interaction sites for other DNA-associated proteins. [ 4 ]


There can be different levels of histone acetylation as well as other types of modifications, allowing the cell to have control over the level of chromatin packing during different cellular events such as replication, transcription, recombination, and repair. Acetylation is not the only regulatory  post-translational modification  to histones that dictates chromatin structure; methylation, phosphorylation, ADP-ribosylation, and ubiquitination have also been reported. [ 4 ] [ 25 ]   These combinations of different covalent modifications on the N-terminal tails of histones have been referred to as the  histone code , and it is thought that this code may be heritable and preserved in the next cell generation. [ 26 ]


H3 and H4 histone proteins are the primary targets of HATs, but H2A and H2B are also acetylated  in vivo . Lysines 9, 14, 18, and 23 of H3 and lysines 5, 8, 12, and 16 of H4 are all targeted for acetylation. [ 4 ] [ 25 ]   Lysines 5, 12, 15, and 20 are acetylated on histone H2B, while only lysines 5 and 9 have been observed to be acetylated on histone H2A. [ 4 ] [ 25 ] [ 26 ]   With so many different sites for acetylation, a high level of specificity can be achieved in triggering specific responses. An example of this specificity is when histone H4 is acetylated at lysines 5 and 12. This acetylation pattern has been seen during histone synthesis. Another example is acetylation of H4K16, which has been associated with dosage compensation of the male X chromosome in  Drosophila melanogaster . [ 2 ] [ 4 ]


Histone modifications modulate the packing of chromatin. The level of packing of the DNA is important for gene transcription, since the transcriptional machinery must have access to the promoter in order for transcription to occur. [ 4 ]   Neutralization of charged lysine residues by HATs allows for the chromatin to decondense so that this machinery has access to the gene to be transcribed. However, acetylation is not always associated with enhanced transcriptional activity. For instance, acetylation of H4K12 has been associated with condensed and transcriptionally inactive chromatin. [ 27 ]  In addition, some histone modifications are associated with both enhanced and repressed activity, in a context-dependent manner. [ 28 ]


HATs act as transcriptional co-activators or gene silencers and are most often found in large complexes made up of 10 to 20 subunits, some of which shared among different HAT complexes. [ 25 ]   These complexes include SAGA (Spt/Ada/Gcn5L acetyltransferase), PCAF, ADA (transcriptional adaptor), TFIID (transcription factor II D), TFTC (TBP-free TAF-containing complex), and NuA3/NuA4 (nucleosomal acetyltransferases of H3 and H4). [ 2 ] [ 25 ]   These complexes modulate HAT specificity by bringing HATs to their target genes where they can then acetylate nucleosomal histones. [ 25 ]   Some HAT transcriptional co-activators contain a  bromodomain , a 110-amino acid module that recognizes acetylated lysine residues and is functionally linked to the co-activators in the regulation of transcription. [ 29 ]


The ability of histone acetyltransferases to manipulate chromatin structure and lay an  epigenetic  framework makes them essential in cell maintenance and survival. The process of chromatin remodeling involves several enzymes, including HATs, that assist in the reformation of nucleosomes and are required for DNA damage repair systems to function. [ 30 ]   HATs have been implicated as accessories to disease progression, specifically in neurodegenerative disorders. For instance,  Huntington's disease  is a disease that affects motor skills and mental abilities. The only known mutation that has been implicated in the disease is in the N-terminal region of the protein  huntingtin (htt) . [ 31 ]   It has been reported that htt directly interacts with HATs and represses the catalytic activity of p300/CBP and PCAF  in vitro .


The human premature aging syndrome Hutchinson Gilford  progeria  is caused by a mutational defect in the processing of  lamin A , a  nuclear matrix  protein.   In a mouse model of this condition, recruitment of  repair  proteins to sites of  DNA damage  is delayed.  The molecular mechanism underlying this delayed repair response involves a histone acetylation defect. [ 32 ]   Specifically,  histone H4  is hypoacetylated at a lysine 16 residue (H4K16) and this defect is due to reduced association of histone acetyltransferase, Mof, to the nuclear matrix [ 32 ]


Spinocerebellar ataxia type 1  is a neurodegenerative disease that arises as a result of a defective mutant  Ataxin-1  protein.  Mutant  Ataxin-1  reduces histone acetylation resulting in repressed histone acetyltransferase-mediated  transcription . [ 33 ]


HATs have also been associated with control of learning and memory functions. Studies have shown that mice without PCAF or CBP display evidence of  neurodegeneration . [ 31 ]   Mice with PCAF deletion are incompetent with respect to learning, and those with CBP deletion seem to suffer from long-term memory loss. [ 34 ]


The misregulation of the equilibrium between acetylation and deacetylation has also been associated with the manifestation of certain cancers. If histone acetyltransferases are inhibited, then damaged DNA may not be repaired, eventually leading to cell death. Controlling the  chromatin remodeling  process within cancer cells may provide a novel drug target for cancer research. [ 35 ]   Attacking these enzymes within cancer cells could lead to increased  apoptosis  due to high accumulation of DNA damage. One such inhibitor of histone acetyltransferases is called garcinol. This compound is found within the rinds of the  garcinia indica  fruit, otherwise known as  mangosteen . To explore the effects of garcinol on histone acetyltransferases, researchers used  HeLa  cells. The cells underwent irradiation, creating double-strand breaks within the DNA, and garcinol was introduced into the cells to see if it influenced the DNA damage response. If garcinol is successful at inhibiting the process of  non-homologous end joining , a DNA repair mechanism that shows preference in fixing double-strand breaks, [ 36 ]  then it may serve as a  radiosensitizer , a molecule that increases the sensitivity of cells to radiation damage. Increases in radiosensitivity may increase the effectiveness of radiotherapy. [ 35 ]


The  histone code  is a  hypothesis  that the transcription of genetic information encoded in  DNA  is in part regulated by chemical modifications (known as  histone marks ) to  histone  proteins, primarily on their unstructured ends.  Together with similar modifications such as  DNA methylation  it is part of the  epigenetic code . [ 1 ]  Histones associate with  DNA  to form  nucleosomes , which themselves bundle to form  chromatin  fibers, which in turn make up the more familiar  chromosome . Histones are globular proteins with a flexible  N-terminus  (taken to be the tail) that protrudes from the nucleosome. Many of the histone tail modifications correlate very well to chromatin structure and both histone modification state and chromatin structure correlate well to gene expression levels. The critical concept of the  histone code hypothesis  is that the histone modifications serve to recruit other proteins by specific recognition of the modified histone via  protein domains  specialized for such purposes, rather than through simply stabilizing or destabilizing the interaction between histone and the underlying DNA. These recruited proteins then act to alter chromatin structure actively or to promote transcription.
For details of gene expression regulation by histone modifications see  table below .


The hypothesis is that  chromatin -DNA interactions are guided by combinations of histone modifications.  While it is accepted that modifications (such as  methylation ,  acetylation ,  ADP-ribosylation ,  ubiquitination ,  citrullination ,  SUMO -ylation [ 2 ]  and  phosphorylation ) to  histone  tails alter chromatin structure, a complete understanding of the precise mechanisms by which these alterations to histone tails influence DNA-histone interactions remains elusive.  However, some specific examples have been worked out in detail.  For example, phosphorylation of  serine  residues 10 and 28 on  histone H3  is a marker for chromosomal condensation.  Similarly, the combination of phosphorylation of  serine  residue 10 and acetylation of a  lysine  residue 14 on histone H3 is a tell-tale sign of active  transcription .


Well characterized modifications to histones include: [ 3 ]


Unlike this simplified model, any real histone code has the potential to be massively complex; each of the four standard histones can be simultaneously modified at multiple different sites with multiple different modifications. To give an idea of this complexity,  histone H3  contains nineteen lysines known to be methylated—each can be un-, mono-, di- or tri-methylated. If modifications are independent, this allows a potential 4 19  or 280 billion different lysine methylation patterns, far more than the maximum number of histones in a human genome (6.4 Gb / ~150 bp = ~44 million histones if they are very tightly packed). And this does not include lysine acetylation (known for H3 at nine residues), arginine methylation (known for H3 at three residues) or threonine/serine/tyrosine phosphorylation (known for H3 at eight residues), not to mention modifications of other histones. [ citation needed ]


Every  nucleosome  in a cell can therefore have a different set of modifications, raising the question of whether common patterns of histone modifications exist. A study of about 40 histone modifications across human gene promoters found over 4000 different combinations used, over 3000 occurring at only a single promoter. However, patterns were discovered including a set of 17 histone modifications that are present together at over 3000 genes. [ 16 ]   Mass spectrometry -based  top-down proteomics  has provided more insight into these patterns by being able to discriminate single molecule co-occurrence from co-localization in the genome or on the same nucleosome. [ 17 ]  A variety of approaches have been used to delve into detailed biochemical mechanisms that demonstrate the importance of interplay between histone modifications. Thus, specific patterns of histone modifications are more common than others. These patterns are functionally important but they are intricate and challenging to study. We currently have the best biochemical understanding of the importance of a relatively small number of discrete modifications and a few combinations.


Structural determinants of histone recognition by readers, writers, and erasers of the histone code are revealed by a growing body of experimental data. [ 18 ]


Histone methylation  is a process by which  methyl groups  are transferred to  amino acids  of  histone  proteins that make up  nucleosomes , which the  DNA  double helix wraps around to form  chromosomes . Methylation of histones can either increase or decrease transcription of genes, depending on which amino acids in the histones are methylated, and how many methyl groups are attached. Methylation events that weaken chemical attractions between histone tails and DNA increase transcription because they enable the DNA to uncoil from nucleosomes so that  transcription factor  proteins and  RNA polymerase  can access the DNA. This process is critical for the regulation of  gene expression  that allows different cells to express different genes.


Histone methylation, as a mechanism for modifying  chromatin  structure is associated with stimulation of neural pathways known to be important for formation of  long-term memories  and learning. [ 1 ]  Histone methylation is crucial for almost all phases of  animal embryonic development . [ 2 ]


Animal models have shown methylation and other epigenetic regulation mechanisms to be associated with conditions of aging,  neurodegenerative diseases , and intellectual disability [ 1 ]  ( Rubinstein–Taybi syndrome ,  X-linked intellectual disability ). [ 3 ]  Misregulation of H3K4, H3K27, and H4K20 are associated with  cancers . [ 4 ]  This modification alters the properties of the  nucleosome  and affects its interactions with other proteins, particularly in regards to gene transcription processes.


The fundamental unit of  chromatin , called a  nucleosome , contains DNA wound around a protein  octamer . This octamer consists of two copies each of four histone proteins:  H2A ,  H2B ,  H3 , and  H4 . Each one of these proteins has a tail extension, and these tails are the targets of nucleosome modification by methylation. DNA activation or inactivation is largely dependent on the specific tail residue methylated and its degree of methylation. Histones can be methylated on lysine (K) and arginine (R) residues only, but methylation is most commonly observed on lysine residues of histone tails H3 and H4. [ 7 ]  The tail end furthest from the nucleosome core is the  N-terminal  (residues are numbered starting at this end). Common sites of methylation associated with gene activation include H3K4, H3K48, and H3K79. Common sites for gene inactivation include H3K9 and H3K27. [ 8 ]  Studies of these sites have found that methylation of histone tails at different residues serve as markers for the recruitment of various proteins or protein complexes that serve to regulate chromatin activation or inactivation.


Lysine and arginine residues both contain amino groups, which confer basic and hydrophobic characteristics. Lysine is able to be mono-, di-, or trimethylated with a methyl group replacing each hydrogen of its NH3+ group. With a free NH2 and NH2+ group, arginine is able to be mono- or dimethylated. This dimethylation can occur asymmetrically on the NH2 group or symmetrically with one methylation on each group. [ 9 ]  Each addition of a methyl group on each residue requires a specific set of protein enzymes with various substrates and cofactors. Generally, methylation of an arginine residue requires a complex including protein arginine methyltransferase (PRMT) while lysine requires a specific  histone methyltransferase  (HMT), usually containing an evolutionarily conserved SET domain. [ 10 ]


Different degrees of residue methylation can confer different functions, as exemplified in the methylation of the commonly studied H4K20 residue. Monomethylated H4K20 ( H4K20me 1) is involved in the compaction of chromatin and therefore transcriptional repression. However, H4K20me2 is vital in the repair of damaged DNA. When dimethylated, the residue provides a platform for the binding of protein 53BP1 involved in the repair of double-stranded DNA breaks by non-homologous end joining. H4K20me3 is observed to be concentrated in heterochromatin and reductions in this trimethylation are observed in cancer progression. Therefore, H4K20me3 serves an additional role in chromatin repression. [ 10 ]   Repair of DNA double-stranded breaks  in  chromatin  also occurs by  homologous recombination  and also involves histone methylation ( H3K9me3 ) to facilitate access of the repair enzymes to the sites of damage. [ 11 ]


The genome is tightly condensed into chromatin, which needs to be loosened for  transcription  to occur. In order to halt the transcription of a gene the DNA must be wound tighter. This can be done by modifying histones at certain sites by methylation.  Histone methyltransferases  are enzymes which transfer methyl groups from  S-Adenosyl methionine  (SAM) onto the lysine or arginine residues of the H3 and H4 histones. There are instances of the core globular domains of histones being methylated as well.


The histone methyltransferases are specific to either lysine or arginine. The lysine-specific transferases are further broken down into whether or not they have a SET domain or a non-SET domain. These domains specify exactly how the enzyme catalyzes the transfer of the methyl from SAM to the transfer protein and further to the histone residue. [ 12 ]  The methyltransferases can add 1-3 methyls on the target residues.


These methyls that are added to the histones act to regulate transcription by blocking or encouraging DNA access to transcription factors. In this way the integrity of the genome and epigenetic inheritance of genes are under the control of the actions of histone methyltransferases. Histone methylation is key in distinguishing the integrity of the genome and the genes that are expressed by cells, thus giving the cells their identities.


Methylated histones can either repress or activate transcription. [ 12 ]  For example, while  H3K4me2 ,  H3K4me3 , and  H3K79me3  are generally associated with transcriptional activity, whereas  H3K9me2 ,  H3K9me3 ,  H3K27me2 ,  H3K27me3 , and  H4K20me3  are associated with transcriptional repression. [ 13 ]


Modifications made on the histone have an effect on the genes that are expressed in a cell and this is the case when methyls are added to the histone residues by the histone methyltransferases. [ 14 ]  Histone methylation plays an important role on the assembly of the heterochromatin mechanism and the maintenance of gene boundaries between genes that are transcribed and those that aren’t. These changes are passed down to progeny and can be affected by the environment that the cells are subject to.  Epigenetic  alterations are reversible meaning that they can be targets for therapy.


The activities of histone methyltransferases are offset by the activity of histone demethylases. This allows for the switching on or off of transcription by reversing pre-existing modifications. It is necessary for the activities of both histone methyltransferases and histone demethylases to be regulated tightly. Misregulation of either can lead to gene expression that leads to increased susceptibility to disease. Many cancers arise from the inappropriate epigenetic effects of misregulated methylation. [ 15 ]  However, because these processes are at times reversible, there is interest in utilizing their activities in concert with anti-cancer therapies. [ 15 ]


In female organisms, a sperm containing an  X chromosome  fertilizes the egg, giving the embryo two copies of the X chromosome. Females, however, do not initially require both copies of the X chromosome as it would only double the amount of protein products transcribed as shown by the hypothesis of dosage compensation. The paternal X chromosome is quickly inactivated during the first few divisions. [ 16 ]  This inactive X chromosome (Xi) is packed into an incredibly tight form of chromatin called  heterochromatin . [ 17 ]  This packing occurs due to the methylation of the different lysine residues that help form different histones. In humans X inactivation is a random process, that is mediated by the non-coding RNA XIST. [ 18 ]


Although methylation of lysine residues occurs on many different histones, the most characteristic of Xi occurs on the ninth lysine of the third histone (H3K9). While a single methylation of this region allows for the genes bound to remain transcriptionally active, [ 19 ]  in heterochromatin this lysine residue is often methylated twice or three times,  H3K9me2  or  H3K9me3  respectively, to ensure that the DNA bound is inactive. More recent research has shown that  H3K27me3  and  H4K20me1  are also common in early embryos. Other methylation markings associated with transcriptionally active areas of DNA, H3K4me2 and H3K4me3, are missing from the Xi chromosome along with many acetylation markings. Although it was known that certain Xi histone methylation markings stayed relatively constant between species, it has recently been discovered that different organisms and even different cells within a single organism can have different markings for their X inactivation. [ 20 ]  Through histone methylation, there is  genetic imprinting , so that the same X  homolog  stays inactivated through chromosome replications and cell divisions.


Due to the fact that histone methylation regulates much of what genes become transcribed, even slight changes to the methylation patterns can have dire effects on the organism. Mutations that occur to increase and decrease methylation have great changes on gene regulation, while mutations to enzymes such as  methyltransferase  and demethyltransferase can completely alter which proteins are transcribed in a given cell. Over methylation of a chromosome can cause certain genes that are necessary for normal cell function, to become inactivated. In a certain yeast strain,  Saccharomyces cerevisiae , a mutation that causes three lysine residues on the third histone, H3K4, H3K36, and H3K79, to become methylated causes a delay in the mitotic cell cycle, as many genes required for this progression are inactivated. This extreme mutation leads to the death of the organism. It has been discovered that the deletion of genes that will eventually allow for the production of histone methyltransferase allows this organism to live as its lysine residues are not methylated. [ 21 ]


In recent years it has come to the attention of researchers that many types of cancer are caused largely due to epigenetic factors. Cancer can be caused in a variety of ways due to differential methylation of histones. Since the discovery of  oncogenes  as well as  tumor suppressor genes  it has been known that a large factor of causing and repressing cancer is within our own genome. If areas around oncogenes become unmethylated these cancer-causing genes have the potential to be transcribed at an alarming rate. Opposite of this is the methylation of tumor suppressor genes. In cases where the areas around these genes were highly methylated, the tumor suppressor gene was not active and therefore cancer was more likely to occur. These changes in methylation pattern are often due to mutations in methyltransferase and demethyltransferase. [ 22 ]  Other types of mutations in proteins such as isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) can cause the inactivation of histone demethyltransferase which in turn can lead to a variety of cancers, gliomas and leukemias, depending on in which cells the mutation occurs. [ 23 ]


In one-carbon metabolism, the amino acids  glycine  and  serine  are converted via the  folate  and  methionine  cycles to nucleotide precursors and SAM. Multiple nutrients fuel one-carbon metabolism, including  glucose , serine, glycine, and  threonine . High levels of the methyl donor SAM influence histone methylation, which may explain how high SAM levels prevent malignant transformation. [ 24 ]


Histone methyltransferases  ( HMT ) are histone-modifying  enzymes  (e.g., histone-lysine N-methyltransferases and histone-arginine N-methyltransferases), that  catalyze  the transfer of one, two, or three  methyl  groups to  lysine  and  arginine  residues of  histone   proteins . The attachment of methyl groups occurs predominantly at specific lysine or arginine residues on histones H3 and H4. [ 1 ]   Two major types of histone methyltranferases exist, lysine-specific (which can be SET ( S u(var)3-9,  E nhancer of Zeste,  T rithorax)  domain  containing or non-SET domain containing) and arginine-specific. [ 2 ] [ 3 ] [ 4 ]   In both types of histone methyltransferases,  S-Adenosyl methionine  (SAM) serves as a  cofactor  and methyl donor group. [ 1 ] [ 5 ] [ 6 ] [ 7 ]
The genomic DNA of eukaryotes associates with histones to form  chromatin . [ 8 ]  The level of chromatin compaction depends heavily on histone methylation and other post-translational modifications of histones. [ 9 ]  Histone methylation is a principal epigenetic modification of chromatin [ 9 ]  that determines gene expression, genomic stability, stem cell maturation, cell lineage development, genetic imprinting, DNA methylation, and cell mitosis. [ 2 ]


The class of lysine-specific histone methyltransferases is subdivided into SET domain-containing and non-SET domain-containing.  As indicated by their monikers, these differ in the presence of a SET domain, which is a type of protein domain.


Human genes encoding proteins with histone methyltransferase activity include:


The structures involved in methyltransferase activity are the SET domain (composed of approximately 130 amino acids), the pre-SET, and the post-SET domains.  The pre-SET and post-SET domains flank the SET domain on either side.  The pre-SET region contains cysteine residues that form triangular zinc clusters, tightly binding the zinc atoms and stabilizing the structure.  The SET domain itself contains a catalytic core rich in β-strands that, in turn, make up several regions of β-sheets.  Often, the β-strands found in the pre-SET domain will form β-sheets with the β-strands of the SET domain, leading to slight variations to the SET domain structure. These small changes alter the target residue site specificity for methylation and allow the SET domain methyltransferases to target many different residues. This interplay between the pre-SET domain and the catalytic core is critical for enzyme function. [ 1 ]


In order for the reaction to proceed,  S-Adenosyl methionine  (SAM) and the lysine residue of the substrate histone tail must first be bound and properly oriented in the catalytic pocket of the SET domain.  Next, a nearby tyrosine residue deprotonates the ε-amino group of the lysine residue. [ 10 ]   The lysine chain then makes a nucleophilic attack on the methyl group on the sulfur atom of the SAM molecule, transferring the methyl group to the lysine side chain.


Instead of SET, non-SET domain-containing histone methyltransferase utilizes the enzyme Dot1.  Unlike the SET domain, which targets the lysine tail region of the histone, Dot1 methylates a lysine residue in the globular core of the histone, and is the only enzyme known to do so. [ 1 ]  A possible homolog of Dot1 was found in archaea which shows the ability to methylate archaeal histone-like protein in recent studies.


The N terminal of Dot1 contains the active site.   A loop serving as the binding site for SAM links the N-terminal and the C-terminal domains of the Dot1 catalytic domain.  The C-terminal is important for the substrate specificity and binding of Dot1 because the region carries a positive charge, allowing for a favorable interaction with the negatively charged backbone of DNA. [ 11 ]   Due to structural constraints, Dot1 is only able to methylate histone H3.


There are three different types of  protein arginine methyltransferases  (PRMTs) and three types of methylation that can occur at arginine residues on histone tails.  The first type of PRMTs ( PRMT1 ,  PRMT3 ,  CARM1 ⧸PRMT4, and Rmt1⧸Hmt1) produce  monomethylarginine  and  asymmetric dimethylarginine  (Rme2a). [ 12 ] [ 13 ] [ 14 ]   The second type (JBP1⧸ PRMT5 ) produces monomethyl or  symmetric dimethylarginine  (Rme2s). [ 5 ]   The third type (PRMT7) produces only monomethylated arginine. [ 15 ]  The differences in methylation patterns of PRMTs arise from restrictions in the arginine binding pocket. [ 5 ]


The catalytic domain of PRMTs consists of a SAM binding domain and substrate binding domain (about 310 amino acids in total). [ 5 ] [ 6 ] [ 7 ]   Each PRMT has a unique N-terminal region and a catalytic core.  The arginine residue and SAM must be correctly oriented within the binding pocket.  SAM is secured inside the pocket by a hydrophobic interaction between an adenine ring and a phenyl ring of a phenylalanine. [ 7 ]


A glutamate on a nearby loop interacts with nitrogens on the target arginine residue. This interaction redistributes the positive charge and leads to the deprotonation of one nitrogen group, [ 16 ]  which can then make a nucleophilic attack on the methyl group of SAM.  Differences between the two types of PRMTs determine the next methylation step: either catalyzing the dimethylation of one nitrogen or allowing the symmetric methylation of both groups. [ 5 ]   However, in both cases the proton stripped from the nitrogen is dispersed through a histidine–aspartate proton relay system and released into the surrounding matrix. [ 17 ]


Histone methylation  plays an important role in  epigenetic   gene regulation . Methylated histones can either repress or activate transcription as different experimental findings suggest, depending on the site of methylation. For example, it is likely that the methylation of lysine 9 on histone H3 (H3K9me3) in the promoter region of genes prevents excessive expression of these genes and, therefore, delays cell cycle transition and/or proliferation. [ 18 ]  In contrast, methylation of histone residues H3K4, H3K36, and H3K79 is associated with transcriptionally active euchromatin. [ 2 ]


Depending on the site and symmetry of methylation, methylated arginines are considered activating (histone H4R3me2a, H3R2me2s, H3R17me2a, H3R26me2a) or repressive (H3R2me2a, H3R8me2a, H3R8me2s, H4R3me2s) histone marks. [ 15 ]
Generally, the effect of a histone methyltransferase on gene expression strongly depends on which histone residue it methylates. See  Histone#Chromatin regulation .


Abnormal expression or activity of methylation-regulating enzymes has been noted in some types of human cancers, suggesting associations between  histone methylation  and malignant transformation of cells or formation of tumors. [ 18 ]    In recent years, epigenetic modification of the histone proteins, especially the methylation of the histone H3, in cancer development has been an area of emerging research.  It is now generally accepted that in addition to genetic aberrations, cancer can be initiated by epigenetic changes in which gene expression is altered without genomic abnormalities.  These epigenetic changes include loss or gain of methylations in both DNA and histone proteins. [ 18 ]


There is not yet compelling evidence that suggests cancers develop purely by abnormalities in  histone methylation  or its signaling pathways, however they may be a contributing factor. For example, down-regulation of methylation of lysine 9 on histone 3 (H3K9me3) has been observed in several types of human cancer (such as colorectal cancer, ovarian cancer, and lung cancer), which arise from either the deficiency of H3K9 methyltransferases or elevated activity or expression of H3K9 demethylases. [ 18 ] [ 19 ] [ 20 ]


The methylation of histone  lysine  has an important role in choosing the pathway for  repairing DNA double-strand breaks . [ 21 ]   As an example,  tri-methylated H3K36  is required for  homologous recombinational  repair, while dimethylated H4K20 can recruit the  53BP1  protein for repair by the pathway of  non-homologous end joining .


Histone methyltransferase may be able to be used as biomarkers for the diagnosis and prognosis of cancers.  Additionally, many questions still remain about the function and regulation of histone methyltransferases in malignant transformation of cells, carcinogenesis of the tissue, and tumorigenesis. [ 18 ]


(Histone-H3)-lysine-36 demethylase  ( EC   1.14.11.27 ,  JHDM1A ,  JmjC domain-containing histone demethylase 1A ,  H3-K36-specific demethylase ,  histone-lysine (H3-K36) demethylase ,  histone demethylase ,  protein-6-N,6-N-dimethyl-L-lysine,2-oxoglutarate:oxygen oxidoreductase ) is an  enzyme  with  systematic name   protein-N6,N6-dimethyl-L-lysine,2-oxoglutarate:oxygen oxidoreductase . [ 1 ]  This enzyme  catalyses  the following  chemical reaction


(Histone-H3)-lysine-36 demethylase contains  iron (II).


Histone-modifying enzymes  are  enzymes  involved in the  modification  of  histone  substrates after  protein translation  and affect cellular processes including  gene expression . [ 1 ] [ 2 ]  To safely store the  eukaryotic   genome ,  DNA  is wrapped around four core  histone   proteins  (H3, H4, H2A, H2B), which then join to form  nucleosomes . These nucleosomes further fold together into highly condensed  chromatin , which renders the organism's genetic material far less accessible to the factors required for  gene transcription ,  DNA replication ,  recombination  and  repair . [ 3 ] [ 4 ]  Subsequently, eukaryotic organisms have developed intricate mechanisms to overcome this repressive barrier imposed by the chromatin through  histone modification , a type of  post-translational modification  which typically involves  covalently attaching  certain groups to histone residues. Once added to the histone, these groups (directly or indirectly) elicit either a loose and open histone conformation,  euchromatin , or a tight and closed histone conformation,  heterochromatin . Euchromatin marks active  transcription  and  gene expression , as the light packing of histones in this way allows entry for proteins involved in the transcription process. As such, the tightly packed heterochromatin marks the absence of current gene expression. [ 4 ]


While there exist several distinct post-translational modifications for  histones , the four most common histone modifications include  acetylation , [ 5 ]   methylation , [ 6 ]   phosphorylation [ 7 ]  and  ubiquitination . [ 8 ]  Histone-modifying enzymes that induce a modification (e.g., add a  functional group ) are dubbed writers, while enzymes that revert modifications are dubbed erasers. Furthermore, there are many uncommon histone modifications including  O -GlcNAcylation , [ 9 ]   sumoylation , [ 10 ]   ADP-ribosylation , [ 11 ]   citrullination [ 12 ] [ 13 ] [ 14 ]  and  proline isomerization . [ 15 ]  For a detailed example of histone modifications in transcription regulation see  RNA polymerase control by chromatin structure  and table " Examples of histone modifications in transcriptional regulation ".


The four common histone modifications and their respective writer and eraser enzymes are shown in the table below. [ 16 ] [ 17 ] [ 18 ] [ 19 ] [ 20 ] [ 21 ] [ 22 ] [ 23 ]


Histone acetylation , or the addition of an  acetyl group  to histones, is facilitated by  histone acetyltransferases (HATs)  which target  lysine (K)  residues on the  N-terminal  histone tail.  Histone deacetylases (HDACs)  facilitate the removal of such groups. The positive charge on a histone is always neutralized upon acetylation, creating  euchromatin  which increases transcription and expression of the target gene. [ 16 ]  Lysine residues 9, 14, 18, and 23 of core histone H3 and residues 5, 8, 12, and 16 of H4 are all targeted for acetylation. [ 17 ] [ 18 ]


Histone methylation  involves adding  methyl groups  to histones, primarily on  lysine (K)  or  arginine (R)  residues. The addition and removal of methyl groups is carried out by  histone methyltransferases (HMTs)  and histone  demethylases  (KDMs) respectively. Histone methylation is responsible for either activation or repression of genes, depending on the target site, and plays an important role in development and learning. [ 19 ]


Histone phosphorylation  occurs when a  phosphoryl group  is added to a  histone .  Protein kinases (PTKs)  catalyze the phosphorylation of histones and  protein phosphatases (PPs)  catalyze the dephosphorylation of histones. Much like histone acetylation, histone phosphorylation neutralizes the positive charge on histones which induces  euchromatin  and increases gene expression. [ citation needed ]  Histone phosphorylation occurs on  serine (S) ,  threonine (T)  and  tyrosine (Y)   amino-acid  residues mainly in the  N-terminal  histone tails. [ citation needed ]


Additionally, the phosphorylation of histones has been found to play a role in  DNA repair  and  chromatin condensation  during  cell division . [ 20 ]  One such example is the phosphorylation of S139 on  H2AX  histones, which is needed to repair  double-stranded breaks  in the DNA. [ 20 ]


Ubiquitination  is a post-translational modification involving the addition of  ubiquitin proteins  onto target proteins. Histones are often ubiquitinated with one ubiquitin molecule (monoubiquitination), but can also be modified with ubiquitin chains (polyubiquitination), both of which can have variable effects on gene transcription. [ 21 ]   Ubiquitin ligases  add these ubiquitin proteins while  deubiquitinating enzymes (DUBs)  remove these groups. [ 22 ]  Ubiquitination of the H2A core histone typically represses gene expression as it prevents methylation at H3K4, while H2B ubiquitination is necessary for H3K4 methylation and can lead to both gene activation or repression. [ citation needed ]  Additionally, histone ubiquitination is related to genomic maintenance, as ubiquitination of  histone H2AX  is involved in  DNA damage  recognition of DNA  double-strand breaks . [ 23 ]


Additional infrequent histone modifications and their effects are listed in the table below. [ 24 ] [ 25 ] [ 26 ] [ 27 ] [ 28 ] [ 29 ] [ 30 ] [ 31 ] [ 32 ] [ 33 ] [ 34 ] [ 35 ] [ 36 ] [ 37 ]


The presence of  O-GlcNAcylation (O-GlcNAc)  on  serine (S)  and  threonine (T)  histone residues is known to mediate other post-transcriptional histone modifications. The addition and removal of  GlcNAc  groups are performed by  O-GlcNAc transferase (OGT)  and  O-GlcNAcase (OGA)  respectively. While our understanding of these processes is limited, GlcNAcylation of S112 on core histone H2B has been found to promote monoubiquitination of K120. [ 24 ]  Similarly, OGT associates with the  HCF1  complex which interacts with  BAP1  to mediate deubiquitination of H2A. OGT is also involved in the trimethylation of H3K27 and creates a co-repressor complex to promote histone deacetylation upon binding to  SIN3A . [ 25 ]


SUMOylation  refers to the addition of  Small Ubiquitin-like Modifier (SUMO) proteins  onto histones. SUMOylation involves covalent attachments between SUMO proteins and  lysine (K)  residues on histones and is carried out in  three main steps  by three respective enzymes: activation via SUMO E1,  conjugation  via SUMO E2, and  ligation  via SUMO E3. In humans, SUMO E1 has been identified as the  heterodimer   SAE1 / SAE2 , SUMO E2 is known as  UBE2I , and the SUMO E3 role may be a  multi-protein complex  played by a handful of different enzymes. [ 26 ]


SUMOylation affects the chromatin status (looseness) of the histone and influences the assembly of  transcription factors  on genetic promoters, leading to either transcriptional repression or activation depending on the substrate. [ 27 ]  SUMOylation also plays a role in the major DNA repair pathways of  base excision repair ,  nucleotide excision repair ,  non-homologous end joining  and  homologous recombination  repair. Additionally, SUMOylation facilitates error prone  translesion synthesis . [ 28 ]


ADP-ribosylation (ADPr)  defines the addition of one or more  adenosine diphosphate ribose (ADP-ribose) groups  to a  protein . [ 29 ]  ADPr is an important mechanism in  gene regulation  that affects chromatin organization, the binding of  transcription factors , and  mRNA processing  through  poly-ADP ribose polymerase (PARP) enzymes . There are multiple types of PARP proteins, but the subclass of DNA-dependent PARP proteins including PARP-1, PARP-2, and PARP-3 interact with the histone. [ 30 ]  The PARP-1 enzyme is the most prominent of these three proteins in the context of gene regulation and interacts with all five histone proteins. [ 31 ]


Like PARPs 2 and 3, the catalytic activity of PARP-1 is activated by discontinuous  DNA fragments , DNA fragments with  single-stranded breaks . PARP-1 binds histones near the axis where DNA enters and exits the nucleosome and additionally interacts with numerous chromatin-associated proteins which allow for indirect association with chromatin. [ 30 ]  Upon binding to chromatin, PARP-1 produces repressive histone marks that can alter the conformational state of histones and inhibit gene expression so that  DNA repair  can take place. Other avenues of transcription regulation by PARP-1 include acting as a  transcription coregulator , regulation of  RNA  and modulation of  DNA methylation  via inhibiting the  DNA methyltransferase   Dnmt1 . [ 30 ] [ 32 ]


Citrullination , or deimination, is the process by which the  amino acid   arginine (R)  is converted into  citrulline .  Protein arginine deiminases (PADs)  replace the  ketimine  group of arginine with a  ketone  group to form the citrulline. [ citation needed ]  PAD4 is the deaminase involved in histone modification and converts arginine to citrulline on histones H3 and H4; because arginine  methylation  on these histones is important for transcriptional activation, citrullination of certain residues can cause the eventual loss of methylation, leading to decreased gene transcription; [ 33 ]  specific citrullination of H3R2, H3R8, H3R17, and H3R26 residues have been identified in breast cancer cells. [ 34 ]  As of research conducted in 2019, this process is thought to be irreversible. [ 35 ]


Isomerization  involves transforming a molecule so that it adopts a different structural conformation;  proline isomerization  plays an integral role in the modification of histone tails. [ 36 ]  Fpr4 is the  prolyl isomerase enzyme (PPIase)  which converts the  amino acid   proline (P)  on histones between the  cis and trans conformations . While Fpr4 has  catalytic activity  on a number of prolines on the  N-terminal region  of core histone H3 (P16, P30 and P38), it most readily binds to P38. [ 37 ]


H3P38 lies near the  lysine (K)  residue H3K36, and changes in P38 can affect the  methylation  status of K36. The two possible P38 isomers available, cis and trans, cause differential effects that are opposite of each other. The cis position induces compact histones and decreases the ability of proteins to bind to the DNA, thus preventing methylation of K36 and decreasing gene transcription. Conversely, the trans position of P38 promotes a more open histone conformation, allowing for K36 methylation and leading to an increase gene transcription. [ 36 ]


Alterations in the functions of histone-modifying enzymes deregulate the control of chromatin-based processes, ultimately leading to  oncogenic  transformation and  cancer . [ 38 ]  Both  DNA methylation  and histone modifications show patterns of distribution in cancer cells. [ 39 ] [ 40 ]  These  epigenetic  alterations may occur at different stages of  tumourigenesis  and thus contribute to both the development and/or progression of cancer. [ 40 ]


Vitamin B12  deficiency in mice has been shown to alter expression of histone modifying enzymes in the brain, leading to behavioral changes and  epigenetic  reprogramming. [ 41 ]  Evidences also show the importance of  HDACs  in regulation of  lipid metabolism  and other metabolic pathways playing a role in the  pathophysiology  of metabolic disorders. [ 42 ]


HSV epigenetics  is the  epigenetic  modification of  herpes simplex virus  (HSV) genetic code.


As of 2012, an estimated 3.7 billion people globally were infected with oral herpes simplex virus (HSV-1), and 417 million were living with genital herpes (HSV-2) worldwide (World Health Organization, 2018). In severe cases, HSV infection can cause corneal scarring and blindness from herpes keratitis, central nervous system infections, and even death in immunocompromised individuals such as neonates and HIV patients. [ 1 ]


The  life cycle  of a virus typically consists of its  entry ,  replication , and eventual  shedding . Upon initial infection, herpes simplex virus (HSV) causes acute lytic infection of epithelial cells, usually at either genital or orolabial mucous membranes. [ 2 ]  During this initial infection, the virus also infects local nerve cells, such as in the trigeminal ganglion in the case of HSV-1. HSV enters the cell when its membrane fuses with the cellular membrane, releasing tegument proteins and the naked capsid into the cytoplasm. The capsid travels to a nuclear pore, likely along cytoskeletal microtubules. The HSV genome is then injected into the nucleus where it is assembled with histones and undergoes chromatin remodeling, thus inducing latency.  Lysogenic  DNA viruses in particular make use of cellular epigenetic mechanisms in their life cycle, particularly prior to replication, during a virus'  latent infection . [ 3 ]  During latency, most viral genes are silenced, and infected individuals are asymptomatic. More importantly, during latency, proviral DNA is hidden from the immune system, making treatment extremely difficult, if not impossible.


Latent infection is described as the dormancy of a virus, by which proliferation of viral particles cease, yet the viral genome still remains incorporated as reservoirs within the cell. Although diversity among viruses' latent phase exist, ranging from selective viral gene expression (e.g., HSV expression of latency associated transcripts) to complete lack of expression, incorporation into the cell's genome (such as in  retroviruses ), and  episomal  persistence, common generalities too can be ascribed. Generally, the long-term latency of a mammalian virus is a dynamic interaction between it and the host cell's antiviral immunity. [ 4 ]  The establishment, maintenance, and reactivation of the infection cycle (reversibility) largely relies on cellular epigenetic processes in this dynamic [ 5 ]  and thus opens a discussion for viral treatment via an epigenetic perspective.


Antiviral drug development has been a complex process utilizing several different fields to synthesize single methods of treatment. [ 6 ]  As of May 2018 there are about 50 drugs approved for human use against viruses such as  HSV ,  HIV ,  cytomegalovirus ,  influenza virus ,  HBV  and  HCV . [ 6 ]  A large amount of viral research in the field of epigenetics has been done on specifically on HSV.


HSV infections have been medically recorded in literature for centuries, but its antiviral treatment has been developed only over the past 50 years. [ 7 ]  The framework for antiviral HSV treatment began with the experimental use of  DNA inhibitors , which interrupt the viral replication cycle. These drugs since have been developed commercially and are subsequently prescribed depending on factors such as the immune status of the host, the site of infection, and whether the infection is primary (initial infection) or recurrent (recurring symptoms). [ 7 ]


Popular, current antiviral agents include  aciclovir ,  penciclovir ,  foscarnet , and  docosanol . [ 7 ]   Aciclovir  acts as a nucleoside analog which specifically target herpes virus-infected cells using viral  thymidine kinase  (TK). Viral TK  phosphorylates  aciclovir into its monophosphate form, which is subsequently phosphorylated to active aciclovir triphoshate by  cellular kinases , thus selectively inhibiting viral  DNA polymerase . The related  valaciclovir  Aciclovir triphosphate is the L-valine ester  prodrug  of aciclovir, which is thus converted to aciclovir in the liver. [ citation needed ]


Penciclovir is similar to aciclovir in mechanism, except that penciclovir is a guanosine analog. Penciclovir triphosphate is 100-fold less potent in inhibiting viral DNA polymerase, yet also remains in infected cells at higher concentrations and for longer periods of time.  Famciclovir  is the diacetyl ester prodrug of penciclovir. [ citation needed ]


Foscarnet does not rely on TK phosphorylation, and is thus potent against TK-deficient HSV isolates which otherwise form a resistant to TK-dependent drug mechanisms. Docosanol (Abreva) blocks initial viral entry via inhibition of fusion between the host plasma membrane and HSV envelope. [ citation needed ]


As seen by most mainstream antiviral agents, the mechanism of treatment for HSV historically is via the inhibition of viral replication using nucleotides that inhibit DNA polymerase. The specificity in targeting for viral polymerases is dependent on the drug used, which in turn is dependent on the severity of the infection.


Epigenetic down-regulation of viral gene expression is likely a result, at least in part, of the evolutionary arms race in which a cellular host attempts to silence viral DNA, such as through chromatin remodeling. However, some viruses have evolved to twist this process to their own advantage by establishing latency. Latent proviral DNA exists as heterochromatin in the host nucleus, evading cellular defenses. [ 3 ]  Four epigenetic processes have been well described: chromatin assembly, histone modifications, DNA methylation, and regulatory RNA. [ 4 ]


Upon initial infection, viral genomes are not associated with histones. Typical double-stranded DNA viruses that infect eukaryotic cells insert their genome into the cell nucleus, where cellular machinery necessary for DNA replication and transcription is present (the notable exceptions are  pox viruses , which remain in the cytoplasm). Once viral DNA enters the nucleus, it colocalizes with promyelocytic leukaemia nuclear bodies (PML-NBs), regions rich in proteins associated with antiviral pathways such as  PML ,  SP100 ,  Daxx , and ATRX. In particular, Daxx and ATRX assemble viral DNA around histone 3, forming heterochromatin and silencing viral gene expression. [ 3 ] [ 8 ]  Viral responses have evolved to either degrade or modify this heterochromatin, such as repurposing to induce latency. [ 3 ]


There are several known examples of viral recruitment of cellular proteins to chromatin for histone modification, particularly in herpesviridae. [ 3 ]  (Polycomb complex recruitment by LAT, VP16 recruits OCT1 and HCF1, which in turn recruit histone demethylases JMJD2s and LSD1, etc).


DNA methylation can occur on viral genomes through recruitment of cellular machinery such as  DNA methyltransferase 3A (DNMT3A) . DNMT3A is a  de novo  methyltransferase which can methylate previously unmethylated DNA, such as that of viral genomes. DNMT3A is recruited to viral genomes by viral proteins such as the Kaposi's Sarcoma herpesvirus (KSHV) protein LANA, and VP26 and VP5 in herpes simplex virus 1 (HSV-1). Methylation of gene promoters represses gene expression, which has several functions in maintaining latency and even reactivation in  Epstein-Barr virus (EBV) . [ 3 ]


In HSV and  Kaposi's Sarcoma-associated herpesvirus (KSHV) , the Latency-Associated Transcripts (LATs), which are non-coding RNAs, are thought to play an important role in recruiting host  Polycomb  proteins to lytic cycle-associated genes on the viral genome. [ 3 ]  This results in the methylation of cytosines within the promoter region of genes associated with lytic cycle processes. In the  gammaherpesvirus   Epstein-Barr virus (EBV) , the non-coding RNA EBER2 has been implicated in gene regulation. EBER2 recruits PAX5, a transcription factor, to immediate-early genes to promote latency. [ 9 ]


Reactivation in HSV requires the stress response enzyme  c-Jun N-terminal kinase (JNK) , which is responsive to stressors such as heat shock, ultraviolet radiation, and cytokine production. Activation of JNK leads to the expression of viral genes of all temporal classes (immediate early, early, and late) by phosphorylating H3, effectively bypassing the latent repression markers (methylation). [ 3 ] [ 10 ]  Next, the  de novo  translation of VP16 results in the temporal regulation of these genes; VP16 associates with Oct-1 and with viral promoters to regulate transcription. [ 3 ] [ 11 ]  Reactivation results in lytic infection of proximal epithelial cells.


Mammalian viral treatment within the scope of epigenetics is a relatively novel approach that has only seen theoretical or laboratory significance, and as such, advancements in chromatin-based viral therapy relies on advancement in knowledge of viral-host chromatin dynamics and interplay. [ 12 ]  Two major therapies that target epigenetic machinery have been proposed for treatment of viral infections: inhibition of Ezh1/2 and m 6 A addition to viral mRNA.


Ezh1  and  Ezh2  (Ezh1/2) are two homologous enzymes of Ezh  histone methyltransferase , and are part of the  Polycomb  repression complex 2 ( PRC2 ), which functions to repress gene transcription by propagating a H3K27 trimethylation. Ezh1/2 has been implicated in suppression of viral replication during lytic infection. [ 13 ]


The Ezh1/2 inhibitors GSK126, GSK343, and UNC1999 as well as astemizole, which interferes with Ezh1/2 association in PRC, were shown by Arbuckle et al. [ 13 ]  to limit HSV immediate early gene expression and the number of transcriptionally active viral genomes during lytic infection. This resulted in suppression of lytic infection.


Ezh1/2 inhibitors also activate cellular antiviral pathways. Specifically, in cells treated with Ezh1/2 inhibitors, greater numbers of promyelocytic leukemia (PML) foci, which are involved with repression of  gene expression, were seen, along with upregulation of  interferon-alpha (IFN-α)  and  interleukin-8 (IL-8) . These pathways are not specific to HSV; treatment with Ezh1/2 inhibitors reduced infection of multiple viral pathogens, including human cytomegalovirus, adenovirus, and  Zika virus . [ 13 ]


Methylation on the N 6  position of adenosine (m 6 A) on  messenger RNA (mRNA)  affects gene expression by changing mRNA splicing, stability, and translation. [ 14 ]  These changes favor viral gene expression, though the mechanism for this effect is not known. Importantly, m 6 A has been proposed to inhibit host immune recognition of viral RNAs. [ 14 ] [ 15 ] [ 16 ]  Inhibition of m 6 A addition by 3-deazaadenosine (DAA) (an  S-adenosylhomocysteine  hydrolase inhibitor) has been shown to have broad antiviral applications. [ 14 ] [ 17 ] [ 18 ]  Therapies utilizing specific m 6 A may be very effective against nuclear viruses without toxicity to the host, though this research is new. [ 14 ]


The  International Human Epigenome Consortium  (IHEC) is a scientific organization, founded in 2010, that helps to coordinate global efforts in the field of  Epigenomics . [ 1 ] [ 2 ] [ 3 ] [ 4 ]  The initial goal was to generate at least 1,000 reference (baseline)  human epigenomes  from different types of normal and disease-related human  cell types . [ 5 ] [ 6 ] [ 7 ]


IHEC's operations are funded by its full members (national and regional scientific funding agencies), and staffed largely on a volunteer basis by scientists and other experts from participating funding agencies and epigenome mapping projects. [ 8 ]


In addition, countries and agencies supportive of IHEC goals are organizations that have not yet made a full financial contribution to the project, but whose members provide time and expertise: [ 22 ]


Oversight of IHEC is provided by an executive committee, whose members are nominated by Full Member organizations. [ 23 ]  This committee works closely with an International Scientific Steering Committee, whose members are the scientific leaders of participating projects and other leaders in the field of epigenetics, as well as a Data Coordination Center. Additional expertise is contributed by workgroups composed of members of participating research projects.


IHEC interacts and coordinates its efforts with other large-scale international genomics projects, such as the  International Cancer Genome Consortium  (ICGC), [ 24 ]   ENCODE ., [ 25 ] [ 26 ]  and the Global Alliance for Genomics and Health. [ 27 ]  Committee and workgroup members, as well as other individuals involved in IHEC, meet annually at an event hosted by member countries on a rotating basis. [ 28 ]  Most meetings are hosted in conjunction with a scientific symposium, some of which are open to non-IHEC scientists and sometimes members of the public.


The ultimate objective of IHEC is to determine how the  Epigenome  has shaped human populations over generations and in response to the environment. [ 25 ] [ 29 ]  The first phase of IHEC's operations involves coordinating the production of at least 1,000 reference epigenomes from healthy and diseased human cells, as well as a limited number of  model organisms  relevant to specific human diseases. The initial focus is on cellular states including stemness, immortality,  proliferation ,  differentiation , senescence, and stress.
The reference epigenome for each sample comprises high resolution maps of  DNA methylation  and key regulatory  histone  modifications, with corresponding information about the type and expression level of all transcribed genes ( protein  coding as well as  non-coding  / small RNAs). [ 30 ]
The data produced are made freely available to the research community via the IHEC Data Portal, [ 31 ] [ 32 ]   European Genome-phenome Archive  (EGA), [ 33 ]  and other venues. [ 34 ] [ 25 ] [ 35 ]


In addition, participating research projects are engaged in developing new epigenomics and associated  bioinformatics  methods.


In November 2016 IHEC members from Canada, the European Union's BLUEPRINT Consortium, the German Epigenome Program “DEEP”, Japan, Singapore, and the United States published a group of 41 coordinated papers in  Cell Press  and other journals. [ 36 ] [ 37 ] [ 38 ] [ 39 ]  The papers included descriptions of molecular biology and computational methods as well as new research on normal and disease biology. [ 40 ] [ 41 ] [ 42 ] [ 43 ]


The  International Human Epigenomics Consortium (IHEC)  was launched in 2010 to coordinate global efforts in the field of  epigenomics . [ 1 ] [ 2 ]  IHEC aims to generate at least 1,000 reference baseline human  epigenomes  from different types of normal and disease-related human cell types.


2AGH ,  2J2S ,  2JYI ,  2KKF ,  2KU7 ,  2KYU ,  2LXS ,  2LXT ,  2MTN ,  2W5Y ,  2W5Z ,  3EG6 ,  3EMH ,  3LQI ,  3LQJ ,  3P4F ,  3U85 ,  3U88 ,  4ESG ,  4GQ6 ,  4NW3 ,  5F6L ,  5F5E


NM_001197104 NM_005933 NM_024891


NM_001081049 NM_001357549


NP_001184033 NP_005924


Histone-lysine  N -methyltransferase 2A , also known as  acute lymphoblastic leukemia 1  ( ALL-1 ),  myeloid/lymphoid or mixed-lineage leukemia   1  ( MLL1 ), or  zinc finger protein HRX  ( HRX ), is an  enzyme  that in humans is encoded by the  KMT2A   gene . [ 5 ]


MLL1 is a histone  methyltransferase  deemed a positive global regulator of  gene transcription . This protein belongs to the group of  histone-modifying enzymes  comprising  transactivation  domain 9aaTAD [ 6 ]  and is involved in the  epigenetic  maintenance of transcriptional memory. Its role as an epigenetic regulator of neuronal function is an ongoing area of research.


KMT2A gene encodes a transcriptional coactivator that plays an essential role in regulating gene expression during early development and  hematopoiesis . The encoded protein contains multiple conserved functional domains. One of these domains, the  SET domain , is responsible for its  histone H3  lysine 4 (H3K4) methyltransferase activity which mediates  chromatin  modifications associated with epigenetic transcriptional activation. Enriched in the nucleus, the MLL1 enzyme trimethylates H3K4 ( H3K4me3 ). It also upregulates mono- and dimethylation of H3K4. [ 7 ]  This protein is processed by the enzyme  Taspase 1  into two fragments, MLL-C (~180 kDa) and MLL-N (~320 kDa). [ 8 ] [ 9 ]  These fragments then assemble into different multi-protein complexes that regulate the transcription of specific target genes, including many of the  HOX genes .


Transcriptome profiling after deletion of MLL1 in cortical neurons revealed decreased promoter-bound H3K4me3 peaks at 318 genes, with 31 of these having significantly decreased expression and promoter binding. [ 10 ]  Among them were  Meis2 , a homeobox transcription factor critical for development of forebrain neurons [ 11 ] [ 12 ]  and  Satb2 , a protein involved in neuronal differentiation. [ 13 ]


Multiple chromosomal translocations involving this gene are the cause of certain  acute lymphoid leukemias  and  acute myeloid leukemias . Alternate splicing results in multiple transcript variants. [ 14 ]


MLL1 has been shown to be an important epigenetic regulator of complex behaviors. Rodent models of MLL1 dysfunction in forebrain neurons showed that conditional deletion results in elevated anxiety and defective cognition.  Prefrontal cortex -specific knockout of MLL1 results in the same phenotypes, as well as working memory deficits. [ 10 ]


MLL1 has been found to be an important regulator of  epiblast-derived stem cells , post-implantation epiblast derived stem cells which display pluripotency yet many recognizable differences from the traditional  embryonic stem cells  derived from inner cell mass prior to implantation. Suppression of MLL1 expression was shown to be adequate for inducing ESC-like morphology and behavior within 72 hours of treatment. It has been proposed that the small molecule inhibitor MM-401, which was used to inhibit MLL1, changes the distribution of  H3K4me1 , the single  methylation  of the  histone H3  lysine 4, to be significantly downregulated at MLL1 targets thus leading to decreased expression of MLL1 targets, rather than a direct regulation of pluripotency core markers. [ 15 ]


KMT2A gene has 37  exons  and resides on  chromosome 11  at q23. [ 14 ]


KMT2A has over a dozen binding partners and is cleaved into two pieces, a larger  N-terminal fragment , involved in gene repression, and a smaller  C-terminal fragment , which is a transcriptional activator. [ 16 ]  The cleavage, followed by the association of the two fragments, is necessary for KMT2A to be fully active. Like many other  methyltransferases , the KMT2 family members exist in multisubunit nuclear complexes (human COMPASS), where other subunits also mediate the enzymatic activity. [ 17 ]


Abnormal H3K4 trimethylation has been implicated in several neurological disorders such as autism. [ 18 ]  Humans with cognitive and neurodevelopmental disease often have dysregulation of H3K4 methylation in  prefrontal cortex  (PFC) neurons. [ 18 ] [ 19 ] [ 20 ]  It also may participate in the process of  GAD67  downregulation in  schizophrenia . [ 19 ]


MLL1 is required for the expression of  senescence-associated secretory phenotype  (SASP)-related genes and promotes increased inflammation. [ 21 ]


Rearrangements of the MLL1 gene are associated with aggressive acute  leukemias , both lymphoblastic and myeloid. [ 22 ]  Despite being an aggressive leukemia, the MLL1 rearranged sub-type had the lowest mutation rates reported for any cancer. [ 23 ]


Mutations in MLL1 cause  Wiedemann-Steiner syndrome  and  acute lymphoblastic leukemia . [ 24 ]  The leukemia cells of up to 80 percent of infants with ALL-1 have a chromosomal rearrangement that fuses the MLL1 gene to a gene on a different chromosome. [ 23 ]


MLL (gene) has been shown to  interact  with:


A  locus control region  ( LCR ) is a long-range  cis-regulatory element  that enhances expression of  linked genes  at distal  chromatin  sites. It functions in a copy number-dependent manner and is tissue-specific, as seen in the selective expression of  β-globin  genes in  erythroid cells . [ 1 ]  Expression levels of genes can be modified by the LCR and gene-proximal elements, such as  promoters ,  enhancers , and  silencers . The LCR functions by recruiting  chromatin-modifying ,  coactivator , and  transcription  complexes. [ 2 ]  Its sequence is  conserved  in many vertebrates, and conservation of specific sites may suggest importance in function. [ 2 ]  It has been compared to a  super-enhancer  as both perform long-range  cis  regulation via recruitment of the transcription complex. [ 3 ]


The β-globin LCR was identified over 20 years ago in studies of  transgenic  mice. These studies determined that the LCR was required for normal  regulation  of beta-globin gene expression. [ 4 ]  Evidence of the presence of this additional regulatory element came from a group of patients that lacked a 20 kb region upstream of the β-globin cluster that was vital for expression of any of the β-globin genes. Even though all of the genes themselves and the other regulatory elements were intact, without this domain, none of the genes in the β-globin cluster were expressed. [ 5 ]


Although the name implies that the LCR is limited to a single region, this implication only applies to the β-globin LCR ( HBB-LCR ). Other studies have found that a single LCR can be distributed in multiple areas around and inside the genes it controls. The β-globin LCR in mice and humans is found 6–22 kb upstream of the first globin gene ( epsilon ). It controls the following genes: [ 1 ] [ 2 ]


There is an  opsin  LCR ( OPSIN-LCR ) controlling the expression of  OPN1LW  and the first copies of  OPN1MW  on the human X chromosome, upstream of these genes. [ 6 ]  A dysfunctional LCR can cause loss of expression of both opsins, leading to  blue cone monochromacy . [ 7 ]  This LCR is also conserved in  teleost fishes  including  zebrafish . [ 8 ]


As of 2002, there are 21 LCR areas known in human. [ 1 ]  As of 2019, 11 human LCRs are recorded in the NCBI database. [ 9 ]


Although studies have been conducted to attempt to identify a model of how the LCR functions, evidence for the following models is not strongly supported or precluded. [ 1 ]


Transcription factors bind to  hypersensitive site  cores and cause the LCR to form a loop that can interact with the promoter of the gene it regulates. [ 1 ]


Transcription factors bind to the LCR to form a complex. The complex moves along the DNA helix until it can bind to the promoter of the gene it regulates. Once bound, the transcriptional apparatus increases gene expression. [ 1 ]


This hypothesis combines the looping and tracking models, suggesting that the transcription factors bind to the LCR to form a loop, which then seeks and binds to the promoter of the gene it regulates. [ 1 ]


Transcription factors bind to DNA from the LCR to the promoter in an orderly fashion using non- DNA-binding proteins  and chromatin modifiers. This changes chromatin conformation to expose the transcriptional domain. [ 1 ]


Studies in transgenic mice have shown that deletion of the β-globin LCR causes the region of chromosome to condense into a  heterochromatic  state. [ 1 ] [ 2 ]  This leads to decreased expression of β-globin genes, which can cause  β-thalassemia  in humans and mice.


Metabolic imprinting  refers to the long-term physiological and metabolic effects that an offspring's prenatal and postnatal environments have on them. [ 1 ]  Perinatal nutrition has been identified as a significant factor in determining an offspring's likelihood of it being predisposed to developing  cardiovascular disease ,  obesity , and  type 2 diabetes  amongst other conditions. [ 2 ]


During  pregnancy , maternal  glucose  can cross the  blood-placental barrier [ 3 ]  meaning maternal hyperglycaemia is associated with foetal hyperglycaemia. [ 4 ]  Despite maternal glucose being able to cross the blood-placental barrier, maternal  insulin  is not able and the foetus has to make its own. [ 5 ] [ 6 ]  As a result, if a mother is hyperglycaemic the foetus is likely to be hyperinsulinaemic which leads to it having increased levels of growth and adiposity. [ 4 ]


Maternal undernutrition has been linked with low birth weight and also a number of diseases, including  Cardiovascular disease ,  stroke ,  hypertension  and  diabetes . [ 7 ]  When a foetus is in the womb and is not receiving sufficient nutrition, it can adapt to prioritize organ growth and increased metabolic efficiency to prepare itself for life in an energy deficient environment. Postnatally, when given the correct nutrition, babies exhibit ‘catch up growth’, potentially leading to obesity and other related complications. Studies based around restricting animals food intake throughout gestation have discovered that a reduction of just 30% of normal intake can cause low birth weight and increase sensitivity to high-fat-diet induced obesity. [ 8 ]


In animal models, intrauterine undernutrition has been shown to be associated with hypertension later in life. This is because the formation of the  kidneys  is inhibited, which decreases filtration and flow rate through the nephrons, leading to increased blood pressure. [ 9 ]


More extreme prenatal conditions such as famine have been shown to have effects on the neurodevelopment of a foetus. [ 7 ]  After the  Dutch Famine  of the winter of 1944–1945, it was found that the risk of  schizophrenia  was significantly higher in those conceived at the height of the famine, as was the prevalence of  schizoid personality . [ 10 ]


Maternal overnutrition can have detrimental effects on the health of the offspring later in life. This area is less well studied and understood but some progress has been made in identifying specific  genes  that are affected. [ 7 ]  Studies have investigated hypermethylation of  DNA  and found it to be higher in  obese  mothers to those of a healthy  BMI . [ 11 ]  More specific studies have investigated  Leptin  (LEP) as a possible gene which is altered via metabolic  imprinting  in response to overnutrition in  utero , and found hypermethylation of LEP in the placenta of those born to overly nourished mothers. [ 12 ]  This hypermethylation has been found to cause changes in the levels of circulating Leptin, as well as to leptin sensitivity and the development of neural circuits involved in the control of homeostasis which causes the higher risk of metabolic disease.


Upon investigation it was found that a mother who was obese before conception was likely to have a higher level of placental LEP than the placenta of a mother of a healthy weight. [ 12 ]  One strategy for overcoming obesity is the use of gastric bypass and other such surgeries, while this does not entirely alleviate the risk of altered metabolic imprinting it has been found that siblings born post maternal surgery are less likely to have as high body fat percentages than over nutrition as siblings born before the surgery. [ 13 ]


Paternal overnutrition can also have a detrimental effect and new-borns have shown changes in methylation of DNA generally, with substantial hypomethylation at the gene Insulin-like Growth factor 2 (IGF2). However, this topic is much less studied than maternal nutrition. [ 13 ]


An increase in certain hormones such as  oestrogen ,  progesterone , human placental  lactogen , human placental growth hormone and  cortisol  during the second and third trimester of pregnancy cause an increase in  insulin resistance . This increase in insulin resistance and following increase in insulin secretion ensures that the foetus develops a normal glucose tolerance. [ 14 ]   Gestational Diabetes Mellitus (GDM)  arises when  beta cells  do not secrete enough insulin to adopt to the insulin resistance triggered by pregnancy, which leads to mild hyperglycaemia. [ 15 ]