Small molecule modulators of histone acetylation a

Biochimica et Biophysica Acta xxx (2010) xxx–xxx

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Review

Small molecule modulators of histone acetylation and methylation:A disease perspective

B. Ruthrotha Selvi, D.V. Mohankrishna, Yogesh B. Ostwal, Tapas K. Kundu ⁎Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur (P.O.), Bangalore 560 064, India

⁎ Corresponding author.E-mail address: [email protected] (T.K. Kundu).

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a b s t r a c t
a r t i c l e i n f o
Article history:Received 18 May 2010Received in revised form 18 September 2010Accepted 24 September 2010Available online xxxx

Keywords:Histone acetylationLysine methylationEpigeneticsArginine methylationInflammationModulator

Chromatin modifications have gained immense significance in the past few decades as key regulators of geneexpression. The enzymes responsible for these modifications along with the other non-histone proteins,remodeling factors and small RNAs modulate the chromatin dynamicity, which in turn directs the chromatinfunction. A concerted action of different modifying enzymes catalyzes these modifications, which are read byeffector modules and converted to functional outcomes by various protein complexes. Several smallmolecules in the physiological system such as acetyl CoA, NAD+, and ATP are actively involved in regulatingthese functional outcomes. Recent understanding in the field of epigenetics indicate the possibility of theexistence of a network, ‘the epigenetic language’ involving cross talk among different modifications that couldregulate cellular processes like transcription, replication and repair. Hence, these modifications are essentialfor the cellular homeostasis, and any alteration in this balance leads to a pathophysiological condition ordisease manifestation. Therefore, it is becoming more evident that modulators of these modifying enzymescould be an attractive therapeutic strategy, popularly referred to as ‘Epigenetic therapy.’ Although this field iscurrently monopolized by DNA methylation and histone deacetylase inhibitors, this review highlights themodulators of the other modifications namely histone acetylation, lysine methylation and argininemethylation and argues in favor of their therapeutic potential.

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1. Introduction

The nucleoprotein complex of DNA wrapped around the histones,non-histone proteins and small RNAs is referred to as chromatin ofwhichthe nucleosome is the fundamental unit. Each nucleosome is an octamerof four core histones namely H3, H4, H2A, and H2B aroundwhich DNA iswrapped and histone H1 is involved in chromatin packaging [1,2]. Thehistones undergo various post-translational modifications. Around 200post-translational modifications of histones have been reported occur-ring at approximately 60 different sites most of which are clustered intheir unstructuredN-terminal tails [3,4]. Thesemodifications provide thecell with an array of regulatory options for the DNA dependent processessuch as transcription, replication, repair, etc. [4].

The current understanding of the role of chromatin and itsmodifications during transcription dictates the spatial and temporalorderly appearance and disappearance of histone modifications alongthe gene so as to regulate the entire process of transcription [5]. Thesehistone modifications not only serve as regulatory switches along theentire process of transcription but also form combinatorial patterns thatgive rise to a ‘Language’ that leads to distinct gene expression patterns.Several other factors such as DNA methylation, small nuclear RNAs,

histone variants also work in conjunction with these histone modifica-tions to form a robust regulatory network which is self-propagating innature and hence is also termed as ‘epigenetic network’. One distinctfeature of this epigenetic language is a mechanism in which modifica-tions initiate transcription activation and reinstate repression [6]. Thisorderly signaling leaves behind the ‘transcriptionalmemory’ in the formof memory marks and establish a particular chromatin state [7].

Thus, chromatin modifications regulate the transcriptional compe-tence of chromatin and thereby influence gene expression. This reviewwill focus not just on the role of chromatin modifications ontranscription regulation but will also highlight their role in severaldisease manifestations and thereby would set the stage for thepossibility of epigenetic therapy. Several modifications are known, butthe review would essentially revolve around lysine acetylation, lysinemethylation and argininemethylation. The mechanism of each of thesemodifications in normal physiological and abnormal pathophysiologicalconditions will be discussed along with an insight into the physiolog-ically relevant small molecules that modulate these enzyme function aswell as the small molecule inhibitors of these enzymes. The final sectionsheds light on thefield of epigenetic therapy, its past, present and future.

1.1. Chromatin modifications: enzymemachineries, substrates and outcome

Modifications that play an important role in transcription orinfluence the transcriptional outcome include acetylation, methylation,

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phosphorylation, ubiquitination, etc. The histone modifications exhibitsome level of specificity with respect to the state of the chromatin.Certain histone modifications are exclusively associated with particularchromatin state, form global chromatin environments and divide theentire genome into euchromatic and heterochromatic regions.

1.1.1. Chromatin acetylationChromatin acetylation is a reversible process catalyzed by the

histone/lysine acetyltransferases (HATs/KATs) and deacetylases(HDACs/KDACs) utilizing the cofactor acetyl CoA. These proteinshelp in maintaining a balance of the steady state acetylation levelsthereby establishing cellular homeostasis [8]. The acetyltransferasesare mainly classified on the basis of their cellular localization asnuclear HATs (type A HATs) or cytoplasmic HATs (type B HATs) andsub-classified based on their structural and functional differences intofive families [9]. These are the GNAT family members represented byGCN5, PCAF, the second family dominated by the p300/CBP enzymes.The major family is the MYST family comprising of MOZ, MOF, andTIP60 with distinct functional outcomes. Yet another important classof nuclear HATs is the nuclear receptor associated proteins like SRC3,ACTR and finally the transcription factor related HATs like TFIIIC,TAFII250. The cytoplasmic HATs characterized so far are HAT1 andHAT2 involved in acetylation of newly synthesized histones. Otherproteins with acetyltransferase activity which do not belong to any ofthese families have also been reported.

There are 18 HDACs in humans and they are classified into threemain groups based on their homology to yeast proteins [8]. Class Iincludes HDAC1, HDAC2, HDAC3, and HDAC8 and have homology toyeast RPD3. HDAC4, HDAC5, HDAC7, and HDAC9 belong to class II andhave homology to yeast HDA1. HDAC6 and HDAC10 contain twocatalytic sites and are classified as class IIa, whereas HDAC11 hasconserved residues in its catalytic center that are shared by both class Iand class II deacetylases and is sometimes placed in class IV. The classIII HDACs include sirtuins, have homology to yeast Sir2, and have anabsolute requirement for NAD+. They do not contain zinc in thecatalytic site and are not inhibited by compounds like TSA orvorinostat. The mechanism of acetylation and its functional signifi-cance have been extensively reviewed in few other chapters in thisissue.

The acetyltransferases and deacetylases are involved in theacetylation of various residues within the nucleosome namely H3(K9, K14, K18, K23, K56), H4 (K5, K8, K12, K16), H2A K5 and H2B K5,as well as involved in the acetylation ofmany transcription factors andnon-histone chromatin associated proteins thus regulating geneexpression [10,11].

1.1.2. Chromatin methylationChromatin methylation is brought about by the methyltrans-

ferases which transfer the methyl group from the methyl cofactordonor S-Adenosyl Methionine (SAM), onto either cytosine residue ofDNA (DNA methylation), to lysine residue of histones and non-histone proteins (lysine methylation) or to arginine residue inhistones and non-histone proteins (arginine methylation). Thus,based on the residue modified, the enzymes are referred to as DNAmethyltransferases (DNMTs), lysine methyltransferases (KMTs) orarginine methyltransferases (PRMTs). Although the DNA methylationis an important modification with well established role in transcrip-tional repression and gene silencing [12], we would be concentratingmore on the protein modifications, lysine and arginine methylation.

1.1.2.1. Lysine methylation. Lysine methylation of histones is catalyzedby enzymes known as lysine methyltransferases, which transfermethyl group from S-Adenosyl Methionine (SAM) to lysine residuesof proteins. All the lysine methyltransferases, with few exceptions(DOT1) possess a unique SET domain (Suppressors of variegationEnhancers of zeste and Tristae), which is sufficient for their enzymatic

Please cite this article as: B.R. Selvi, et al., Small molecule modulators of hBiophys. Acta (2010), doi:10.1016/j.bbagrm.2010.09.005

activity. Human genome encodes 73 SET domain containing proteins,many of which have been assigned the enzymatic activity. Lysinemethylation of histones occurs on H3 (K4, K9, K27, K36, K79), H4(K20), H2B (K5), H1 (K26), etc. Unlike other modifications, which areexclusively associated with either activation or repression, methyla-tion is associated with both, depending on the residues that aremodified [13].

The first methyltransferase to be discovered was the H3K9methyltransferase, SUV39 which belongs to the SUV39 family [14].Although SET domain is sufficient for its enzymatic activity, PRE-SETand POST-SET domains are required for their specificity. This familyincludes SUV39H1/H2, GLP, G9a, ESET, and CLLL8 proteins. SUV4–20H1 and SUV4–20H2 are members of SUV39 related family and areknown to catalyze H4K20 trimethylation [13–15]. These proteins arerecruited to the chromatin via HP1 proteins. Yeast has singleH3K4methyltransferase SET1, whereas in mammals, at least 10known or predicted H3K4 methyltransferases exist, which includesix MLL complexes, SET1A, SET1B, ASH1, SET7/9, SMYD3, andMeisetz.These enzymes are not functionally redundant and this might be dueto their differential expression, differential recruitment, distinct setsof non-histone substrates, etc. MLL proteins exist as complexes andshare 3 subunits among them namely, WDR5, RbPB5 and ASH2 [16].Among all proteins in this family, methyltransferase activity has beenconfirmed for MLL1–4, SET1A, SMYD3, SET7/9 andMeisetz, whereas itis predicted for SET1B and ASH1. MLL1–4 and SET1A show H3K4mono, di and trimethylation activity. SMYD3 display di andtrimethylation activity, which is enhanced by HSP90A. SET7/9 notonly shows H3K4 monomethylation activity but also methylates non-histone substrates such as TAF10 and p53. Mouse Meisetz isspecifically known to trimethylate H3K4 during meiosis [16].

SET2/NSD family proteins exhibit methyltransferase activity, buttheir specificity depends on the nature of the substrate, the major sitein the nucleosomal context being H3K36. The other sites are H3K4,H3K27 and H4K20. This family consists of three proteins NSD1, NSD2and NSD3 all of which are homologous to the yeast SET 2 whichinteracts with elongating RNA polymerase II and displays co-transcriptional H3K36 trimethyltransferase activity [17]. Anotherimportant family of histone lysine methyltransferases belongs to thepolycomb group of proteins discovered in Drosophila melanogastercatalyze H3K27 trimethylation required for Hox gene repression.Human homologue of this protein, EED-EZ (H)2 complex, comprisesof four proteins namely, SUZ12, EED, RbAp48 and AEBP2. It functionsin the silencing of developmentally regulated genes and differentia-tion in embryonic stem cells [18]. RIZ (retinoblastoma-interactingzinc finger) family includes three proteins namely RIZ, BLIMP andPFM1. They all have a SET domain towards amino terminus, lack PRE-and POST-SET domains and possess a number of zinc finger motifs inthe C-terminus. RIZ protein can bind to estrogen receptor andstimulate its activity. It catalyzes H3K9 methylation thereby playinga role in transcription repression. BLIMP is also a transcriptionalrepressor but very little is known about PFM1 [13].

Non-SET domain containing lysine methyltransferases includesDOT1 and its homologues [19]. Human DOT1L (disruptor of telomeresilencing) catalyzes H3K79 methylation which is localized in thecoding regions of actively transcribing genes and telomeres. Thisresidue resides in the globular domain of H3 but is located in theaccessible surface of the nucleosomes. Loss of this mark causes loss oftelomeric silencing. It presumably acts by restricting ‘Sir’ proteins tochromatin [20].

Thus, lysine methylation can be associated with either activation(H3K4 trimethylation for transcription initiation, H3K36 methylationfor transcriptional elongation, and H3K79 methylation) or withrepression (H3K9 dimethylation and H3K9 trimethylation). Thesedifferent modifications (different sites and different degrees), lead tospecific protein complex interactions and thereby influence thechromatin function globally. This modification was considered to be

istone acetylation and methylation: A disease perspective, Biochim.


Table 1Histone modifications, enzyme machineries and functional outcome.

Modificationsite

Enzyme Function

AcetylationH2AK5 TIP60, p300/CBP, Hat1 Transcriptional activationH3K9 SRC1 Receptor Signaling

PCAF, p300/CBP Transcriptional activationH3K14 TAFII250, p300/CBP, PCAF, TIP60 Transcriptional activationH3K18 P300/CBP, PCAF Transcription activationH3K23 CBP Transcription activationH3K56 p300/CBP Transcription activationH4K5 Hat1, HBO1, Histone deposition

p300/CBP, ATF2, TIP60 Transcription activationH4K8 HBO1, ATF2, TIP60, PCAF Transcription activationH4K12 p300/CBP Hat1, HBO1 Transcription activationH4K16 p300/CBP, PCAF, TIP60, Mof, ATF2 Chromatin structure and

Transcription activation

Lysine methylationH3K4 MLL1–6 Trithorax activation

SET7–9H3K9 G9a, EuHMTase1, ESET Transcriptional repression

SUV39H1 Rb mediated silencingAsh1 Trithorax activationRIZ1 Tumor repression

H3K27 EZH2 Early B-cell development,X-chromosome inactivation

H3K36 SET2, NSD1, SYMD2 Transcriptional activationH3K79 DOT1 Transcriptional activationH4K20 Pr-SET 7/8 Transcriptional silencing,

mitotic condensation

Arginine methylationH3R2 CARM1, PRMT2 Gene expressionH3R8 PRMT5 Transcriptional repressionH3R17 CARM1 Transcriptional activationH3R26 CARM1H4R3 PRMT1 Transcriptional activationH4R3 PRMT5 Transcriptional repression

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a stable mark for a long time but the discovery of lysine specificdemethylase (LSD1) in 2006 changed the entire outlook of the field[21]. LSD1 was initially thought to be a demethylase of H3K9methylation but was subsequently also found to act on H3K4methylation in a context dependent manner [22,23]. This discoverysparked off the search for demethylases for other modifications.Several jumonji domain containing proteins have been identifiedwithdemethylating activities [24]. Recent evidences also suggest the roleof these demethylases in processes like stem cell renewal; stem celldifferentiation, etc., indicating a major role of histone modifications inthe early developmental stages [25].

1.1.2.2. Arginine methylation. Protein arginine methylation wasidentified almost five decades ago [26]. Protein arginine methyl-transferases (PRMTs) catalyze the addition of methyl group from themethyl cofactor, SAM to the guanidino-nitrogen of the arginineresidue on proteins. There are 9 PRMTs identified so far, classified intotwo groups based on symmetric and asymmetric arginine dimethyla-tion; PRMT5/JBP1, PRMT7 and PRMT9 are known to bring aboutsymmetric modification which leads to transcriptional repression. Incontrast, all the other PRMTs, PRMT1–4 and PRMT6, 8 are asymmetricdimethyl arginine methyltransferases resulting in transcriptionalactivation. There have been reports of PRMT10 and PRMT11 as well;however, their substrate specificities have not been identified so far[27]. These enzymes can thus lead to the formation of monomethy-lated, asymmetric dimethylated or symmetric dimethylated arginineresidues inmammalian cells. Thewell characterized PRMTs, PRMT1, 3,4, 5, 6 and 8 have been shown to be coded for by six distinct genes.However, the not so well characterized PRMTs, PRMT2, 7 and 9 havealso been mapped to three different genes. The PRMTs arecharacterized by a characteristic motif consisting of seven β-strandsaswell as the PRMT family specific double E and THW sequencemotifs[28]. Two proteins FBXO10 and FBXO11 which lack the characteristicPRMT motifs (described above), have been proposed to havemethyltransferase activity [29].

PRMT1 is responsible for 80% methylation of all protein methyl-ation in the cellular system. PRMT1 and other PRMTs except PRMT4recognize glycine arginine rich motifs for methylation whereasPRMT4 recognizes XXPRXX motif for methylation. The argininemethyltransferases also have other non-histone protein substrates.All these modifications result in various functional processes like RNAprocessing, DNA damage and repair, cell signaling and mostimportantly in transcription [27]. In the case of RNA processing, theRNA splicing machinery is known to be regulated by argininemethylation. So far, the identified histone argininemethyltransferasesare PRMT1 that methylates histone H4R3 [30], PRMT6 that methylatesH3R2 [31], and PRMT4/CARM1 which catalyzes H3R17 and R26 invitro as well as in vivo [32]. PRMT5 and PRMT1 share the samemodification site, histone H4R3, however based on the symmetric orasymmetric modification, the transcriptional state differs. PRMT5 is awell characterized type II arginine methyltransferase, which has beenshown to exist in repressor complexes containing DNA methylationmachinery and NURD complex [33]. One of the major unansweredquestions in the field of PRMT biology is the extent of modulation ofthe enzyme specificity and function by the various interactingproteins. There are a few indications to prove that this might be animportant level of regulation of arginine methyltransferase activitysince these proteins in spite of ubiquitous expression, have veryspecific activities.

Arginine methylation is also a partly reversible modificationwherein two distinct mechanisms have been shown to be operationalin removal of the modification. The first mechanism is an indirectreversal mechanism involving the deimination reaction by peptidylarginine deiminase PAD4, which converts monomethyl arginine tocitrulline [34]. Since this is an intermediary form, the completedemethylation has not been shown for this case. However, a jumonji


domain containing protein JMJD6 has been shown to be a bona-fidearginine demethylase [35]. Thus, although it is clear that the field ofarginine demethylation is still in its infancy, there are indications tosuggest that this reaction might also have physiological relevance,since non-histone proteins like p53 and NPM1 have been shown tohave the citrulline modification [36,37].

In comparison to the research on acetylation and lysine methyl-ation, arginine methylation is still an unexplored modification. Unlikethe former two modifications whose reader modules and effectordomains have beenwell characterized, such effector proteins have notyet been discovered for arginine methylation. Due to this lack ofinformation, it has been difficult to assign a sequential andmechanistic insight for the role of arginine methylation in transcrip-tion. However, singularly these modifications have been studied withrespect to transcription regulation, which will be discussed in thesubsequent section.

2. Chromatin modifications and transcription regulation

The histone modifications exhibit a localized pattern acrosspromoters and open reading frames and thereby play importantroles in the process of transcription as depicted in Table 1 (adaptedfrom [205]). The specific modifications with distinct functions arebriefly described below.

2.1. Role of chromatin modifications in transcription regulation

Histone acetylation at various lysine residues in promoter regionspromotes transcription through various mechanisms such as histone




removal, general transcription factor recruitment, chromatin remo-deling, etc. [38,39]. Histone lysine (K4) trimethylation is enriched inthe 5′ ORFs of active genes [40]. In fact, this modification mediatestranscriptional activation via multiple pathways. It not only leads tothe disruption of repressor complexes (NuRD complex), but is alsorecognized by Royal family domains and PHD family domains presentin various chromatin modifying enzymes and remodeling complexes[41–43]. H3K4 trimethylation is considered to be a determinant of thetranscriptional competence of chromatin and is also correlated withenhanced acetylation kinetics. Modifications such as H2B ubiquitina-tion and H3K79 trimethylation are required in promoter regions aswell as coding regions of genes for transcription to occur [44,45].H3K36 trimethylation is another important polymerase dependentelongation mark specifically present in the 3′ ORFs of active genes[46]. H3S10 phosphorylation and H4K16 acetylation have been shownto have an important role in the elongation process [47]. In fact,phosphorylation of H3Y41 has been shown to exclude HP1 fromchromatin [48]. Apart from these, many other modifications areenriched in promoter regions and ORFs of active genes whose rolesremain elusive. Transcription activation is an active process and forsuccessful transcription to occur, many regulatory checkpoints needto be crossed. There exists a unique language of these chromatinmarks, which is associated with transcription repression. DNAhypermethylation has already been shown to be associated withrepressed chromatin states. Di and trimethylation of H3K9, trimethy-lation of H3K27 and H4K20 is also associated with transcriptionalsilencing and heterochromatinization.

2.1.1. Role of acetylation in chromatin transcriptionAcetylation occurs at multiple lysine residues and each of this

modification has distinct functional consequences associated withactive gene transcription and thus is a mark of euchromatin. Thismodification results in a change in the electrostatic charge of histoneswhich alters the structural properties of histone and its binding to theDNA. It assists in loosening of inter- or intra-nucleosomal DNA–histone interactions. Also, modifications create binding surfaces forprotein recognition modules and thus recruit specific functionalcomplexes to their proper sites of action. Considering the repertoire ofmodifications possible on the histones it is clear that the combinationof events are immense and not just an acetylation mediated ‘switchon’ or ‘switch off’ of gene transcription. Each of these modificationscan be ‘read’ by other proteins that would then influence chromatindynamicity. Indeed, protein domains like the ‘bromodomain’, presentin ATP dependent remodeling complexes like Swi/Snf recognize theacetylated lysine and assist in binding of the complex to acetylatedchromatin activating nucleosome remodeling and transcription [49].The switch to a repressed chromatin involves deacetylation andcompaction of the chromatin fiber. Thus, histone acetylation is centralto establishing a permissive or repressive chromatin. For any gene, inmost of the cases, acetylation of histones sets the positive epigeneticstate along with other modifications (methylation, phosphorylation,etc.) for transcriptional activation. Thus, the overall effect ofacetylation on the different H3 and H4 lysine residues seems tohave an activation effect partly due to the structural alteration of thechromatin and also due to the recognition by the effector modulespresent in remodeling complexes. However, there are signal specificacetylation events, such as with histone H4 wherein acetylation at itsK8 and K12 residues is preferred while the other two potential targetsH4 K5 and K16 are relatively spared [50].

The effect of histone acetylation on chromatin transcription isimmense but a further level of action is evident wherein theacetylation of chromatin proteins facilitates better transcription. Ithas been shown that acetylation of histone chaperone NPM1 by p300enhances transcription [51]. Increasing evidences implicate theautoacetylation of the acetyltransferases as important regulators ofenzyme activity thereby indicating that the acetylation of the protein


itself might have an important role in determining the efficiency oftranscription as described in another article in the same issue. Apartfrom this several transcription factors like p53, NF-κB, c-myc, HIF1α,MEF2C, etc. get acetylated and the acetylated proteins have also beendemonstrated to have better transcriptional activity [10,52].

2.1.2. Role of lysine methylation in chromatin transcriptionLysine methylation is unique due to its multiple modification

status. The residue of modification determines whether it will recruitthe activator complexes or repressor complexes. Most of the lysinemethyltransferases have recognition domains that facilitate thedifferential recruitment of determining factors. SUV39 containschromodomain, which might have a role in recognizing methylatedlysines. G9a and GLP possess ankyrin repeats that are required forprotein–protein interactions and might play an important role insignaling. Interestingly, ESET and CLL8 have methyl-binding domain(MBD) which can potentially recognize methylated DNA [14]. H3K27trimethylation helps in the recruitment of PRC1 complex via bindingof polycomb chromodomain proteins which may aid in chromatincondensation [53].

The methylation status of the lysine residue determines theinteraction and recognition of the HP1 isoforms thereby resulting infacultative or constitutive heterochromatin. H3K9 dimethylation isrecognized by HP1γ resulting in facultative chromatin [54,55]. On theother hand, H3K9 trimethylation is a mark associated with HP1βthereby forming constitutive heterochromatin. Such a structure is alsomarked by H3K27 trimethylation and H4K20 trimethylation. TheH3K4 trimethylation is a mark closely linked with transcriptionalactivation. There are many compelling evidences linking thismodification through the process of transcription. In fact across thebody of a gene, this modification has been shown to decrease from atrimethylated state to di to a mono methylated form. The promoterregions show an increased H3K4 trimethylation enrichment whichsubsequently decreases across the gene implicating an activedemethylation process. Yet another mark associated with transcrip-tional activation is the H3K79 methylation which is better recognizedfor its role in telomere functioning wherein the loss of this mark hasbeen linked with telomere silencing [20].

The elongation associated H3K36 methylation has been shown tobe essential for the process of transcription. This mark comes into playafter the transcription is set into process. The Ser2 phosphorylation ofthe RNA Pol II CTD acts as the signal for the recruitment of the enzymeresponsible for this modification. Independent studies have demon-strated the recruitment of a deacetylase complex into this system [56].Apart from histones, many non-histone protein substrates such as p53and NF-κB are known to influence transcription.

2.1.3. Role of arginine methylation in chromatin transcriptionFor a long time it was considered that the asymmetric arginine

methylation is associated with transcriptional activation and thesymmetric dimethylation is a mark of transcriptional repression.Recent reports have challenged the understanding of asymmetricmodification and transcription. PRMT1 and PRMT4 together havebeen shown to be involved in several cellular processes like repair andsignal transduction wherein the methylated proteins have betteractivity, thus leading to the conclusion that these enzyme mediatedmodifications lead to activation of transcription. The histone modi-fication patterns have also been correlated with the transcriptionallyactive states. Namely, the histone H4R3 methylation by PRMT1 hasbeen shown to be a prerequisite for p300 mediated histoneacetylation [57]. The histone H3K18 and K23 acetylation are alsoshown to be essential for CARM1mediated H3R17methylation for theestrogen responsive gene expression [58].

PRMT1 and PRMT4 mediated histone modifications have beenvery closely linked with the hormone receptor function which led tothe speculation that possibly these modifications are essential for




signal dependent nuclear hormone receptor associated gene expres-sion. However, the past decade has observed an increase in thenumber of transcription factors regulated by arginine methylationwhich include p53, NF-κB, CITED, etc., thereby implicating a possiblerole of arginine methylation in general transcription as well [59,60].Few instances indicate that the asymmetric methylation might not bean exclusively activation associated mark. The SRC3 co-activatorassembly and disassembly are intricately regulated by CARM1wherein the methylation has been shown to signal for disassembly[61]. This intriguing observation could be explained as an effect ofarginine methylation on the remodeling complexes favoring aremodeling event; however in terms of transcriptional output, thisevent is associated with repression. Yet some evidence implicates thepresence of H3R17 methylation on genes that are in a repressed statewhich are turned on by the estrogen signaling in an ER responsivesystem [62].

The symmetric arginine methylation catalyzed by PRMT5 has beenlinked with transcriptional repression. This enzyme has two modifi-cation sites on histones, H3R8 and H4R3 which coincidentally is alsothe site for PRMT1. The H4R3 site seems to be a major site of argininemethylationwhose role in the context of transcription probably needsto be addressed more carefully since it is a mark associated with bothtranscriptional activation and repression based on the enzymemediating the modification [63]. PRMT1 mediated modification is aprerequisite for the acetylation whereas the vice versa is not trueimplicating that possibly there is an exclusion event associatedpossibly due to the presence of a deacetylase. Detailed investigationsinto the role of arginine methylation in chromatin transcription areessential to understand this process holistically.

2.2. Crosstalk of chromatin acetylation and methylation in transcriptionregulation

The recent spate of discoveries in the field of chromatinmodifications has led to the unraveling of a possible networkamong these modifications, the epigenetic network which seems tobe operational even on non-histone proteins such as p53, thusregulating the overall gene expression. Many crosstalk and regulationamong these modifications have been studied, but we would behighlighting only the lysine acetylation, methylation and argininemethylation crosstalk.

2.2.1. Lysine acetylation and lysine methylationThe crosstalk among these two modifications is the best studied

network operational in establishing both transcriptional activationand repression. The interplay of acetylation of H3 and H4 and lysinemethylation of H3K4 facilitate activation whereas the crosstalk ofDNA methylation, deacetylation and lysine methylation of H3K9 ondistinct residues results in transcriptional repression [64]. Thedifferent modifications and the network that regulates transcriptionalactivation are detailed below. Modifications such as H2B ubiquitina-tion and H3K79 trimethylation are required in promoter regions aswell as coding regions of genes for transcription to occur. In fact, theyare required for di and trimethylation of H3K4 residue. H3K36trimethylation is another important polymerase dependent elonga-tion mark specifically present in the 3′ ORFs of active genes. This leadsto the recruitment of histone deacetylase complexes that maintainchromatin in the deacetylated state in the coding regions to preventspurious intragenic transcription initiation [56]. In fact, phosphory-lation of H3S10 and H3Y41 has been shown to exclude HP1 fromchromatin. Phosphorylation of H3S10 is considered to be the triggerleading to acetylation of H3K14 residue at the promoter proximalhistones. This is then followed by demethylation of H3K9 andsubsequently, its acetylation. H3K4 methylation (di- and tri-) acts asa tag for recruiting various modulatory factors that involve complexeslike SAGA (in yeast); in which case histone acetylation is further


increased and transcription is enhanced [5]. A recent report indicatesthe interplay of this modification with DNAmethylation as well as thedeacetylase in the NURD complex implicating the overall effect ontranscription in the form of silencing. This modification has beenclosely linked with DNA methylation. Especially the H3K9 methyla-tion in conjunction with the DNA methylation has been shown to beinvolved in heterochromatinization involving the heterochromatinprotein HP1. H3K9 trimethylation is a mark associated with HP1βthereby forming constitutive heterochromatin. Such a structure is alsomarked by H3K27 trimethylation and H4K20 trimethylation. Thesemodifications are also actively supported by the deacetylationmachinery, essentially which occurs prior to methylation. Thus, therecruitment of the deacetylases and the subsequent removal ofacetylation act as a signal for the methylation at these residues. Thissequential recognition of these marks and the subsequent higherorder chromatinization has been extensively investigated.

2.2.2. Lysine acetylation and arginine methylationThemostwell studied example of such a crosstalk is the network that

exists between acetyltransferase p300/CBP, PRMT1 and PRMT4. Thenuclear hormone receptor associated transcriptional activation is onesuchmolecular system that emphasizes theneed for a co-operative effectof acetylation and arginine methylation. The NR system includes thesteroid receptors, estrogen receptor, androgen receptor, glucocorticoidreceptor, and thyroid receptors. Each of these receptors is a mediator ofsignal dependent gene expression by specific receptor–ligand interac-tions. Since these events are associated with transcriptional activationfollowed by gene expression, they are also associated with chromatinremodeling as well as the involvement of chromatin modifyingmachineries. Apart from the general transcriptional activators, theseevents alsouse secondary co-activators suchasp300/KAT3BandCARM1/PRMT4. This is possible because of the co-activation property as well asthe enzymatic property of these enzymes. The various domain mappingled to the identification of the exact interaction regions wherein p300/CBPwas found to interactwithAD1 regionof p160 co-activatorGRIP. TheAD2 region was found to be the primary site of interaction with theenzyme CARM1 [65]. Another independent study showed the existenceof yet another activation domain in GRIP, i.e. AD3 which interactedspecifically with another arginine methyltransferase PRMT1. Theexistence of all these co-activators led to several fold induction intranscription [66]. Very little is known about the interplay of lysinemethylation andargininemethylation, except for thenegative regulatoryrole of H3R2 methylation on H3K4 methylation [67]. The exactphysiological outcome of this regulation has still not been worked out.

The cross talk ofmodifications is not limited to histones. Several non-histone chromatin proteins and transcription factors are reported toundergo multiple modifications, which have important regulatory roleson their function [10]. The acetylation of p53 leads to activation oftranscription by multiple mechanisms such as enhancement of specificDNA binding, inhibition of non-specific DNA binding, aswell as acting astags for recognitionmotifs such as the bromodomain present in differentrecognition modules. Apart from the structural intricacies, p53 acetyla-tion also acts as a regulator of its stability. Many of the acetylation sitesare also ubiquitination targets which finally lead to the proteindegradation. The acetylation precludes any other modification at thesame site and hence helps in conferring protein stability. Recent studiesrevealed that K370, K372 and K382 can also bemethylated [68–70]. p53acetylationmay also crosstalk with neighboringmodifications similar tothe histone code. For example, p53 acetylation at K370 and K372mightcommunicate with Ser 215 phosphorylation [71]. Strikingly, a recentstudy revealed that K372 methylation is required for p53 acetylation atmultiple sites. The methylation likely recruits Tip60 via its chromodo-main and promotes K120 acetylation, which might then form dockingsites for PCAF and p300 mediated acetylation. p53 also undergoesarginine methylation mediated by PRMT5 that inhibits p53 dependentapoptosis. Recently, it has been shown that peptidyl arginine deiminase




(PAD4) which is an arginine demethylase acts on p53 resulting in theformation of citrullinated p53 which results in repression of target geneexpression [36].

3. Small molecule regulators of chromatin function(transcription regulation)

It is clear that the chromatin modifications regulate transcriptionand thereby greatly influence gene expression. Hence, the factorsassociated with the establishment of these modifications such as thechromatin modifying enzymes also influence gene expression.However, a significant role is also played by the small moleculepseudo-substrates of these modification reactions, such as acetyl CoAand SAM which intricately link the process of physiological metab-olism and gene expression. A brief overview of these significant smallmolecules in the physiological system is discussed below.

3.1. Acetyl CoA

Like ATP, acetyl CoA lies at the core of a number of critical cellularpathways, and its intracellular concentration is therefore a potentialreadout of the processes involved. Changes in the availability of acetylCoA could directly affect the acetylation status of critical substrates, asobserved for the core histone proteins H2A, H2B, H3, and H4. Thus, oneof the longstanding hypotheses that there exists a link betweenmetabolic pathways and gene expression has been proven in modelsystems ranging from bacteria to yeast to mouse, indicating theuniversality of this process [72,73].

3.2. NAD+

Acetylation and deacetylation are reversible processes thatmaintaina balance of the total cellular acetylation levels. Although deacetylationinvolves just the removal of the acetyl group, the class III deacetylases,the sirtuins are NAD+ dependent deacetylases and require this smallmolecule to bring about deacetylation [10]. Perturbations of NAD+

metabolism alter sirtuin catalytic activity in yeast and in human cellsand implicate NAD+ and related metabolites as regulators of geneticevents in the cell nucleus during stress and toxicity [74]. NAD+ is anabundant metabolite. The concentration of this metabolite is normallyin the range of 400 to 700 μM in human cells. NAD+ also potentiates thebiological effects of sirtuin. Yet another physiological process thatutilizesNAD+ is theDNArepair process [75]. Specifically, the poly (ADP-ribose) polymerases (PARPs), particularly PARP-1, are activated by DNAstrand breaks. The PARPs consume NAD+ as an adenosine diphosphateribose (ADPR) donor and synthesize poly (ADP-ribose) onto nuclearproteins such as histones and PARP itself [76]. Over-activation of PARPcan cause significant depletion of cellular NAD+, leading to cellularnecrosis. Thus, the cellular NAD+ levels regulate important processesinvolved in DNA repair as well as longevity apart from its wellestablished role in transcriptional repression.

3.3. S-Adenosyl Methionine (SAM)

SAM (S-Adenosyl Methionine, also known as AdoMet) occurs as aconjugate of methionine and the adenosinemoiety of ATP, catalyzed bySAM synthetase. SAM alongwith ATP is one of the vitalmetabolites thatis used as source/substrate in a wide array of reactions. SAM is thecofactor for all the methyltransferases, and the reaction results in theformation of SAH which is subsequently hydrolysed to homocysteine.The latter is the substrate for methionine synthase, which uses aderivative of folate, MTHF, as a methyl donor to regenerate methionine,producing THF (tetrahydrofolate). Thus, the folate metabolism and theSAMbiosynthesis reaction are very closely linked and hence the cellularvitamin B12 levels and the dietary folate status influence the SAMbiosynthesis. More than 40metabolic reactions involve the transfer of a


methyl group from SAM to various substrates such as nucleic acids,proteins, phospholipids and small molecules such as arsenic, poly-amines, etc. [77].

3.4. miRNA

The identification of these small RNAs, referred to as micro RNAswas a revolutionary change in the area of gene expression. It wasdiscovered that in Caenorhabditis elegans, there were few RNAs whichwere, involved in inhibiting translation [78]. Subsequently, suchmechanism was found to be functional even in other species such asplants, fruit flies, mice, and humans, and all seem to play a similarregulatory role. miRNA is structurally different than mRNA. miRNAsfold back on themselves to create a double-stranded structure knownas a stem–loop, which acts as a tag for the recruitment of the RNAprocessing machinery such as Dicer and the RNA-Induced SilencingComplex (RISC) and assumes a linear single-stranded shape. mRNAand miRNA both have single-stranded structures in the cell. Thisproperty allows miRNA to seek out and bind specific nucleotidesequences in the target mRNA molecule. There are two modes ofinhibiting the mRNA translation; most often the miRNA–mRNAhybrid is degraded by the processing machinery, whereas in othercases themiRNA stalls themRNA from undergoing translation. In bothcases, however, the gene coding for the mRNA is not expressed. Theinitial discovery of miRNAwas as a development associated molecule,but the recent discoveries suggest the role of miRNA in cancer,differentiation, growth and development [79]. Modulation of histonedeacetylases using respective inhibitors also alter microRNA profilessuggesting a link between histone acetylation dynamics and micro-RNAs [80]. Moreover it is also known that microRNA plays a role inheterochromatin assembly through recruitment of histone lysinemethyltransferases. This brings to fore the intricate link existingbetween histone post-translational modification and microRNAs.

Apart from these physiological small molecules, there are severalothers too which intricately modulate the process of gene expression.The most important being ATP, since it modulates the cellular signalingprocess, aswell as regulates the remodeling events associatedwith geneexpression. However, since the focus of the review is solely onacetylation andmethylation, only those small moleculeswhich regulatethese events have been highlighted.

4. Chromatin modifications in disease manifestationand progression

The role of chromatin modifications in transcription and thereby ingene expression is unquestionable. Thesemodifications on histones andother chromatin proteins help in fine tuning the essential cellularprocesses. As a corollary, it is evident that any alteration would havesignificant effects on these processes. This altered state of modificationsand altered gene expression is what leads finally to disease manifes-tation i.e. abnormal functioning of the systemwhich could be loss of cellcycle control and thereby increased proliferation as in cancer, orincreased inflammatory reactions leading to severalmetabolic disordersor allergies and several more as represented in Table 2. Hence, itbecomes essential to understand the role of these modifications in theabnormal states so as to increase the diagnostic and therapeuticpossibilities. Described below are few disease states wherein theinvolvement of the histone modifications has been clearly demonstrat-ed for the manifestation and progression of the disease.

4.1. Chromatin modifications in cancer

The six hallmarks of cancer are unlimited replication potential,evasion of apoptosis, evasion of growth inhibitory signals, angiogen-esis, metastasis and growth signal autonomy [81]. There are evidencessuggesting the role of chromatin modifications both in a positive and



Table 2Altered histone modifications in pathophysiological conditions.

Disease Altered histone modifications Reference

CancerSolid cancers Decreased H4K16ac, H4K20me3,

mistargeting of H3K27me3[83,84]

Prostate cancer Decreased H3K18ac, H4K12ac [85]Hepatocellular carcinoma,oral cancer

Histone hyperacetylation [86,87]

Breast cancer Increased H3R17me2 [96]Inflammatory disorders Increased H4K8ac, H4K12ac at

specific promoters[50]

Increased H3R17me2 and H4R3me2 [131]Increased H3K4me2 [130]Decreased H3K9me2

Diabetes Increased histone acetylation [121]Increased H3K9me2 at IL 2 andNF-κB promoters in lymphocytes

[123]

Cardiac hypertrophy Increased histone acetylation [136–138]Increased H3K4me [144]Increased asyymetric argininedimethylation

[145]

Retroviral infections Increased H3K4me3 at viralintegration site

[153]

Increased H3K9me3 duringviral latency

[154]


negative regulatory mode on cancer which will be individuallydiscussed below in the context of the three important modifications:acetylation; lysine and arginine methylation.

4.1.1. Acetylation and cancerChromatin (hypo)acetylation and its effect on cancerwere recognized

very early. Thiswas the concept thatwas prevalent till the last decade andhence histone deacetylase inhibitors are currently in clinical trials [82].The reason that this conceptwas inculcatedwas due to the fact that therewas an increased repression of tumor suppressor genes by DNAmethylation, histone methylation and deacetylation [83]. Importantly,HDAC activity is crucial to prepare the histone template for methyl-transferases by removing acetyl groups obstructing methylation. Thusdeacetylase inhibitors were tried to rescue the tumor suppressors. Also,p300mis-sensemutationswere identified in glioblastomas, breast cancerand colorectal cancer [84]. These necessitated investigation into thealteration of acetyltransferase function in cancers. An elegant observationby Kurdistani's group in samples of prostate cancer indicated that thereare several residues like H3K18 whose hypocetylation is positivelycorrelated with increased grades of prostate cancer and its recurrence[85]. However histone hyperacetylation also is observed in certaincancers. Hepatocellular carcinoma is one such example, indicated toexhibit hyperacetylation [86]. Recent work from our group have alsoshown the positive relation of acetylation and oral cancer manifestationwhich is discussed in another chapter [87,88]. These differential roles ofacetylation in cancer suggest the need for an extensive investigation intothe different histone modifications in relation to various cancers.

4.1.2. Lysine methylation and cancerAberrant gene expression leads to various diseases including cancer.

The major cause of cancer is either silencing of tumor suppressor genesor activation of oncogenes. Since histone methylation plays animportant role in transcription, dysfunctioning of the enzymescatalyzing these modifications is expected to lead to such diseases.This may include overexpression, underexpression or misexpression ofany of these enzymes.

Mice lacking SUV39H1 and SUV39H2, or both, develop leukemiasuggesting the importance of these enzymes in regulation of cellproliferation [89,90]. G9a is involved in silencing of tumor suppressorgenes and its knockdown inhibits cancer cell growth [91]. Global lossof H4K20 trimethylation is one of the hallmarks of cancer [92]. MLL is


translocated in most of the hematological malignancies and depend-ing on the fusion partner, it leads to dysregulation of target genes. Hoxgenes are one of the most important targets of MLL members and arefrequently upregulated in most of the leukemias [93]. Mammalianpolycomb group proteins are known to repress transcription viarecruitment of histone deacetylases. Human homologue of EZ(H)2 isfrequently upregulated in prostate cancer and lymphomas. In fact,mice lacking this protein show impairment of B-cell development,suggesting its role in development and cell proliferation. PRC2,responsible for silencing of tumor suppressor genes in early stagesof cancer contains EZ(H)2 that catalyze H3K27 trimethylation, whichis recognized by chromodomain containing proteins of PRC1 toestablish the epigenetic repressive states [92]. One of themistargets ofthis pathway in early stages of cancer is p16, which drasticallydisrupts cell cycle control allowing clonal expansion. EZH2 can serveas a marker in early breast cancer growth, and promotes proliferationand invasiveness of prostate cancer cells. NSD family members havealso been implicated in various cancers. Chromosome translocationsof NSD1 or NSD3 to NUP98 result in acute myeloid leukemia [94].NSD2 overexpression leads to multiple myelomawhereas reducing itslevels suppresses cancer growth. NSD3 is also known to beupregulated in breast cancer cell lines and primary breast carcinomas.Knock down of DOT1L inhibits growth of cancer cells having MLLtranslocations. Constitutively active MLL-DOT1L may misregulatetranscription of MLL regulated genes [95]. Mice lacking RIZ1 proteindevelop diffuse large B-cell lymphomas and other tumors. PFM1mapsto chromosomal regions that are frequently deleted in tumors. Allthese reports suggest that there exists a cause and effect relationshipbetween lysine methyltransferases and cancer.

4.1.3. Arginine methylation and cancerArgininemethyltransferases PRMT1, PRMT2 and PRMT4 have been

shown to have direct roles in several cancers. PRMT1 forms a complexwith the lysine methyltransferase MLL (mixed lineage leukemia), andregulates the MLL downstream targets, thereby playing an importantrole in oncogenesis [96]. The lysine methyltransferase MLL is a wellknown candidate that forms fusion proteins (by gene fusion) whichlead to leukemia. The involvement of the PRMTs along with MLL is anindication of the role of arginine methylation in leukemia. On theother hand, PRMT1 methylation regulates the AML1/Runx1 activitywhich is essential in differentiation and is also involved in acutemyeloid leukemia. PRMT4 methylates histone H3R17 and R26.Estrogen receptor alpha (ERα) dependent breast cancer shows anincrease in H3R17 methylation indicating the role of this transcrip-tional co-activator in ER-dependent breast cancers [97]. It has beenobserved that the H3R17 dimethylation is concomitant with ERαrecruitment on the E2F1 promoter on estrogen induction. Themethylation has been shown to be dependent on the presence ofthe oncogenic co-activator AIB1. Thus the presence of CARM1 and theincreased H3R17 methylation have been directly implicated in theonset of ER alpha dependent breast cancer [97]. The essential role ofPRMT4 in SRC3/AIB stability and complex assembly and disassemblyindicates that there could be important contribution of PRMT4/CARM1 and H3R17 methylation in breast cancer manifestation andprogression [61]. Furthermore, PRMT1 and PRMT4 have been shownto cooperate synergistically along with the histone acetyltransferasep300, thereby resulting in the transcriptional activation of the tumorsuppressor, p53 responsive genes [57]. Furthermore, there are fewreports of prostate cancer and arginine methylation wherein theincreased levels of PRMT1 and PRMT4 have been shown [98–100].Although these indicate a possible role of these enzymes in prostatecancer manifestation/progression, the exact mechanism has not beenidentified. Since these two proteins are also co-activators of nuclearhormone receptors like androgen and estrogen receptor, it is possiblethat there exists a direct transcriptional activation due to the co-




activation property of these two enzymes in both prostate and breastcancers.

4.2. Chromatin modifications in inflammatory disorders

One of the main mediators of inflammatory response is thetranscription factor NF-κB, which is regulated by acetylation, lysinemethylation and arginine methylation. Direct evidences of acetylationlinked to inflammatory disorders have been shown in the case ofacetylation with very minimal and indirect evidences linking thelatter two modifications.

4.2.1.1. Role of acetylation in inflammation and fibrosis

Histone H4 hyperacetylation is a well known inflammationassociated epigenetic mark [50]. IL-1β causes increased histoneacetylation on H4 at K8 and K12 residues. This increase is due to therecruitment of HATs like CBP and PCAF, to promoters of inflammatorygenes, by a very important transcription factor controlling expressionof inflammatory genes, nuclear factor-κB (NF-κB). Family membersinclude p50, p52, p65 (RelA), c-Rel, and RelB. In vivo, they exist ashomo or hetero-dimers with distinct DNA binding specificities [101].Their response is regulated at multiple levels: synthesis, subcellularlocalization, postranslational modification, differential dimerization,DNA binding, and interaction with a combination of receptorsdepending on the given context [102]. Upon activation by phorbol12-myristate 13-acetate, H2O2, cigarette smoke condensate, interleu-kin-1beta, lipopolysaccharide, and okadaic acid, etc., NF-κB getsderepressed and translocates into the nucleus. Acetylation of distinct

Fig. 1. Role of acetylation in the NF-κB inflammatory response: interleukins and TNF-αdissociation of NF-κB and the inhibitory kinase complex, IκBα is subsequently phosphorylateacetylation by p300, followed by the recruitment of the other acetyltransferases and the tranenlisted as the inflammatory genes. HAT inhibitors such as EGCG, anacardic acid and plumb


lysine residues of RelA regulates different functions of NF-kappa B,including transcriptional activation, DNA binding affinity, IκB alphaassembly and subcellular localization [103,104]. Acetylation of NF-κBby p300/CBP inhibits its interaction with IκB and retains it within thenucleus. HDACs like HDAC3 deacetylate p65 resulting in its binding toIκB and thus translocation to cytoplasm. HDAC1 and HDAC2 are alsofound in p65-HAT complex.

NF-κB regulates a wide variety of genes responsible for inflam-mation like cytokines (e.g., IL-1,2,6,12, GM-CSF, TNF-α, etc.);chemokines (e.g., RANTES, MCP1, IL-8, Eotaxin, MIP1-a); cell adhesionmolecules (e.g., ICAM, VCAM, E-Selectin); acute phase proteins (e.g.,SAA); inducible enzymes (e.g., iNOS, COX-2), some antimicrobialpeptides; and genes responsible for regulation of apoptosis andproliferation [105]. NF-κB dependent transcriptional complexesrequire multiple co-activators like p300 or its close homologue CBP,P/CAF and the p160 family of steroid receptor co-activators, whichalso are acetyltransferases, thus facilitating rapid formation of the pre-initiation complex and re-initiation which facilitate multiple roundsof transcription by histone acetylation [106,107]. Along with NF-κB,Poly (ADP-ribose) polymerase-1 (PARP-1) has been demonstrated toplay a pathophysiological role in a number of inflammatory disorders[108]. PARP-1 is a promoter-specific co-activator of NF-κB in vivo.However this function is independent of its enzymatic activity [109].PARP-1 directly interacts with p300 and also with both subunits ofNF-κB (p65 and p50) and synergistically co-activates NF-κB depen-dent transcription. p300 in turn acetylates PARP-1 at specific lysineresidues upon inflammatory stimulation in a variety of cell types.Acetylation of PARP-1 at these residues is required for the interactionof PARP-1 with p50 and synergistic co-activation of NF-κB by p300

on interaction with their respective ligands and stress associated ROS, stimulate thed and ubiquitinated followed by degradation. The NF-κB subunits, p50 and p65 undergoscription machinery leading to the expression of the NF-κB dependent genes which areagin have been shown to inhibit the NF-κB pathway by inhibiting histone acetylation.


image of Fig.�1



and the mediator complex in response to stimuli [110]. NF-κBmediated activation of histone H3, H4 acetylation specifically bycytokines IL-β, TNF-α or endotoxins has shown to increase expressionof inflammatory genes such as Granulocyte–macrophage colonystimulating factor (GM-CSF) [105]. The role of acetylation in the NF-κB signaling pathway is depicted in Fig. 1.

Infiltration of cellular mediators of inflammation needs chemo-tactic agents such as eotaxin (eosinophil chemoattractant), secretedat the site of inflammation [111]. The human eotaxin promoterconsists of several binding sites for transcription factors including NF-κB [112]. Inflammatory signaling by TNF-α induces selective histoneH4 acetylation and binding of p65 to the eotaxin promoter, thusregulating its transcription. It has been shown that beta(2)-agonistsand glucocorticoids, which are mainstream therapies for allergic andinflammatory conditions were able to reduce transcription of eotaxinby inhibiting both TNF-α-induced histone H4 acetylation andrecruitment of p65 to the eotaxin promoter, without altering thecapability of NF-κB to translocate to the nucleus [113]. Thus reversibleacetylation of transcription factors like NF-κB, proteins like PARP andhistones plays pivotal roles in regulating gene expression duringinflammation. Pharmacological agents used in the therapy ofinflammatory diseases like Corticosteroids and β-2 antagonists albeitindirectly reduce histone hyperacetylation in the affected cells.

HATs like p300 play a role in themanifestation of fibrotic disorderstoo. Deregulated expression of collagen genes underlies the patho-genesis of fibrotic disorders. Fibroblasts are responsible for theproduction and accumulation of extracellular matrix (ECM), com-posed of largely of collagen, and other macromolecules. Aberrantfibroblast activation and differentiation to myofibroblasts lead to anexcessive accumulation of collagens and other ECM proteins andpathological fibrosis [114]. Transforming growth factor-β (TGF-β) is amultifunctional cytokine that regulates cellular growth, differentia-tion, and survival as well as the profibrotic tissue response [115]. TGF-β transduces its signal from surface receptors to the nucleus via Smadsand interacting cofactors. One of the cofactors that is implicated inTGF-β induced signaling is p300. Upon activation of the type II cellsurface receptors, activated type I TGF-β receptor phosphorylatesSmad2/3 which heterodimerize with Smad4 and translocate to thenucleus wherein they stimulate collagen gene transcription [116].However, for maximal collagen synthesis, interaction of activatedSmad2/3 with Sp1, along with p300 and other transcriptional co-activators is required. Interestingly, the p300 homologue, CBP doesnot play a role in this process.

It is intriguing to note that p300 also plays a role in IFN-γmediateddecrease in collagen gene transcription that involves a different set oftranscription factors like activated STAT1α [117]. This antagonisticresponse is due to competition for limited availability of p300 by TGF-β induced Smads and IFN-γ induced STAT1α and others. This isproved by the fact that ectopic expression of p300 overcomes theinhibition by IFN-γ and rescued stimulation by TGF-β, thus rescuingcollagen gene transcription. p300 thus acts as an integrator of IFN-γ/STAT1α and TGF-β/Smad2/3 signals.

Another pro-inflammatory cytokine, tumor necrosis factor-alpha(TNF-α), also inhibits basal collagen gene expression and blocks itsTGF-β induced stimulation [118,119]. Again, a different set oftranscription factors is implicated. Activation of c-jun and JunBinduced by TNF-α results in competition with TGF-β-activatedSmad3 for limiting p300. Even in this case, overexpression of p300overcomes the inhibition. Hence p300 plays a key role as an integratorof signals in both positive and negative modulation of fibroticresponse mediated by its co-activation as well as acetyltransferaseactivity. This modulation is based on the balance of intracellular signaltransduction molecules that are decided by profibrotic or antifibroticcues.

Other HATs like PCAF have also been implicated in fibrosis bymodulating the stability of Fli1 by acetylation [120]. Fli1 is a member


of Ets transcriptional factors which acts as a negative regulator ofcollagen gene expression in dermal fibroblasts. Fli1 downregulation isimplicated in conditions such as cutaneous fibrosis in scleroderma.PCAF-dependent acetylation abolishes the repressor function of Fli1with respect to collagen gene expression. Thus, the role of HATs in themanifestation of inflammatory disorders seems to be multifaceted.

4.2.1.2. Role of acetylation in diabetes

It is well known that both type I and type II forms of diabetes areintricately linked to inflammation [121]. Recent reports implicate arole for HATs in the manifestation of diabetes. High glucose canaugment the expression of NF-κB dependent genes such as TNF-α[122]. Transcriptional activities of p65 as well as co-activator effects ofCBP, p300, or PCAF are clearly enhanced under high glucoseconditions in THP cells. Very interestingly, the promoter bound CBPand HDAC1 show different dynamics in the presence of high glucose.As the level of CBP increased, a concomitant decrease in HDAC1 hasbeen observed. Presently, HUVECs incubated in varied glucoseconcentrations were investigated for their acetylation status follow-ing p300 siRNA transfection, p300 overexpression, or incubation withthe p300 HAT inhibitor, curcumin [123]. These cells on treatment withhigh glucose showed increased p300 and hyperacetylation atpromoters of inflammatory genes like Endothelin-1 and fibronectin.The p300 overexpression showed glucose like effects with an increasein transcription of Endothelin-1, Fibronectin and VEGF. Silencing ofp300 abrogated the effects of glucose in cells and the p300 specificHAT inhibitor; curcumin prevented the effects of glucose. It has to benoted that hyperacetylation is not the only epigenetic changeoccurring at the promoters. Transient hyperglycemia induces persis-tent mobilization of SET7 to the p65 promoter that results in H3K4monomethylation and activates p65 downstream genes [124]. It isonly logical to conclude that inflammation which is associated withmost diseases, results in an increase in HAT activity and HATinhibitors could therefore be novel anti-inflammatory agents.

4.2.1.3. Inflammatory conditions of the lung

Lung inflammatory conditions involve expression of multiplegenes in the lungs that encode chemokines, cytokines, receptors ofchemical mediators of inflammation, cell adhesion molecules, etc.Infiltration of cellular mediators of inflammation results in extensivedestruction of lung architecture initially due to tissue necrosis andlater due to fibrosis that occurs as a part of the healing process.Complex epigenetic mechanisms orchestrate expression of subsets ofgenes at various time points during disease progression resulting intissue damage and healing. Bronchial asthma is a common allergiccondition of small airways. Hyper responsive and hypertrophic airwaysmooth muscle cells and mucosal hypertrophy are the majorpathologic findings. There is a marked increase in HAT activity inboth tissues and alveolar macrophages isolated from bronchoalveolarlavage in patients [125]. A small decrease in HDAC activity due toreduced HDAC1 is seen albeit no changes in HDAC2 and 3 [126].Interestingly, an increase in SIRT1 (class III HDAC) levels and activityis found to be responsible for OVA (Ovalbumin) induced bronchialhyper reactivity in mouse models necessitating further investigationinto the role of other histone modifying enzymes [127].

COPD (Chronic Obstructive Pulmonary Disease), associated inva-riably with smoking occurs as a destruction of lung alveoli. It has beencorrelated to reduced HDAC levels in peripheral lung especiallyHDAC2 with a lesser extent of HDAC5 and HDAC8 reduction [128].Reduction in HDAC2 follows severity of disease, with 95% decrease inStage 4 disease. Reduction in HDAC2 levels also directly correlateswith airway inflammation as evidenced by an increase in IL-8 and thenumber of inflammatory cells in small airway. Thus a decrease in thedeacetylation status implies a constant level of acetylation within




tissues that leads to prolonged expression of inflammation associatedgenes resulting in chronic and widespread lung damage. Inhibition ofHAT could in theory be able to limit inflammatory gene expressionand thus reduce the damage.

4.2.2. Role of lysine methylation in inflammation associated diseases

Apart from cancer, alteredmethylation patterns are also observed indiabetes and other inflammatory diseases. Glucose induced insulintranscription is regulated by PDX1 which recruits SET9methyltransfer-ase to the insulin promoter. Several PDX1 mutations associated withdiabetes that lead to epigenetic changes are known [129]. Hyperglyce-mia also leads to changes in epigenetic patterns on the genes involved invascular inflammation. H3K4 methyltransferase SET7/9 influencesrecruitment of NF-κB p65 to promoters and thereby regulate expressionof pro-inflammatory genes [130]. Vascular smoothmuscle cells of db/dbmice show increased H3K4 dimethylation and decreased H3K9dimethylation at the promoters of inflammatory genes. Interestingly,overexpression of SUV39H1 in vascular smooth muscle cells reversesdiabetic phenotype, whereas silencing this protein in normal smoothmuscle cells increases the expression of inflammatory genes [131]. Intype I diabetes, IL-1β and NF-κB promoters show increase in H3K9dimethylation occupancy in lymphocytes. SET7 is known to be involvedin regulating epigenetic changes at NF-κB promoter in response totransient glycemia. Recently, SET7/9 was shown to play an importantrole in TNF-α expression in monocytes implicating a possible role inmanifestation of diabetes and inflammatory disorders where TNF-α isover expressed [130].

4.2.3. Role of arginine methylation in inflammatory disorders

Arginine methylation has not been directly implicated withdiabetes manifestation; however, important members in the glucosemetabolism are methylated by PRMTs. Further investigation in thiscontext might reveal new roles for the PRMTs in diabetes too. BothPRMT1 and PRMT4 are known transcriptional co-activators of NF-κB[132].

4.3. Chromatin modifications in cardiovascular disease conditions

Similar to the inflammatory disorders, the information availableabout acetylation and cardiovascular disease is much more than theother twomodifications. Described below are the compelling evidencesof these modifications on different cardiac disease conditions.

4.3.1. Acetylation and cardiovascular disordersCardiac hypertrophy is associated with increase in cardiac muscle

size, re-induction of fetal cardiac gene program (α to β isoforms ofMyosin Heavy Chain), increased protein synthesis and sarcomerereorganisation resulting in decreased cardiac performance. The role ofseveral signaling pathways including the chromatin modifyingenzymes, has been implicated in cardiac hypertrophy. The acetyl-transferase p300 acetylates GATA-4 and induces expression of geneslike ANF, ET-1, and α-MHC [133]. Acetylated GATA-4 has increasedDNA binding ability and also increased expression of GATA-4regulated genes as mentioned above [134]. Cardiac overexpressionof p300 results in increasedmortality [135]. p300 alongwithMEF2D isknown to activate cardiac genes in differentiated cardiac myocytes[136]. p300 mediated acetylation of MEF2C affects MEF2C DNAbinding and transactivation function of MEF2C [137]. MEF2 basalactivity is very low and tightly regulated by deacetylationmediated byHDAC5 and 9. Even a small increase in myocardial p300 can switchMEF2 from a basal deacetylated state to an active acetylated state.Recent studies argue whether MEF2 or GATA-4 mediated activation isimportant for p300 mediated synergic effects on transcription [136].However, all these studies underscore the critical role of p300


mediated hyperacetylation of these factors and histones [138–140].Recent studies with p300 inhibitors such as anacardic acid andcurcumin have been shown to prevent the induction of hypertrophyin isolated neonatal rat cardiomyocytes, whichwill be discussed in thesubsequent section [141,142]. Interestingly, HDACi treatment has alsobeen shown to repress cardiac hypertrophy, but through anacetylation independent mechanism, possibly by blocking the HATand MEF2C interactions [143]. HDACs may also directly or indirectlymodulate hypertrophic signal transduction cascades like the HAT-GATA4-hypertrophy axis [144]. The delicate balance between HATsand HDACs in normal physiology implicates a careful investigation ofthe role of each of these components in the disease manifestation.

4.3.2. Lysine methylation and cardiovascular disordersThere is an active effort towards identifying the possible alteration

in lysine methylation associated changes and cardiovascular diseaseonset, but very less success has been achieved in this field. One of thenotable studies done on a rat model of cardiac disease signifies thealteration of H3K4 methylation across the stages of disease progres-sion. Another mark, H3K9methylation that was also analyzed showedvery minimal changes across the different disease states. The exactrole of lysine methylation on these disorders also remains to beelucidated, but the fact that inflammation and diabetic complicationsshow the involvement in these disorders indicates a possible role oflysine methylation in cardiovascular disorders [145].

4.3.3. Arginine methylation and cardiovascular diseasesAll the class I PRMTs are involved in the formation of asymmetric

dimethyl arginine (ADMA), which is a competitive inhibitor of nitricoxide synthase (NOS), and thus decreases nitric oxide (NO)availability in the cells [146]. Decreased NO levels are a hallmark ofcardiovascular diseases. Thus, ADMA levels play an important role inthe cardiovascular diseases as well as conditions related to oxidativestress like hyperlipidemia, diabetes, arterial hypertension, hyperho-mocysteinemia and heart failure. ADMA levels are also considered tobe indicative of atherosclerosis [147]. Thus, all the class I enzymes areresponsible for the oxidative stress induced disease manifestation viathe ADMA intermediate [148].

4.4. Chromatin modifications in retroviral diseases

Retroviruses are RNA viruses which use the reverse transcriptaseto form DNA and get integrated into the host genome upon infection.These have also been shown to form chromatinized templates withproperly positioned nucleosomes as in the case of HIV. One of themajor retroviral diseases of global concern is AIDS caused by HIV,wherein the epigenetic mechanisms regulating several steps of viralintegration and replication have been well studied. Other retroviraldiseases are indirect causes of retroviral infections as in the case ofretroviral induced cancer. Rous sarcoma virus (RSV) and mousemammary tumor viruses are two such viruses wherein the src gene isa (RSV) viral oncogene.

4.4.1. Role of acetylation in retroviral infectionThemajor challenge in the field of retroviral infections is due to the

integration of the viral genome into the host genome resulting in virallatency [149]. An actively replicating virus can be targeted by theenzyme inhibitors such as protease and reverse transcriptaseinhibitors; however, the latent viruses are difficult to eradicate.Hence, various strategies have been devised to break this latency; oneof which is increasing the acetylation status. HDAC inhibitors havebeen tried for this purpose since these can lead to indirect activationof the viral genome and once the virus is active, it can be targeted bythe inhibitors [150].

This indicates that acetylation plays an important role in the viralgene expression. The HATs p300 and PCAF acetyltransferases have




been shown to be absolutely essential for the transcription of the viralgenome especially in the case of HIV. The nucleosome organization ofthe integrated HIV genome has been well characterized. PCAF hasbeen shown to be one of the initial acetyltransferases involved for theactivation [151]. The transactivation protein Tat is a p300 substrateand is activated in an acetylation dependent manner [152]. Themultiple acetylation events are known to bring about remodelingevents which leads to the formation of the TAR (transactivationresponse element), which is bound by the Tat and p-TEFb complex.These together facilitate the recruitment of CDK9 to mediate RNApolymerase II phosphorylation thereby initiating transcription [153].Thus, targeting the Tat acetylation and the subsequent acetylationevents are attractive therapeutic strategies which will be discussed inthe last section.

4.4.2. Role of lysine methylation in retroviral infectionHistone lysine methylation is also known to play a role in

modulation of gene expression upon viral infection. Recent reportssuggest that integration site selection by HIV depends on thechromatin environment of the site. Integration is particularly favoredin regions having transcription associated histone modifications suchas H3K4 trimethylation but disfavored in regions associated withtranscription-inhibitory marks such as H3K27 trimethylation [154].One of the major hurdles in the treatment of HIV is its latency whichprevents its complete eradication. One of the mechanisms responsiblefor HIV latency involves SUV39H1, HP1 and H3K9 trimethylationwhich are reversibly associated with HIV-1 and help in themaintenance of latency [155]. Apart from silencing, Menin, acomponent of MLL1 complex, H3K4 methyltransferase is shown tobe required for HIV-1 tat transactivation [156]. These observationssuggest that targeting lysine methyltransferases to combat HIV canprovide a hope for treatment of HIV.

4.4.3. Role of arginine methylation in retroviral pathogenesisArginine methylation has both positive and negative modulatory

effects on viral pathogenesis. PRMT1 methylates the adenoviral 100 kprotein which is essential for the synthesis of late proteins and theestablishment of the viral infection [157]. PRMT1 also methylates theEpstein Barr viral EBNA1 protein which is required for the replicationand transcriptional activation of the viral genes [158]. Yet anotherviral target of PRMT1 is the small antigen of the hepatitis delta virus[159]. The methylation is required for the viral DNA replication.However, PRMT6 mediated arginine methylation is a negativeregulator of viral pathogenesis [160]. PRMT6 is the only argininemethyltransferase that methylates HIV-Tat protein and the HIVnucleocapsid. Tat methylation leads to its decreased binding to TARand therefore the viral transactivation is impaired. The methylation ofnucleocapsid reduces the initiation of reverse transcription [161].

4.5. Chromatin modifications in neurodegenerative diseases

Another important area of research that has an epigenetic link todisease is the neurodegenerative disorders. Although epigeneticregulation of gene expression in the brain has been linked to neuraldevelopment and differentiation, circadian rhythmicity, memoryformation, and synaptic plasticity, its role in neurologic disorderslike seizures and psychiatric disorders like depression and schizo-phrenia has been recently identified [162]. There are enoughconvincing evidences which suggest that chromatin modifyingmachineries have a role in the CNS disorders. Acetylation of histonesmediated by CBP is known to be vital in neuronal cell survival [163]. Afine balance between acetylation and deacetylation of histones isexpected to be pivotal in post mitotic neuron physiology [164]. Theabsence of acetylated histones is conspicuous in neurodegenerativestates like Alzheimer's, Parkinson's and Huntington's diseases.Multiple factors that affect normal function of histone acetyltrans-


ferases (especially CBP) like decreased expression, degradation, andsequestration of CBP into compartments that do not allow histoneacetylation finally lead to hypoacetylation of histones inducingneuronal apoptosis. Inhibition of histone deacetylases increases thepool of acetylated histones and hence, is an attractive approach fortreatment of neurodegenerative disorders. However, the pleotropiceffects inherent to these inhibitors, that include apoptosis induction inneurons (primarily due to lack of specificity to any one class ofHDACs), are a cause of concern [165]. Activation of enzyme functionsof CBP and p300 in the neurons could be a more specific approach anda potential alternative to the problem, thereby reducing pleotropiceffects associated with indiscriminate hyperacetylation associatedwith HDACi. In addition to diseased states, importance of histoneacetylation is also recognized now in the process of synaptic plasticityand has been extrapolated to learning and memory [162]. Yet again,CBP mediated histone acetylation is implicated but it is also knownthat p300 and PCAF HATs are essential for establishment of long termmemory [166]. Recent studies report specific increase in H2B and H4acetylation in the rat dorsal hippocampus, while spatial memory wasbeing consolidated, which was associated with a global increase inHAT activity as well as increase of CBP, p300, and PCAF expres-sion [166]. The role of acetylation in neuronal physiology andpathophysiology has been discussed extensively in another article inthis same issue (Selvi BR et al.).

Other modifications of histones like methylation and phosphory-lation have also been identified to be important for various neuronalfunctions. It was recently reported that misregulation of the H3K4 tri-demethylase SMCX, that acts as a transcriptional repressor on sodiumchannel type 2A (SCN2A) and synapsin1 promoters, results in X-linked mental retardation [167]. Another independent study has alsoshown that postnatal neuron-specific deficiency of the complexcomposed by the H3-K9 dimethyltransferases G9a/GLP results in de-repression of non-neuronal (e.g. Serpinb1b, Serpinb5) and earlyneuronal progenitor (e.g. Dach2) genes [168]. This decrease in H3K9methylation reflects on complex behavioural abnormalities, such asdefects in learning, motivation, and environmental adaptation.

5. Epigenetics as a therapeutic target

5.1. Modulators of chromatin modifying enzymes

The above examples clearly indicate the immense significance ofchromatin modifications in transcription and gene expression. It alsoillustrates the role of altered modifications in abnormal states, i.e.diseased conditions. These altered modifications are a consequence ofaltered enzyme activity of the chromatin modifying machineries.Hence, if these activities could be modulated, the alterations couldalso be minimized. There are several ways of modulating an enzymefunction but the successful examples so far implicate the use of smallmolecules which can inhibit or activate the enzymes. The field ofepigenetic modifications also is abound with such examples but themonopoly is of the DNA methylation and histone deacetylationinhibitors [169]. However, of late, several histone acetylationinhibitors have also been identified. Furthermore, protein methyla-tion inhibitors have also been identified which will be brieflydiscussed below. These inhibitors and their targets are representedin Fig. 2.

5.1.1. HAT inhibitors

5.1.1.1. Synthetic inhibitors. The very first class of inhibitors identifiedfor the HATs, p300 and PCAF were the bi-substrate inhibitors whichshowed remarkable selectivity. Lys-CoA was specific to p300 whileH3-CoA was specific for PCAF [170]. The major drawback of theseinhibitors was their lack of cell permeability. Hence, a coenzyme Aanalogue conjugated to a cell permeabilizing oligo-arginine peptide



Fig. 2. Histone modification inhibitors and their targets: the figure represents the histone modifications on the core histone substrate with the different histone acetyltransferasesand the methyltransferases which bring about the histone acetylation, histone lysine methylation and arginine methylation. Since the inhibitors enlisted are for p300, PCAF, andGCN5, these are represented in green. The available inhibitors of these enzymes and the inhibited modification residue are indicated alongside. The acetylation inhibitors are enlistedin the blue box, whereas the histone methylation inhibitors are represented in the yellow box. Briefly, HATi such as lysyl CoA, H3-CoA, γ-butyrolactones, isothiazolones, Spd-CoA,anacardic acid, curcumin, CTK7A, C646, garcinol, plumbagin, EGCG, and gallic acid inhibit different acetylation sites. The histone lysine methylation inhibitors BIX-01294 andchaeotocin inhibit H3K9 methylation. H3K27 methylation is inhibited by BIX-01294 and DZNep. The latter also inhibits H4K20 methylation. The well characterized argininemethylation inhibitors include RM-65 and AMI-1 which target the H4R3 methylation whereas TBBD is an inhibitor of H3R17 methylation.


via disulfide linkage was synthesized, which could block cellularhistone acetylation and transcription using p300 reporter assays.GCN5 specific inhibitors, γ-butyrolactones have been synthesizedbased on the catalytic site [171]. High throughput screening has led tothe identification of PCAF specific inhibitors, isothiazolones [172],which inhibit acetylation of histones in the cells in a time and dose-dependent manner. Many analogues for isothiazolones have beensynthesized raising hope for more better and specific inhibitors. Theisothiazolone functional group can easily bemodifiedwith a variety ofsubstitutions that will enhance binding to the enzyme active site.Importantly, isothiazolone core structure has been used as a startingpoint to explore PCAF inhibition [173]. HAT inhibition in vitro has beeninitially reportedwith a bi-substrate adduct, spermidine-CO-CH2-CoA(abbreviated as Spd-CoA) [174]. It is formed by joining spermidine(Spd) covalently to the S atom of coenzyme A (CoA) through athioglycolic acid linkage. Each of the two isomeric forms of Spd-CoA,linking the N1 or N8 atom of spermidine to CoA, respectively, hassubsequently been shown to be HAT inhibitors in vitro. Negatively-charged CoA moiety is considered to be a hindrance to cellularpermeability. Though there have been doubts about this issue,banking on the fact that polyamines such as spermidine are efficientlytransported across cellular membranes, it has been hypothesized thatSpd-CoA could also be internalized into whole cells and thatinternalization would lead to inhibition of HATs. In line with thehypothesis, bi-substrate HAT inhibitor, Spd(N1)-CoA, acts on cellsbringing about inhibition of histone acetylation [175]. Bi-substrateinhibitors of MYST family enzymes like Esa1 and TIP60 have also beensynthesized [176].


An elegant study from Cole P.A.'s group, reported a commerciallyavailable pyrazolone-containing small molecule; C646 as a potentp300 HAT inhibitor. Incidentally, C646 is a competitive p300 inhibitorwith low micromolar inhibitory constant [177]. However, naturalproducts having HAT inhibitory properties have drawn attention andraised expectations of having better cell permeability and moreselectivity for HATs, which are summarized below.

5.1.1.2. Natural products. Anacardic acid (AA) is a simple salicylic acidderivative (nonadecyl salicylic acid), purified fromcashewnut shell liquid.It is a potent inhibitor of p300 and p300/CBP-associated factor histoneacetyltransferase activities [178]. Anacardic acid non-specifically inhibitsboth p300 and PCAFwith an IC50 less than 10 μM. Since anacardic acid is anon-specific inhibitor, the search for specific inhibitors led to theidentification of the polyphenolic compound from Curcuma longarhizome, curcumin, as a p300 specific HAT inhibitor and does not inhibitPCAF [179].However, curcuminhas its limitations inpossessingpleotropiceffects [180]. Recently, a water soluble curcumin derivative, CTK7A hasbeen synthesizedwhich has a broad spectrumHAT inhibitory property ascompared to curcumin [88]. A naturally occurring polyisoprenylatedbenzophenone, garcinol, isolated from Garcinia indica or kokum fruit (indigenous to southern parts of India) as a potent inhibitor of both PCAFand p300 [181]. IC50 of garcinol for p300 acetyltransferase activity was7 μMwhile it was 5 μMfor PCAF. However, by virtue of its non-specificity,garcinol is found to be immensely cytotoxic when treated to HeLa cells. Itcaused apoptosis of HeLa cells and caused downregulation of majority ofgenes. The potential use of garcinol to target p300 for understanding thephysiological role of HATs or to develop a therapeutic molecule is less


image of Fig.�2



likely due to the same. The limitations of garcinol necessitated a search fornon-toxic but specific derivative with garcinol as a leadmolecule. Amongnearly 50 derivatives from garcinol, three have been found to be highlyspecific for HAT activity of p300 [182]. Isogarcinol (IG) is a product ofintramolecular cyclisation of garcinol. Controlled modification andmonosubstitution at “14” position yields 14-isopropoxy IG (LTK-13) and14-methoxy IG (LTK-14). Di-substitution of Isogarcinol resulted in 13, 14disulfoxy IG (LTK-19). The mono substituted IG derivatives LTK-13, -14,and di-substituted LTK-19 specifically inhibit the p300 HAT activity butnot the PCAF HAT activity. Selective nature of one such derivative LTK-14hasbeenused inunderstanding the regulationof gene expressionbyp300HAT. The mechanism behind LTK-14 mediated HAT inhibition has alsobeen elucidated [183]. Plumbagin has been identified as yet anothernaturally occurring p300 specific HAT inhibitor [184]. Plumbagin is ahydroxynaphthoquinone isolated from the roots of Plumbago rosea(known in Indian ayurvedic medicine as Chitraka). Most lysineacetyltransferase inhibitors possess poly-hydroxy functional groups. Therole of these poly-hydroxy functional groups in HAT inhibition propertywas unknown. Plumbagin has a single hydroxyl group. The substitution ofthis group with other moieties results in a complete loss of the p300inhibitory activity. Thus it is now known for the first time that such achemical entity (-hydroxyl; functional group) is responsible for p300inhibition. EGCG, a major phenol in green tea also adds to the list ofgrowing naturally occurring HAT inhibitors [185].

5.1.2. Lysine methyltransferase inhibitorsLysine methyltransferases represent one of the valid targets for

discovery of small molecule inhibitors in what is called as ‘epigenetictherapy’. These chemical inhibitors will be instrumental not only intargeted therapy of various cancers but also for testing the variousmodels proposed to describe the role of these enzymes in chromatinstructure. As of now, analogues of cofactor SAM namely Sinefungin,S-Adenosyl homocysteine, Methylthio-adenosine and adenosinedialdehyde have been used, but the main disadvantage of usingthese is lack of specificity, as SAM is also a cofactor for DNAmethyltransferases (DNMTs), protein arginine methyltransferases(PRMTs), O, and N-methyltransferases.

Few specific inhibitors for these HMTases have been recentlydiscovered. A fungal derived toxin, chaeotocin was found to inhibit Su(var)3–9, a Drosophila melanogasterH3K9methyltransferase. It showedIC50 of 0.6 μM and acted as a competitive inhibitor for SAM. In fact, athigher concentrations chaeotocin was also found to inhibit human Su(var)3–9 in vivo, but at the same time revealed cytotoxic effects [186].HMTs that do not belong to the SU(VAR)3–9 family such as E(Z), PRSET7or SET7/9, are not inhibited, despite having recognizable SET domain.BIX-01294, another small molecule inhibitor of euchromatic histonemethyltransferase G9a discovered from screening of chemical librarycomprising 125,000 compounds, was shown to reverse H3K9 dimethy-lation marks in mouse ES cells and fibroblasts, but did not affect monoand trimethylation levels. In vitro, it showed IC50 of 1.7 μMand inhibitedG9a in a non-competitive manner [187]. The same study also identifieddual inhibitor of lysine and arginine methyltransferase, BIX-01338.DZNep, 3-deazoneplanocin A was reported to inhibit trimethylation ofH3K27 and H4K20, and shown to reactivate silenced genes in cancercells [188].

5.1.3. Arginine methyltransferase inhibitorsThe field of arginine methylation inhibitor is relatively less

populated as compared to the other inhibitors. One of the firstinhibitors identified was way back in 1986, Sinefungin which wasshown to inhibit the arginine methyltransferase activity associatedwith myelin basic protein methylation [189]. However, not muchprogress has occurred since then. Many SAM and SAH analogues havebeen tested, but since this is the commonmethyl cofactor donor for allthe methylation reactions, there is no specificity observed. A properlydesigned small molecule screen identified AMI-1 as a potent arginine


methylation specific inhibitor with absolutely no effect on the lysinemethyltransferases [190]. Subsequently, the carboxy analogues ofAMI-1 were also found to inhibit the arginine methyltransferases[191]. Yet another PRMT1 substrate with very low micromolar IC50value is RM-65 [192], however no further development has occurredin this area. Several docking based approaches have been used toidentify potential inhibitors but a lot needs to be done. One of theintriguing yet novel inhibitor is ellagic acid (TBBD) which has beenfound to be specific to CARM1/PRMT4 activity both in vitro and in vivo.This inhibitor binds to the enzyme–substrate complex based on thesubstrate sequence and thereby inhibits the enzymemodification on asingle site alone [193]. This has not only led to the identification of anovel mechanism of enzyme inhibition but also has provided a tool tomonitor the single residue, H3R17 methylation specifically. Byemploying a similar approach, a specific inhibitor of PRMT1 hasbeen identified wherein the inhibition of enzyme function is broughtabout by binding to the substrate [194]. The increasing evidence ofarginine methylation in several important cellular processes andthereby in diseases necessitates the immediate action towardsidentifying more modulators of arginine methylation [195].

5.2. Epigenetic therapy

The concept of epigenetics and epigenetic modifications hasundergone a tremendous change in the past few decades. It is nolonger considered as simple covalent modifications facilitatingopening and closing of chromatin. Rather the modifications are nowconsidered as platforms that help in recruiting complexes, as signalsthat facilitate the entire orchestration of gene expression. In fact,along with the genome, there is a hypothesis that an epigenome alsoexists. Since these modifications are so essential and the deregulationof the enzymes responsible for thesemodifications have been causallyrelated to several diseases as described here, the modulators of thesemodifying enzymes have gained immense significance. Epigenetictherapy using these modulators has also gained an impetus asobserved by the increasing number of DNAmethylation inhibitors andhistone deacetylase inhibitors going into clinical trials. The followingsectionwould highlight the different inhibitors known for acetylation,lysine methylation and arginine methylation and would provide aninsight into the possible therapeutic potential of these inhibitors.

5.2.1. HAT inhibitors and possible therapeutic applicationAnacardic acid potentiates apoptosis induced by cytokine TNF-α and

chemotherapeutic agents like cisplatin, doxorubicin, etc. [196]. Thisincrease in apoptosis correlates with downregulation of various geneproducts that mediate proliferation (cyclin D1 and cyclooxygenase-2),survival (Bcl-2, Bcl-xL, cFLIP, cIAP-1, and survivin), invasion (matrixmetalloproteinase-9 and intercellular adhesion molecule-1), andangiogenesis (vascular endothelial growth factor). Anacardic acidinhibits both inducible and constitutive NF-κB activation. Importantlyanacardic acid has been shown to inhibit TNF mediated p65 acetylationand nuclear translocation of p65. Downregulation of p300 by RNAiabrogated the effect of anacardic acid on NF-κB suppression, substan-tiating the fact that p300 is a critical enzyme for NF-κB mediated geneexpression. Interestingly, anacardic acid also inhibits the activity ofTIP60, a MYST family acetyltransferase and sensitizes tumor cells toionizing radiation [197]. Starting from the natural product lead HATinhibitor anacardic acid, a series of 28 analogues have been synthesized[198]. The compounds inhibited up to 95% HAT activity in vitro. There isalso a clear correlation between their inhibitory potency and cytotoxi-city towards a broad variety of cancer cells. It is interesting to note thatall the compounds were relatively non-toxic to nonmalignant humancell lines, thus promising newer and better HAT inhibitors.

Curcumin (diferuloylmethane), a major curcuminoid in the spiceturmeric, is a specific inhibitor of the p300/CBP histone acetyltrans-ferase (HAT) activity but not of PCAF, in vitro and in vivo. It could




inhibit the acetylation of HIV-Tat protein in vitro by p300 as well asproliferation of the virus, as revealed by the repression in syncytiaformation upon curcumin treatment in SupT1 cells [179]. Thus non-toxic curcumin, which targets p300/CBP HAT activity, may serve as alead compound in combinatorial HIV therapeutics. Curcumin has alsobeen used to prevent and reverse cardiac hypertrophy and heartfailure which depends on its HAT inhibition. Curcumin is a potentinhibitor of NF-κB pathway [199]. Since curcumin has manypleotropic effects, derivatization is essential to realize the truepotential of its inhibitory scaffold. This has been possible with awater soluble curcumin derivative, CTK7A which leads to senescencelike growth arrest in oral cancer cells. Furthermore, CTK7A treatmentto oral cancer xenografted nude mice exhibited a reduction in thetumor size concomitant with a reduction in histone acetylation thusshowing a potent anti-cancer activity [88].

The naturally occurring polyisoprenylated benzophenone garcinolinhibits non-specifically both PCAF and p300. Garcinol inhibitshistone acetylation in cells even when cells are pretreated with theHDAC inhibitors like Trichostatin or sodium butyrate [181]. Garcinolinduces apoptosis and predominantly downregulates global geneexpression in HeLa cells. In an effort to reduce the toxic effects andincrease specificity towards p300, various molecules with alteredstructural group from the parent molecule garcinol have beenderivatized. Garcinol and isogarcinol inhibit p300 and PCAF andexhibit high cytotoxicity. However the derivative LTK-14 inhibitsspecifically p300 and not PCAF in the concentration range studied.LTK-14 is non-toxic to T cells. Very importantly it could inhibit viralsyncytia formation in HIV-infected cells and also inhibit themultiplication of HIV [182]. Plumbagin, isolated from Plumbagorosea root extract, inhibits histone acetyltransferase activity potentlyin vivo [184]. Plumbagin is a potent inhibitor of NF-κB pathway and itsdownstream gene products [200]. It is expected that one of themechanisms of NF-κB inhibition would involve plumbagin mediatedp300 HAT inhibition and thereby hypoacetyation of p65, among othermechanisms. The newly identified p300 specific inhibitor C646 hasalso shown anti-proliferative activity on cancer cell lines implicating apossible anti-cancer property [177].

Epigallocatechin-3-gallate (EGCG) is a major polyphenol found ingreen tea. It is reported to have an anti-NF-κB transactivation activityin a broad range of cancers of the colon, lung, and breast, and also inchronic inflammation [201]. EGCG is a novel HAT inhibitor with globalspecificity for the majority of HAT enzymes. Interestingly it has noactivity towards other epigenetic enzymes including HDAC, SIRT1,and HMTase, but has been shown to be an inhibitor of DNAmethyltransferase [202]. EGCG abrogates p300-induced p65 acetyla-tion in vitro and in vivo, increases the level of cytosolic IκBα, andsuppresses TNF-α induced NF-κB activation [202]. EGCG also preventsTNF-α induced p65 translocation to the nucleus. This highlights theimportance of the balance between HATs and HDACs in the NF-κB-mediated inflammatory signaling pathway. These results, in line withother HAT inhibitors, repeatedly highlight, the crucial role ofacetylation of histones, non-histone proteins and transcription factorsin the development of inflammation related diseases.

S-Substituted coenzyme A (CoA) is an inhibitor of histone acetyla-tion. While exposure of cancer cells to Spd-CoA causes inhibition ofhistone acetylation that correlates with a transient arrest of DNAsynthesis, that is linked to a transient delay in S-phase progression, andan inhibition of nucleotide excision repair and DNA double strand breakrepair [175]. Treatment of this molecule to cancer cells renders themmore susceptible toDNA-targeted chemotherapeutic drugs like cisplatinand5-fluorouracil, to theDNAdamagingdrug, camptothecin, and also toUV-C irradiation. This sensitization effect of Spd-CoA is not observed innormal cells due to a barrier tomolecule uptake. The truncated Spd-CoAderivative displays similar but enhanced chemosensitization effects.Thus, it is evident that theHAT inhibitors could be promising anti-canceragents, anti-inflammatory agents as well as anti-viral agents which can


be used in the HAART therapy for AIDS. Few of the results have beenvalidated in experimental model systems but a lot more has to be donebefore these inhibitors could be used for therapeutic purposes.

5.2.2. Lysine methylation inhibitors and possible therapeutic applicationThe known lysine methylation inhibitors are few and their use for

therapeutic interventions fewer. An isolated study has shown theanti-HIV activity of DZNep in a cellular system as evidenced by adecrease in syncytia formation; however the mechanism of action isnot clear. In another independent study, this SAH inhibitor was shownto possess anti-cancer activity on breast cancer cells with relatively noeffect on normal cells. In fact, it was shown to activate certain PRC2repressed genes thus exhibiting its anti-cancer activity. Another studyusing the SUVar inhibitor chaetocin has shown that it possesses anti-tumor activity on IL-6 dependent and independent myeloma cells by aprocess distinctly different from doxorubicin and dexamethasone.There seems to be an accumulation of chaetocin in the myeloma cellswhich finally substantiates the cytotoxicity. Although not much workhas been done, all the initial data support the possibility of the lysinemethylation inhibitors to be used for therapeutic purposes [203].

5.2.3. Arginine methylation inhibitors and possible therapeutic applicationEllagic acid (TBBD) is the only arginine methylation inhibitor

(CARM1 inhibitor), which has shown some prognostic value. It wasconsidered to be an anti-cancer agent against hormone receptorassociated cancers such as breast cancer and prostate cancer.However, the molecular target for its activity was not known. It hasbeen identified as an inhibitor of CARM1 activity and hence demandsan investigation into its anti-cancer activity in a more systematiccontext in the above mentioned cancers [97–99]. Apart from cancer,the role of asymmetric arginine methylation in cardiovasculardisorders is well recognized. The discovery of the type I PRMTinhibitors indicates a possible therapeutic potential against thecardiovascular disorders. Furthermore, the observation that thearginine methylation of Tat negatively regulates the viral healthimplicates a possible role for any activator of PRMT6. Speculations aremany but the available data is less to conclude that these inhibitorsare good therapeutic agents. However, a more detailed and systematicanalysis can change this concept such that the arginine methylationinhibitors too will be front runners in the field of epigenetic therapy.

6. Future perspectives

Epigenetic therapy as evidenced by the different studies seems tohold immense promise as a new generation therapeutic strategyespecially for diseases such as cancer and inflammation associateddisorders. Although this is gladdening, the broad spectrum action ofthese modulators also rings a warning bell. Thus, a ‘System biology’approach is required to completely understand the effect of thesemodulators on the pathophysiological conditions. The DNA methyl-ation inhibitors and the histone deacetylase inhibitors are in clinicaltrials with a few of them acquiring FDA approval as well. The currentwell established epigenetic therapeutic agents are the DNA methyl-ation and histone deacetylase inhibitors as depicted in Table 3(adapted from [169]) with histone methyltransferase inhibitors asfuture epigenetic modulators.

An overall view of the field of epigenetics and disease clarifies theconcept that these are not individual alterations that finally lead todisease; rather these are multiple events operational at multiple stagesleading to the abnormal pathophysiological state, implicating the needfor combinatorial approach for therapeutic intervention. Combinatorialapproach essentially involves the use of present therapeutic moleculesalong with newly discovered molecules targeting epigenetic modifica-tions. A phase I/II study of the combination of decitabine and valproicacid in patientswith leukemia has shown that valproic acid can be safelyadministered up to doses of 50 mg/kg orally for 10 days in combination



Table 3Current status of epigenetic therapy.

Inhibitor Effective against Current status

DNMTi1) 5-Azacytidine MDS, solid tumors, leukemia FDA approved phase II2) Decitabine MDS Phase II

Leukemia Preclinical3) Zebularine Urinary bladder cancer Preclinical4) Procainamide Prostate cancer Preclinical5) Procaine Breast cancer Preclinical6) EGCG Photocarcinogenesis,

cervical cancerPreclinical

7) DNMT1 antisense oligo Solid tumors Phase I

HDACi1) Trichostatin A Breast cancer, ovarian cancer Preclinical2) SAHA Solid tumors, leukemias Phase I/II3) Depsipeptide Leukemias Phase I/II

Melanoma/colon cancer Preclinical4) Apicidin Leukemia Preclinical5) Valproate Breast and ovarian cancer Preclinical6) Phenyl butyrate MDS, leukemia Phase I7) MS-275 Solid tumors Phase I8) CI-994 Solid tumors Phase I9) Trifluoromethyl ketones Cancer Preclinical10) α-Ketoamides Cancer Preclinical


with decitabine, and that this combination has significant activity inpatients with relapsed/refractory AML andMDS. In a phase II study, thedemethylating hydralazine plus the HDAC inhibitor magnesiumvalproate was tested along with neoadjuvant doxorubicin and cyclo-phosphamide in locally advancedbreast cancer to assess their safety andbiological efficacy [204]. Another advantage of targeting epigeneticchanges is that they are reversible. Thus, understanding the epigeneticchanges associated with changes from normal to diseased phenotypeand targeting them specifically will certainly help to treat such diseases.For example, reversing the epigenetic silencing of tumor suppressorgenes in cancer using specific drugsmayhelp in the reactivation of thesegenes. Understanding the role of cancer stem cells and their growth inthe context of cancer is also important especially the epigeneticsignaling underlying their development.

One of the major challenges of epigenetic therapy is their non-specificity. Thus, understanding the molecular targets of such smallmolecules and their pleotropic effectswill be one of the immediate goalsof thisfield.Most importantly, targeteddelivery of these smallmoleculeswith the help of nanotechnology is required, since the diseases likecancer usually begin as localized abnormalities which later metastasize.Considering the increase in the new age diseases and the understandingbehind themechanismof these diseases,what is essential is to culminatethe field of chemical biology, nanotechnology, and medical biotechnol-ogy to discover, design and deliver smallmolecules targeted towards thecausative mechanisms of gene expression alteration, thereby makingepigenetic therapy a part of mainstream medicine.

Acknowledgements

The work done in the authors' laboratory was supported byJawaharlal Nehru Centre for Advanced Scientific Research and theDepartment for Biotechnology, Government of India. RS and YOacknowledge the Council of Scientific and Industrial Research for SeniorResearch Fellowship and Junior Research Fellowship respectively.

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