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1 BBAGRM-00286; No. of pages: 19; 4C: 8, 12 Biochimica et Biophysica Acta xxx (2010) xxx xxx Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: 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 , India article info abstract Article history: Received 18 May 2010 Received in revised form 18 September 2010 Accepted 24 September 2010 Available online xxxx Keywords: Histone acetylation Lysine methylation Epigenetics Arginine methylation Inflammation Modulator Chromatin modifications have gained immense significance in the past few decades as key regulators of gene expression. 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 chromatin function. A concerted action of different modifying enzymes catalyzes these modifications, which are read by effector modules and converted to functional outcomes by various protein complexes. Several small molecules in the physiological system such as acetyl CoA, NAD +, and ATP are actively involved in regulating these functional outcomes. Recent understanding in the field of epigenetics indicate the possibility of the existence of a network, the epigenetic language involving cross talk among different modifications that could regulate cellular processes like transcription, replication and repair. Hence, these modifications are essential for the cellular homeostasis, and any alteration in this balance leads to a pathophysiological condition or disease manifestation. Therefore, it is becoming more evident that modulators of these modifying enzymes could be an attractive therapeutic strategy, popularly referred to as Epigenetic therapy. Although this field is currently monopolized by DNA methylation and histone deacetylase inhibitors, this review highlights the modulators of the other modifications namely histone acetylation, lysine methylation and arginine methylation and argues in favor of their therapeutic potential Published by Elsevier B.V. 1. Introduction The nucleoprotein complex of DNA wrapped around the histones, non-histone proteins and small RNAs is referred to as chromatin of which the nucleosome is the fundamental unit. Each nucleosome is an octamer of four core histones namely H3, H4, H2A, and H2B around which DNA is wrapped and histone H1 is involved in chromatin packaging [1,2]. The histones undergo various post-translational modifications. Around 200 post-translational modifications of histones have been reported occurring at approximately 60 different sites most of which are clustered in their unstructured N-terminal tails [3,4].Thesemodifications provide the cell with an array of regulatory options for the DNA dependent processes such as transcription, replication, repair, etc. [4]. The current understanding of the role of chromatin and its modifications during transcription dictates the spatial and temporal orderly appearance and disappearance of histone modifications along the gene so as to regulate the entire process of transcription [5]. These histone modifications not only serve as regulatory switches along the entire process of transcription but also form combinatorial patterns that give rise to a Language that leads to distinct gene expression patterns. Several other factors such as DNA methylation, small nuclear RNAs, Corresponding author. address: tapas@jncasr.ac.in (T.K. Kundu). histone variants also work in conjunction with these histone modifications to form a robust regulatory network which is self-propagating in nature and hence is also termed as epigenetic network. One distinct feature of this epigenetic language is a mechanism in which modifications initiate transcription activation and reinstate repression [6]. This orderly signaling leaves behind the transcriptional memory in the form of memory marks and establish a particular chromatin state [7]. Thus, chromatin modifications regulate the transcriptional competence of chromatin and thereby influence gene expression. This review will focus not just on the role of chromatin modifications on transcription regulation but will also highlight their role in several disease manifestations and thereby would set the stage for the possibility of epigenetic therapy. Several modifications are known, but the review would essentially revolve around lysine acetylation, lysine methylation and arginine methylation. The mechanism of each of these modifications in normal physiological and abnormal pathophysiological conditions will be discussed along with an insight into the physiologically relevant small molecules that modulate these enzyme function as well as the small molecule inhibitors of these enzymes. The final section sheds light on the field of epigenetic therapy, its past, present and future Chromatin modifications: enzyme machineries, substrates and outcome Modifications that play an important role in transcription or influence the transcriptional outcome include acetylation, methylation, /$ see front matter 2010 Published by Elsevier B.V. doi: /j.bbagrm

2 2 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx phosphorylation, ubiquitination, etc. The histone modifications exhibit some level of specificity with respect to the state of the chromatin. Certain histone modifications are exclusively associated with particular chromatin state, form global chromatin environments and divide the entire genome into euchromatic and heterochromatic regions Chromatin acetylation Chromatin acetylation is a reversible process catalyzed by the histone/lysine acetyltransferases (HATs/KATs) and deacetylases (HDACs/KDACs) utilizing the cofactor acetyl CoA. These proteins help in maintaining a balance of the steady state acetylation levels thereby establishing cellular homeostasis [8]. The acetyltransferases are mainly classified on the basis of their cellular localization as nuclear HATs (type A HATs) or cytoplasmic HATs (type B HATs) and sub-classified based on their structural and functional differences into five families [9]. These are the GNAT family members represented by GCN5, PCAF, the second family dominated by the p300/cbp enzymes. The major family is the MYST family comprising of MOZ, MOF, and TIP60 with distinct functional outcomes. Yet another important class of 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 and HAT2 involved in acetylation of newly synthesized histones. Other proteins with acetyltransferase activity which do not belong to any of these families have also been reported. There are 18 HDACs in humans and they are classified into three main groups based on their homology to yeast proteins [8]. Class I includes HDAC1, HDAC2, HDAC3, and HDAC8 and have homology to yeast RPD3. HDAC4, HDAC5, HDAC7, and HDAC9 belong to class II and have homology to yeast HDA1. HDAC6 and HDAC10 contain two catalytic sites and are classified as class IIa, whereas HDAC11 has conserved residues in its catalytic center that are shared by both class I and class II deacetylases and is sometimes placed in class IV. The class III HDACs include sirtuins, have homology to yeast Sir2, and have an absolute requirement for NAD +. They do not contain zinc in the catalytic site and are not inhibited by compounds like TSA or vorinostat. The mechanism of acetylation and its functional significance have been extensively reviewed in few other chapters in this issue. The acetyltransferases and deacetylases are involved in the acetylation 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 of many transcription factors and non-histone chromatin associated proteins thus regulating gene expression [10,11] Chromatin methylation Chromatin methylation is brought about by the methyltransferases which transfer the methyl group from the methyl cofactor donor S-Adenosyl Methionine (SAM), onto either cytosine residue of DNA (DNA methylation), to lysine residue of histones and nonhistone proteins (lysine methylation) or to arginine residue in histones and non-histone proteins (arginine methylation). Thus, based on the residue modified, the enzymes are referred to as DNA methyltransferases (DNMTs), lysine methyltransferases (KMTs) or arginine methyltransferases (PRMTs). Although the DNA methylation is an important modification with well established role in transcriptional repression and gene silencing [12], we would be concentrating more on the protein modifications, lysine and arginine methylation Lysine methylation. Lysine methylation of histones is catalyzed by enzymes known as lysine methyltransferases, which transfer methyl group from S-Adenosyl Methionine (SAM) to lysine residues of proteins. All the lysine methyltransferases, with few exceptions (DOT1) possess a unique SET domain (Suppressors of variegation Enhancers of zeste and Tristae), which is sufficient for their enzymatic activity. Human genome encodes 73 SET domain containing proteins, many of which have been assigned the enzymatic activity. Lysine methylation of histones occurs on H3 (K4, K9, K27, K36, K79), H4 (K20), H2B (K5), H1 (K26), etc. Unlike other modifications, which are exclusively associated with either activation or repression, methylation is associated with both, depending on the residues that are modified [13]. The first methyltransferase to be discovered was the H3K9 methyltransferase, SUV39 which belongs to the SUV39 family [14]. Although SET domain is sufficient for its enzymatic activity, PRE-SET and POST-SET domains are required for their specificity. This family includes SUV39H1/H2, GLP, G9a, ESET, and CLLL8 proteins. SUV4 20H1 and SUV4 20H2 are members of SUV39 related family and are known to catalyze H4K20 trimethylation [13 15]. These proteins are recruited to the chromatin via HP1 proteins. Yeast has single H3K4methyltransferase SET1, whereas in mammals, at least 10 known or predicted H3K4 methyltransferases exist, which include six MLL complexes, SET1A, SET1B, ASH1, SET7/9, SMYD3, and Meisetz. These enzymes are not functionally redundant and this might be due to their differential expression, differential recruitment, distinct sets of non-histone substrates, etc. MLL proteins exist as complexes and share 3 subunits among them namely, WDR5, RbPB5 and ASH2 [16]. Among all proteins in this family, methyltransferase activity has been confirmed for MLL1 4, SET1A, SMYD3, SET7/9 and Meisetz, whereas it is predicted for SET1B and ASH1. MLL1 4 and SET1A show H3K4 mono, di and trimethylation activity. SMYD3 display di and trimethylation activity, which is enhanced by HSP90A. SET7/9 not only shows H3K4 monomethylation activity but also methylates nonhistone substrates such as TAF10 and p53. Mouse Meisetz is specifically known to trimethylate H3K4 during meiosis [16]. SET2/NSD family proteins exhibit methyltransferase activity, but their specificity depends on the nature of the substrate, the major site in the nucleosomal context being H3K36. The other sites are H3K4, H3K27 and H4K20. This family consists of three proteins NSD1, NSD2 and NSD3 all of which are homologous to the yeast SET 2 which interacts with elongating RNA polymerase II and displays cotranscriptional H3K36 trimethyltransferase activity [17]. Another important family of histone lysine methyltransferases belongs to the polycomb group of proteins discovered in Drosophila melanogaster catalyze H3K27 trimethylation required for Hox gene repression. Human homologue of this protein, EED-EZ (H)2 complex, comprises of four proteins namely, SUZ12, EED, RbAp48 and AEBP2. It functions in the silencing of developmentally regulated genes and differentiation in embryonic stem cells [18]. RIZ (retinoblastoma-interacting zinc finger) family includes three proteins namely RIZ, BLIMP and PFM1. They all have a SET domain towards amino terminus, lack PREand POST-SET domains and possess a number of zinc finger motifs in the C-terminus. RIZ protein can bind to estrogen receptor and stimulate its activity. It catalyzes H3K9 methylation thereby playing a role in transcription repression. BLIMP is also a transcriptional repressor but very little is known about PFM1 [13]. Non-SET domain containing lysine methyltransferases includes DOT1 and its homologues [19]. Human DOT1L (disruptor of telomere silencing) catalyzes H3K79 methylation which is localized in the coding regions of actively transcribing genes and telomeres. This residue resides in the globular domain of H3 but is located in the accessible surface of the nucleosomes. Loss of this mark causes loss of telomeric silencing. It presumably acts by restricting Sir proteins to chromatin [20]. Thus, lysine methylation can be associated with either activation (H3K4 trimethylation for transcription initiation, H3K36 methylation for transcriptional elongation, and H3K79 methylation) or with repression (H3K9 dimethylation and H3K9 trimethylation). These different modifications (different sites and different degrees), lead to specific protein complex interactions and thereby influence the chromatin function globally. This modification was considered to be

3 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx 3 a stable mark for a long time but the discovery of lysine specific demethylase (LSD1) in 2006 changed the entire outlook of the field [21]. LSD1 was initially thought to be a demethylase of H3K9 methylation but was subsequently also found to act on H3K4 methylation in a context dependent manner [22,23]. This discovery sparked off the search for demethylases for other modifications. Several jumonji domain containing proteins have been identified with demethylating activities [24]. Recent evidences also suggest the role of these demethylases in processes like stem cell renewal; stem cell differentiation, etc., indicating a major role of histone modifications in the early developmental stages [25] Arginine methylation. Protein arginine methylation was identified almost five decades ago [26]. Protein arginine methyltransferases (PRMTs) catalyze the addition of methyl group from the methyl cofactor, SAM to the guanidino-nitrogen of the arginine residue on proteins. There are 9 PRMTs identified so far, classified into two groups based on symmetric and asymmetric arginine dimethylation; PRMT5/JBP1, PRMT7 and PRMT9 are known to bring about symmetric modification which leads to transcriptional repression. In contrast, all the other PRMTs, PRMT1 4 and PRMT6, 8 are asymmetric dimethyl arginine methyltransferases resulting in transcriptional activation. 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 monomethylated, asymmetric dimethylated or symmetric dimethylated arginine residues in mammalian cells. The well 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 have also been mapped to three different genes. The PRMTs are characterized by a characteristic motif consisting of seven β-strands as well as the PRMT family specific double E and THW sequence motifs [28]. Two proteins FBXO10 and FBXO11 which lack the characteristic PRMT motifs (described above), have been proposed to have methyltransferase activity [29]. PRMT1 is responsible for 80% methylation of all protein methylation in the cellular system. PRMT1 and other PRMTs except PRMT4 recognize glycine arginine rich motifs for methylation whereas PRMT4 recognizes XXPRXX motif for methylation. The arginine methyltransferases also have other non-histone protein substrates. All these modifications result in various functional processes like RNA processing, DNA damage and repair, cell signaling and most importantly in transcription [27]. In the case of RNA processing, the RNA splicing machinery is known to be regulated by arginine methylation. So far, the identified histone arginine methyltransferases are PRMT1 that methylates histone H4R3 [30], PRMT6 that methylates H3R2 [31], and PRMT4/CARM1 which catalyzes H3R17 and R26 in vitro as well as in vivo [32]. PRMT5 and PRMT1 share the same modification site, histone H4R3, however based on the symmetric or asymmetric modification, the transcriptional state differs. PRMT5 is a well characterized type II arginine methyltransferase, which has been shown to exist in repressor complexes containing DNA methylation machinery and NURD complex [33]. One of the major unanswered questions in the field of PRMT biology is the extent of modulation of the enzyme specificity and function by the various interacting proteins. There are a few indications to prove that this might be an important level of regulation of arginine methyltransferase activity since these proteins in spite of ubiquitous expression, have very specific activities. Arginine methylation is also a partly reversible modification wherein two distinct mechanisms have been shown to be operational in removal of the modification. The first mechanism is an indirect reversal mechanism involving the deimination reaction by peptidyl arginine deiminase PAD4, which converts monomethyl arginine to citrulline [34]. Since this is an intermediary form, the complete demethylation has not been shown for this case. However, a jumonji domain containing protein JMJD6 has been shown to be a bona-fide arginine demethylase [35]. Thus, although it is clear that the field of arginine demethylation is still in its infancy, there are indications to suggest that this reaction might also have physiological relevance, since non-histone proteins like p53 and NPM1 have been shown to have the citrulline modification [36,37]. In comparison to the research on acetylation and lysine methylation, arginine methylation is still an unexplored modification. Unlike the former two modifications whose reader modules and effector domains have been well characterized, such effector proteins have not yet been discovered for arginine methylation. Due to this lack of information, it has been difficult to assign a sequential and mechanistic insight for the role of arginine methylation in transcription. However, singularly these modifications have been studied with respect to transcription regulation, which will be discussed in the subsequent section. 2. Chromatin modifications and transcription regulation The histone modifications exhibit a localized pattern across promoters and open reading frames and thereby play important roles in the process of transcription as depicted in Table 1 (adapted from [205]). The specific modifications with distinct functions are briefly described below Role of chromatin modifications in transcription regulation Histone acetylation at various lysine residues in promoter regions promotes transcription through various mechanisms such as histone Table 1 Histone modifications, enzyme machineries and functional outcome. Modification site Enzyme Function Acetylation H2AK5 TIP60, p300/cbp, Hat1 Transcriptional activation H3K9 SRC1 Receptor Signaling PCAF, p300/cbp Transcriptional activation H3K14 TAFII250, p300/cbp, PCAF, TIP60 Transcriptional activation H3K18 P300/CBP, PCAF Transcription activation H3K23 CBP Transcription activation H3K56 p300/cbp Transcription activation H4K5 Hat1, HBO1, Histone deposition p300/cbp, ATF2, TIP60 Transcription activation H4K8 HBO1, ATF2, TIP60, PCAF Transcription activation H4K12 p300/cbp Hat1, HBO1 Transcription activation H4K16 p300/cbp, PCAF, TIP60, Mof, ATF2 Chromatin structure and Transcription activation Lysine methylation H3K4 MLL1 6 Trithorax activation SET7 9 H3K9 G9a, EuHMTase1, ESET Transcriptional repression SUV39H1 Rb mediated silencing Ash1 Trithorax activation RIZ1 Tumor repression H3K27 EZH2 Early B-cell development, X-chromosome inactivation H3K36 SET2, NSD1, SYMD2 Transcriptional activation H3K79 DOT1 Transcriptional activation H4K20 Pr-SET 7/8 Transcriptional silencing, mitotic condensation Arginine methylation H3R2 CARM1, PRMT2 Gene expression H3R8 PRMT5 Transcriptional repression H3R17 CARM1 Transcriptional activation H3R26 CARM1 H4R3 PRMT1 Transcriptional activation H4R3 PRMT5 Transcriptional repression

4 4 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx removal, general transcription factor recruitment, chromatin remodeling, etc. [38,39]. Histone lysine (K4) trimethylation is enriched in the 5 ORFs of active genes [40]. In fact, this modification mediates transcriptional activation via multiple pathways. It not only leads to the disruption of repressor complexes (NuRD complex), but is also recognized by Royal family domains and PHD family domains present in various chromatin modifying enzymes and remodeling complexes [41 43]. H3K4 trimethylation is considered to be a determinant of the transcriptional competence of chromatin and is also correlated with enhanced acetylation kinetics. Modifications such as H2B ubiquitination and H3K79 trimethylation are required in promoter regions as well as coding regions of genes for transcription to occur [44,45]. H3K36 trimethylation is another important polymerase dependent elongation mark specifically present in the 3 ORFs of active genes [46]. H3S10 phosphorylation and H4K16 acetylation have been shown to have an important role in the elongation process [47]. In fact, phosphorylation of H3Y41 has been shown to exclude HP1 from chromatin [48]. Apart from these, many other modifications are enriched in promoter regions and ORFs of active genes whose roles remain elusive. Transcription activation is an active process and for successful transcription to occur, many regulatory checkpoints need to be crossed. There exists a unique language of these chromatin marks, which is associated with transcription repression. DNA hypermethylation has already been shown to be associated with repressed chromatin states. Di and trimethylation of H3K9, trimethylation of H3K27 and H4K20 is also associated with transcriptional silencing and heterochromatinization Role of acetylation in chromatin transcription Acetylation occurs at multiple lysine residues and each of this modification has distinct functional consequences associated with active gene transcription and thus is a mark of euchromatin. This modification results in a change in the electrostatic charge of histones which alters the structural properties of histone and its binding to the DNA. It assists in loosening of inter- or intra-nucleosomal DNA histone interactions. Also, modifications create binding surfaces for protein recognition modules and thus recruit specific functional complexes to their proper sites of action. Considering the repertoire of modifications possible on the histones it is clear that the combination of events are immense and not just an acetylation mediated switch on or switch off of gene transcription. Each of these modifications can be read by other proteins that would then influence chromatin dynamicity. Indeed, protein domains like the bromodomain, present in ATP dependent remodeling complexes like Swi/Snf recognize the acetylated lysine and assist in binding of the complex to acetylated chromatin activating nucleosome remodeling and transcription [49]. The switch to a repressed chromatin involves deacetylation and compaction of the chromatin fiber. Thus, histone acetylation is central to establishing a permissive or repressive chromatin. For any gene, in most of the cases, acetylation of histones sets the positive epigenetic state along with other modifications (methylation, phosphorylation, etc.) for transcriptional activation. Thus, the overall effect of acetylation on the different H3 and H4 lysine residues seems to have an activation effect partly due to the structural alteration of the chromatin and also due to the recognition by the effector modules present in remodeling complexes. However, there are signal specific acetylation events, such as with histone H4 wherein acetylation at its K8 and K12 residues is preferred while the other two potential targets H4 K5 and K16 are relatively spared [50]. The effect of histone acetylation on chromatin transcription is immense but a further level of action is evident wherein the acetylation of chromatin proteins facilitates better transcription. It has been shown that acetylation of histone chaperone NPM1 by p300 enhances transcription [51]. Increasing evidences implicate the autoacetylation of the acetyltransferases as important regulators of enzyme activity thereby indicating that the acetylation of the protein itself might have an important role in determining the efficiency of transcription as described in another article in the same issue. Apart from this several transcription factors like p53, NF-κB, c-myc, HIF1α, MEF2C, etc. get acetylated and the acetylated proteins have also been demonstrated to have better transcriptional activity [10,52] Role of lysine methylation in chromatin transcription Lysine methylation is unique due to its multiple modification status. The residue of modification determines whether it will recruit the activator complexes or repressor complexes. Most of the lysine methyltransferases have recognition domains that facilitate the differential recruitment of determining factors. SUV39 contains chromodomain, which might have a role in recognizing methylated lysines. G9a and GLP possess ankyrin repeats that are required for protein protein interactions and might play an important role in signaling. Interestingly, ESET and CLL8 have methyl-binding domain (MBD) which can potentially recognize methylated DNA [14]. H3K27 trimethylation helps in the recruitment of PRC1 complex via binding of polycomb chromodomain proteins which may aid in chromatin condensation [53]. The methylation status of the lysine residue determines the interaction and recognition of the HP1 isoforms thereby resulting in facultative or constitutive heterochromatin. H3K9 dimethylation is recognized by HP1γ resulting in facultative chromatin [54,55]. On the other hand, H3K9 trimethylation is a mark associated with HP1β thereby forming constitutive heterochromatin. Such a structure is also marked by H3K27 trimethylation and H4K20 trimethylation. The H3K4 trimethylation is a mark closely linked with transcriptional activation. There are many compelling evidences linking this modification through the process of transcription. In fact across the body of a gene, this modification has been shown to decrease from a trimethylated state to di to a mono methylated form. The promoter regions show an increased H3K4 trimethylation enrichment which subsequently decreases across the gene implicating an active demethylation process. Yet another mark associated with transcriptional activation is the H3K79 methylation which is better recognized for its role in telomere functioning wherein the loss of this mark has been linked with telomere silencing [20]. The elongation associated H3K36 methylation has been shown to be essential for the process of transcription. This mark comes into play after the transcription is set into process. The Ser2 phosphorylation of the RNA Pol II CTD acts as the signal for the recruitment of the enzyme responsible for this modification. Independent studies have demonstrated the recruitment of a deacetylase complex into this system [56]. Apart from histones, many non-histone protein substrates such as p53 and NF-κB are known to influence transcription Role of arginine methylation in chromatin transcription For a long time it was considered that the asymmetric arginine methylation is associated with transcriptional activation and the symmetric dimethylation is a mark of transcriptional repression. Recent reports have challenged the understanding of asymmetric modification and transcription. PRMT1 and PRMT4 together have been shown to be involved in several cellular processes like repair and signal transduction wherein the methylated proteins have better activity, thus leading to the conclusion that these enzyme mediated modifications lead to activation of transcription. The histone modification patterns have also been correlated with the transcriptionally active states. Namely, the histone H4R3 methylation by PRMT1 has been shown to be a prerequisite for p300 mediated histone acetylation [57]. The histone H3K18 and K23 acetylation are also shown to be essential for CARM1 mediated H3R17 methylation for the estrogen responsive gene expression [58]. PRMT1 and PRMT4 mediated histone modifications have been very closely linked with the hormone receptor function which led to the speculation that possibly these modifications are essential for

5 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx 5 signal dependent nuclear hormone receptor associated gene expression. However, the past decade has observed an increase in the number of transcription factors regulated by arginine methylation which include p53, NF-κB, CITED, etc., thereby implicating a possible role of arginine methylation in general transcription as well [59,60]. Few instances indicate that the asymmetric methylation might not be an exclusively activation associated mark. The SRC3 co-activator assembly and disassembly are intricately regulated by CARM1 wherein the methylation has been shown to signal for disassembly [61]. This intriguing observation could be explained as an effect of arginine methylation on the remodeling complexes favoring a remodeling event; however in terms of transcriptional output, this event is associated with repression. Yet some evidence implicates the presence of H3R17 methylation on genes that are in a repressed state which are turned on by the estrogen signaling in an ER responsive system [62]. The symmetric arginine methylation catalyzed by PRMT5 has been linked with transcriptional repression. This enzyme has two modification sites on histones, H3R8 and H4R3 which coincidentally is also the site for PRMT1. The H4R3 site seems to be a major site of arginine methylation whose role in the context of transcription probably needs to be addressed more carefully since it is a mark associated with both transcriptional activation and repression based on the enzyme mediating the modification [63]. PRMT1 mediated modification is a prerequisite for the acetylation whereas the vice versa is not true implicating that possibly there is an exclusion event associated possibly due to the presence of a deacetylase. Detailed investigations into the role of arginine methylation in chromatin transcription are essential to understand this process holistically Crosstalk of chromatin acetylation and methylation in transcription regulation The recent spate of discoveries in the field of chromatin modifications has led to the unraveling of a possible network among these modifications, the epigenetic network which seems to be operational even on non-histone proteins such as p53, thus regulating the overall gene expression. Many crosstalk and regulation among these modifications have been studied, but we would be highlighting only the lysine acetylation, methylation and arginine methylation crosstalk Lysine acetylation and lysine methylation The crosstalk among these two modifications is the best studied network operational in establishing both transcriptional activation and repression. The interplay of acetylation of H3 and H4 and lysine methylation of H3K4 facilitate activation whereas the crosstalk of DNA methylation, deacetylation and lysine methylation of H3K9 on distinct residues results in transcriptional repression [64]. The different modifications and the network that regulates transcriptional activation are detailed below. Modifications such as H2B ubiquitination and H3K79 trimethylation are required in promoter regions as well as coding regions of genes for transcription to occur. In fact, they are required for di and trimethylation of H3K4 residue. H3K36 trimethylation is another important polymerase dependent elongation mark specifically present in the 3 ORFs of active genes. This leads to the recruitment of histone deacetylase complexes that maintain chromatin in the deacetylated state in the coding regions to prevent spurious intragenic transcription initiation [56]. In fact, phosphorylation of H3S10 and H3Y41 has been shown to exclude HP1 from chromatin. Phosphorylation of H3S10 is considered to be the trigger leading to acetylation of H3K14 residue at the promoter proximal histones. This is then followed by demethylation of H3K9 and subsequently, its acetylation. H3K4 methylation (di- and tri-) acts as a tag for recruiting various modulatory factors that involve complexes like SAGA (in yeast); in which case histone acetylation is further increased and transcription is enhanced [5]. A recent report indicates the interplay of this modification with DNA methylation as well as the deacetylase in the NURD complex implicating the overall effect on transcription in the form of silencing. This modification has been closely linked with DNA methylation. Especially the H3K9 methylation in conjunction with the DNA methylation has been shown to be involved in heterochromatinization involving the heterochromatin protein HP1. H3K9 trimethylation is a mark associated with HP1β thereby forming constitutive heterochromatin. Such a structure is also marked by H3K27 trimethylation and H4K20 trimethylation. These modifications are also actively supported by the deacetylation machinery, essentially which occurs prior to methylation. Thus, the recruitment of the deacetylases and the subsequent removal of acetylation act as a signal for the methylation at these residues. This sequential recognition of these marks and the subsequent higher order chromatinization has been extensively investigated Lysine acetylation and arginine methylation The most well studied example of such a crosstalk is the network that exists between acetyltransferase p300/cbp, PRMT1 and PRMT4. The nuclear hormone receptor associated transcriptional activation is one such molecular system that emphasizes the need for a co-operative effect of acetylation and arginine methylation. The NR system includes the steroid receptors, estrogen receptor, androgen receptor, glucocorticoid receptor, and thyroid receptors. Each of these receptors is a mediator of signal dependent gene expression by specific receptor ligand interactions. Since these events are associated with transcriptional activation followed by gene expression, they are also associated with chromatin remodeling as well as the involvement of chromatin modifying machineries. Apart from the general transcriptional activators, these events also use secondary co-activators such as p300/kat3b and CARM1/ PRMT4. This is possible because of the co-activation property as well as the enzymatic property of these enzymes. The various domain mapping led to the identification of the exact interaction regions wherein p300/ CBP was found to interact with AD1 region of p160 co-activator GRIP. The AD2 region was found to be the primary site of interaction with the enzyme CARM1 [65]. Another independent study showed the existence of yet another activation domain in GRIP, i.e. AD3 which interacted specifically with another arginine methyltransferase PRMT1. The existence of all these co-activators led to several fold induction in transcription [66]. Very little is known about the interplay of lysine methylation and arginine methylation, except for the negative regulatory role of H3R2 methylation on H3K4 methylation [67]. The exact physiological outcome of this regulation has still not been worked out. The cross talk of modifications is not limited to histones. Several nonhistone chromatin proteins and transcription factors are reported to undergo multiple modifications, which have important regulatory roles on their function [10]. The acetylation of p53 leads to activation of transcription by multiple mechanisms such as enhancement of specific DNA binding, inhibition of non-specific DNA binding, as well as acting as tags for recognition motifs such as the bromodomain present in different recognition modules. Apart from the structural intricacies, p53 acetylation also acts as a regulator of its stability. Many of the acetylation sites are also ubiquitination targets which finally lead to the protein degradation. The acetylation precludes any other modification at the same site and hence helps in conferring protein stability. Recent studies revealed that K370, K372 and K382 can also be methylated [68 70].p53 acetylation may also crosstalk with neighboring modifications similar to the histone code. For example, p53 acetylation at K370 and K372 might communicate with Ser 215 phosphorylation [71]. Strikingly, a recent study revealed that K372 methylation is required for p53 acetylation at multiple sites. The methylation likely recruits Tip60 via its chromodomain and promotes K120 acetylation, which might then form docking sites for PCAF and p300 mediated acetylation. p53 also undergoes arginine methylation mediated by PRMT5 that inhibits p53 dependent apoptosis. Recently, it has been shown that peptidyl arginine deiminase

6 6 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx (PAD4) which is an arginine demethylase acts on p53 resulting in the formation of citrullinated p53 which results in repression of target gene expression [36]. 3. Small molecule regulators of chromatin function (transcription regulation) It is clear that the chromatin modifications regulate transcription and thereby greatly influence gene expression. Hence, the factors associated with the establishment of these modifications such as the chromatin modifying enzymes also influence gene expression. However, a significant role is also played by the small molecule pseudo-substrates of these modification reactions, such as acetyl CoA and SAM which intricately link the process of physiological metabolism and gene expression. A brief overview of these significant small molecules in the physiological system is discussed below Acetyl CoA Like ATP, acetyl CoA lies at the core of a number of critical cellular pathways, and its intracellular concentration is therefore a potential readout of the processes involved. Changes in the availability of acetyl CoA could directly affect the acetylation status of critical substrates, as observed for the core histone proteins H2A, H2B, H3, and H4. Thus, one of the longstanding hypotheses that there exists a link between metabolic pathways and gene expression has been proven in model systems ranging from bacteria to yeast to mouse, indicating the universality of this process [72,73] NAD + Acetylation and deacetylation are reversible processes that maintain a balance of the total cellular acetylation levels. Although deacetylation involves just the removal of the acetyl group, the class III deacetylases, the sirtuins are NAD + dependent deacetylases and require this small molecule to bring about deacetylation [10]. Perturbations of NAD + metabolism alter sirtuin catalytic activity in yeast and in human cells and implicate NAD + and related metabolites as regulators of genetic events in the cell nucleus during stress and toxicity [74]. NAD + is an abundant metabolite. The concentration of this metabolite is normally in the range of 400 to 700 μm in human cells. NAD + also potentiates the biological effects of sirtuin. Yet another physiological process that utilizes NAD + is the DNA repair process [75].Specifically, the poly (ADPribose) polymerases (PARPs), particularly PARP-1, are activated by DNA strand breaks. The PARPs consume NAD + as an adenosine diphosphate ribose (ADPR) donor and synthesize poly (ADP-ribose) onto nuclear proteins such as histones and PARP itself [76]. Over-activation of PARP can cause significant depletion of cellular NAD +, leading to cellular necrosis. Thus, the cellular NAD + levels regulate important processes involved in DNA repair as well as longevity apart from its well established role in transcriptional repression S-Adenosyl Methionine (SAM) SAM (S-Adenosyl Methionine, also known as AdoMet) occurs as a conjugate of methionine and the adenosine moiety of ATP, catalyzed by SAM synthetase. SAM along with ATP is one of the vital metabolites that is used as source/substrate in a wide array of reactions. SAM is the cofactor for all the methyltransferases, and the reaction results in the formation of SAH which is subsequently hydrolysed to homocysteine. The latter is the substrate for methionine synthase, which uses a derivative of folate, MTHF, as a methyl donor to regenerate methionine, producing THF (tetrahydrofolate). Thus, the folate metabolism and the SAM biosynthesis reaction are very closely linked and hence the cellular vitamin B12 levels and the dietary folate status influence the SAM biosynthesis. More than 40 metabolic 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, polyamines, etc. [77] mirna The identification of these small RNAs, referred to as micro RNAs was a revolutionary change in the area of gene expression. It was discovered that in Caenorhabditis elegans, there were few RNAs which were, involved in inhibiting translation [78]. Subsequently, such mechanism was found to be functional even in other species such as plants, fruit flies, mice, and humans, and all seem to play a similar regulatory role. mirna is structurally different than mrna. mirnas fold back on themselves to create a double-stranded structure known as a stem loop, which acts as a tag for the recruitment of the RNA processing machinery such as Dicer and the RNA-Induced Silencing Complex (RISC) and assumes a linear single-stranded shape. mrna and mirna both have single-stranded structures in the cell. This property allows mirna to seek out and bind specific nucleotide sequences in the target mrna molecule. There are two modes of inhibiting the mrna translation; most often the mirna mrna hybrid is degraded by the processing machinery, whereas in other cases the mirna stalls the mrna from undergoing translation. In both cases, however, the gene coding for the mrna is not expressed. The initial discovery of mirna was as a development associated molecule, but the recent discoveries suggest the role of mirna in cancer, differentiation, growth and development [79]. Modulation of histone deacetylases using respective inhibitors also alter microrna profiles suggesting a link between histone acetylation dynamics and micro- RNAs [80]. Moreover it is also known that microrna plays a role in heterochromatin assembly through recruitment of histone lysine methyltransferases. This brings to fore the intricate link existing between histone post-translational modification and micrornas. Apart from these physiological small molecules, there are several others too which intricately modulate the process of gene expression. The most important being ATP, since it modulates the cellular signaling process, as well as regulates the remodeling events associated with gene expression. However, since the focus of the review is solely on acetylation and methylation, only those small molecules which regulate these events have been highlighted. 4. Chromatin modifications in disease manifestation and progression The role of chromatin modifications in transcription and thereby in gene expression is unquestionable. These modifications on histones and other chromatin proteins help in fine tuning the essential cellular processes. As a corollary, it is evident that any alteration would have significant effects on these processes. This altered state of modifications and altered gene expression is what leads finally to disease manifestation i.e. abnormal functioning of the system which could be loss of cell cycle control and thereby increased proliferation as in cancer, or increased inflammatory reactions leading to several metabolic disorders or allergies and several more as represented in Table 2. Hence, it becomes essential to understand the role of these modifications in the abnormal states so as to increase the diagnostic and therapeutic possibilities. Described below are few disease states wherein the involvement of the histone modifications has been clearly demonstrated for the manifestation and progression of the disease Chromatin modifications in cancer The six hallmarks of cancer are unlimited replication potential, evasion of apoptosis, evasion of growth inhibitory signals, angiogenesis, metastasis and growth signal autonomy [81]. There are evidences suggesting the role of chromatin modifications both in a positive and

7 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx 7 Table 2 Altered histone modifications in pathophysiological conditions. Disease Altered histone modifications Reference Cancer Solid cancers Decreased H4K16ac, H4K20me3, [83,84] mistargeting of H3K27me3 Prostate cancer Decreased H3K18ac, H4K12ac [85] Hepatocellular carcinoma, Histone hyperacetylation [86,87] oral cancer Breast cancer Increased H3R17me2 [96] Inflammatory disorders Increased H4K8ac, H4K12ac at [50] specific promoters Increased H3R17me2 and H4R3me2 [131] Increased H3K4me2 [130] Decreased H3K9me2 Diabetes Increased histone acetylation [121] Increased H3K9me2 at IL 2 and [123] NF-κB promoters in lymphocytes Cardiac hypertrophy Increased histone acetylation [ ] Increased H3K4me [144] Increased asyymetric arginine [145] dimethylation Retroviral infections Increased H3K4me3 at viral [153] integration site Increased H3K9me3 during viral latency [154] negative regulatory mode on cancer which will be individually discussed below in the context of the three important modifications: acetylation; lysine and arginine methylation Acetylation and cancer Chromatin (hypo)acetylation and its effect on cancer were recognized very early. This was the concept that was prevalent till the last decade and hence histone deacetylase inhibitors are currently in clinical trials [82]. The reason that this concept was inculcated was due to the fact that there was an increased repression of tumor suppressor genes by DNA methylation, histone methylation and deacetylation [83]. Importantly, HDAC activity is crucial to prepare the histone template for methyltransferases by removing acetyl groups obstructing methylation. Thus deacetylase inhibitors were tried to rescue the tumor suppressors. Also, p300 mis-sense mutations were identified in glioblastomas, breast cancer and colorectal cancer [84]. These necessitated investigation into the alteration of acetyltransferase function in cancers. An elegant observation by Kurdistani's group in samples of prostate cancer indicated that there are several residues like H3K18 whose hypocetylation is positively correlated with increased grades of prostate cancer and its recurrence [85]. However histone hyperacetylation also is observed in certain cancers. Hepatocellular carcinoma is one such example, indicated to exhibit hyperacetylation [86]. Recent work from our group have also shown the positive relation of acetylation and oral cancer manifestation which is discussed in another chapter [87,88]. These differential roles of acetylation in cancer suggest the need for an extensive investigation into thedifferenthistonemodifications in relation to various cancers Lysine methylation and cancer Aberrant gene expression leads to various diseases including cancer. The major cause of cancer is either silencing of tumor suppressor genes or activation of oncogenes. Since histone methylation plays an important role in transcription, dysfunctioning of the enzymes catalyzing these modifications is expected to lead to such diseases. This may include overexpression, underexpression or misexpression of any of these enzymes. Mice lacking SUV39H1 and SUV39H2, or both, develop leukemia suggesting the importance of these enzymes in regulation of cell proliferation [89,90]. G9a is involved in silencing of tumor suppressor genes and its knockdown inhibits cancer cell growth [91]. Global loss of H4K20 trimethylation is one of the hallmarks of cancer [92]. MLL is translocated in most of the hematological malignancies and depending on the fusion partner, it leads to dysregulation of target genes. Hox genes are one of the most important targets of MLL members and are frequently upregulated in most of the leukemias [93]. Mammalian polycomb group proteins are known to repress transcription via recruitment of histone deacetylases. Human homologue of EZ(H)2 is frequently 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 stages of cancer contains EZ(H)2 that catalyze H3K27 trimethylation, which is recognized by chromodomain containing proteins of PRC1 to establish the epigenetic repressive states [92]. One of the mistargets of this pathway in early stages of cancer is p16, which drastically disrupts cell cycle control allowing clonal expansion. EZH2 can serve as a marker in early breast cancer growth, and promotes proliferation and invasiveness of prostate cancer cells. NSD family members have also been implicated in various cancers. Chromosome translocations of NSD1 or NSD3 to NUP98 result in acute myeloid leukemia [94]. NSD2 overexpression leads to multiple myeloma whereas reducing its levels suppresses cancer growth. NSD3 is also known to be upregulated in breast cancer cell lines and primary breast carcinomas. Knock down of DOT1L inhibits growth of cancer cells having MLL translocations. Constitutively active MLL-DOT1L may misregulate transcription of MLL regulated genes [95]. Mice lacking RIZ1 protein develop diffuse large B-cell lymphomas and other tumors. PFM1 maps to chromosomal regions that are frequently deleted in tumors. All these reports suggest that there exists a cause and effect relationship between lysine methyltransferases and cancer Arginine methylation and cancer Arginine methyltransferases PRMT1, PRMT2 and PRMT4 have been shown to have direct roles in several cancers. PRMT1 forms a complex with the lysine methyltransferase MLL (mixed lineage leukemia), and regulates the MLL downstream targets, thereby playing an important role in oncogenesis [96]. The lysine methyltransferase MLL is a well known candidate that forms fusion proteins (by gene fusion) which lead to leukemia. The involvement of the PRMTs along with MLL is an indication of the role of arginine methylation in leukemia. On the other hand, PRMT1 methylation regulates the AML1/Runx1 activity which is essential in differentiation and is also involved in acute myeloid leukemia. PRMT4 methylates histone H3R17 and R26. Estrogen receptor alpha (ERα) dependent breast cancer shows an increase in H3R17 methylation indicating the role of this transcriptional co-activator in ER-dependent breast cancers [97]. It has been observed that the H3R17 dimethylation is concomitant with ERα recruitment on the E2F1 promoter on estrogen induction. The methylation has been shown to be dependent on the presence of the oncogenic co-activator AIB1. Thus the presence of CARM1 and the increased H3R17 methylation have been directly implicated in the onset of ER alpha dependent breast cancer [97]. The essential role of PRMT4 in SRC3/AIB stability and complex assembly and disassembly indicates that there could be important contribution of PRMT4/ CARM1 and H3R17 methylation in breast cancer manifestation and progression [61]. Furthermore, PRMT1 and PRMT4 have been shown to cooperate synergistically along with the histone acetyltransferase p300, thereby resulting in the transcriptional activation of the tumor suppressor, p53 responsive genes [57]. Furthermore, there are few reports of prostate cancer and arginine methylation wherein the increased levels of PRMT1 and PRMT4 have been shown [98 100]. Although these indicate a possible role of these enzymes in prostate cancer manifestation/progression, the exact mechanism has not been identified. Since these two proteins are also co-activators of nuclear hormone receptors like androgen and estrogen receptor, it is possible that there exists a direct transcriptional activation due to the co-

8 8 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx activation property of these two enzymes in both prostate and breast cancers Chromatin modifications in inflammatory disorders One of the main mediators of inflammatory response is the transcription factor NF-κB, which is regulated by acetylation, lysine methylation and arginine methylation. Direct evidences of acetylation linked to inflammatory disorders have been shown in the case of acetylation with very minimal and indirect evidences linking the latter two modifications Role of acetylation in inflammation and fibrosis Histone H4 hyperacetylation is a well known inflammation associated epigenetic mark [50]. IL-1β causes increased histone acetylation on H4 at K8 and K12 residues. This increase is due to the recruitment of HATs like CBP and PCAF, to promoters of inflammatory genes, by a very important transcription factor controlling expression of inflammatory genes, nuclear factor-κb (NF-κB). Family members include p50, p52, p65 (RelA), c-rel, and RelB. In vivo, they exist as homo or hetero-dimers with distinct DNA binding specificities [101]. Their response is regulated at multiple levels: synthesis, subcellular localization, postranslational modification, differential dimerization, DNA binding, and interaction with a combination of receptors depending on the given context [102]. Upon activation by phorbol 12-myristate 13-acetate, H 2 O 2, cigarette smoke condensate, interleukin-1beta, lipopolysaccharide, and okadaic acid, etc., NF-κB gets derepressed and translocates into the nucleus. Acetylation of distinct lysine residues of RelA regulates different functions of NF-kappa B, including transcriptional activation, DNA binding affinity, IκB alpha assembly and subcellular localization [103,104]. Acetylation of NF-κB by p300/cbp inhibits its interaction with IκB and retains it within the nucleus. HDACs like HDAC3 deacetylate p65 resulting in its binding to IκB and thus translocation to cytoplasm. HDAC1 and HDAC2 are also found in p65-hat complex. NF-κB regulates a wide variety of genes responsible for inflammation 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 adhesion molecules (e.g., ICAM, VCAM, E-Selectin); acute phase proteins (e.g., SAA); inducible enzymes (e.g., inos, COX-2), some antimicrobial peptides; and genes responsible for regulation of apoptosis and proliferation [105]. NF-κB dependent transcriptional complexes require multiple co-activators like p300 or its close homologue CBP, P/CAF and the p160 family of steroid receptor co-activators, which also are acetyltransferases, thus facilitating rapid formation of the preinitiation complex and re-initiation which facilitate multiple rounds of transcription by histone acetylation [106,107]. Along with NF-κB, Poly (ADP-ribose) polymerase-1 (PARP-1) has been demonstrated to play 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 of NF-κB (p65 and p50) and synergistically co-activates NF-κB dependent transcription. p300 in turn acetylates PARP-1 at specific lysine residues upon inflammatory stimulation in a variety of cell types. Acetylation of PARP-1 at these residues is required for the interaction of PARP-1 with p50 and synergistic co-activation of NF-κB by p300 Fig. 1. Role of acetylation in the NF-κB inflammatory response: interleukins and TNF-α on interaction with their respective ligands and stress associated ROS, stimulate the dissociation of NF-κB and the inhibitory kinase complex, IκBα is subsequently phosphorylated and ubiquitinated followed by degradation. The NF-κB subunits, p50 and p65 undergo acetylation by p300, followed by the recruitment of the other acetyltransferases and the transcription machinery leading to the expression of the NF-κB dependent genes which are enlisted as the inflammatory genes. HAT inhibitors such as EGCG, anacardic acid and plumbagin have been shown to inhibit the NF-κB pathway by inhibiting histone acetylation.

9 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx 9 and the mediator complex in response to stimuli [110]. NF-κB mediated activation of histone H3, H4 acetylation specifically by cytokines IL-β, TNF-α or endotoxins has shown to increase expression of inflammatory genes such as Granulocyte macrophage colony stimulating factor (GM-CSF) [105]. The role of acetylation in the NFκB signaling pathway is depicted in Fig. 1. Infiltration of cellular mediators of inflammation needs chemotactic agents such as eotaxin (eosinophil chemoattractant), secreted at the site of inflammation [111]. The human eotaxin promoter consists of several binding sites for transcription factors including NFκB [112]. Inflammatory signaling by TNF-α induces selective histone H4 acetylation and binding of p65 to the eotaxin promoter, thus regulating its transcription. It has been shown that beta(2)-agonists and glucocorticoids, which are mainstream therapies for allergic and inflammatory conditions were able to reduce transcription of eotaxin by inhibiting both TNF-α-induced histone H4 acetylation and recruitment of p65 to the eotaxin promoter, without altering the capability of NF-κB to translocate to the nucleus [113]. Thus reversible acetylation of transcription factors like NF-κB, proteins like PARP and histones plays pivotal roles in regulating gene expression during inflammation. Pharmacological agents used in the therapy of inflammatory diseases like Corticosteroids and β-2 antagonists albeit indirectly reduce histone hyperacetylation in the affected cells. HATs like p300 play a role in the manifestation of fibrotic disorders too. Deregulated expression of collagen genes underlies the pathogenesis of fibrotic disorders. Fibroblasts are responsible for the production and accumulation of extracellular matrix (ECM), composed of largely of collagen, and other macromolecules. Aberrant fibroblast activation and differentiation to myofibroblasts lead to an excessive accumulation of collagens and other ECM proteins and pathological fibrosis [114]. Transforming growth factor-β (TGF-β)isa multifunctional cytokine that regulates cellular growth, differentiation, and survival as well as the profibrotic tissue response [115]. TGFβ transduces its signal from surface receptors to the nucleus via Smads and interacting cofactors. One of the cofactors that is implicated in TGF-β induced signaling is p300. Upon activation of the type II cell surface receptors, activated type I TGF-β receptor phosphorylates Smad2/3 which heterodimerize with Smad4 and translocate to the nucleus wherein they stimulate collagen gene transcription [116]. However, for maximal collagen synthesis, interaction of activated Smad2/3 with Sp1, along with p300 and other transcriptional coactivators is required. Interestingly, the p300 homologue, CBP does not play a role in this process. It is intriguing to note that p300 also plays a role in IFN-γ mediated decrease in collagen gene transcription that involves a different set of transcription factors like activated STAT1α [117]. This antagonistic response is due to competition for limited availability of p300 by TGFβ induced Smads and IFN-γ induced STAT1α and others. This is proved by the fact that ectopic expression of p300 overcomes the inhibition by IFN-γ and rescued stimulation by TGF-β, thus rescuing collagen 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 its TGF-β induced stimulation [118,119]. Again, a different set of transcription factors is implicated. Activation of c-jun and JunB induced by TNF-α results in competition with TGF-β-activated Smad3 for limiting p300. Even in this case, overexpression of p300 overcomes the inhibition. Hence p300 plays a key role as an integrator of signals in both positive and negative modulation of fibrotic response mediated by its co-activation as well as acetyltransferase activity. This modulation is based on the balance of intracellular signal transduction molecules that are decided by profibrotic or antifibrotic cues. Other HATs like PCAF have also been implicated in fibrosis by modulating the stability of Fli1 by acetylation [120]. Fli1 is a member of Ets transcriptional factors which acts as a negative regulator of collagen gene expression in dermal fibroblasts. Fli1 downregulation is implicated in conditions such as cutaneous fibrosis in scleroderma. PCAF-dependent acetylation abolishes the repressor function of Fli1 with respect to collagen gene expression. Thus, the role of HATs in the manifestation of inflammatory disorders seems to be multifaceted Role of acetylation in diabetes It is well known that both type I and type II forms of diabetes are intricately linked to inflammation [121]. Recent reports implicate a role for HATs in the manifestation of diabetes. High glucose can augment the expression of NF-κB dependent genes such as TNF-α [122]. Transcriptional activities of p65 as well as co-activator effects of CBP, p300, or PCAF are clearly enhanced under high glucose conditions in THP cells. Very interestingly, the promoter bound CBP and HDAC1 show different dynamics in the presence of high glucose. As the level of CBP increased, a concomitant decrease in HDAC1 has been observed. Presently, HUVECs incubated in varied glucose concentrations were investigated for their acetylation status following p300 sirna transfection, p300 overexpression, or incubation with the p300 HAT inhibitor, curcumin [123]. These cells on treatment with high glucose showed increased p300 and hyperacetylation at promoters of inflammatory genes like Endothelin-1 and fibronectin. The p300 overexpression showed glucose like effects with an increase in transcription of Endothelin-1, Fibronectin and VEGF. Silencing of p300 abrogated the effects of glucose in cells and the p300 specific HAT inhibitor; curcumin prevented the effects of glucose. It has to be noted that hyperacetylation is not the only epigenetic change occurring at the promoters. Transient hyperglycemia induces persistent mobilization of SET7 to the p65 promoter that results in H3K4 monomethylation and activates p65 downstream genes [124]. It is only logical to conclude that inflammation which is associated with most diseases, results in an increase in HAT activity and HAT inhibitors could therefore be novel anti-inflammatory agents Inflammatory conditions of the lung Lung inflammatory conditions involve expression of multiple genes in the lungs that encode chemokines, cytokines, receptors of chemical mediators of inflammation, cell adhesion molecules, etc. Infiltration of cellular mediators of inflammation results in extensive destruction of lung architecture initially due to tissue necrosis and later due to fibrosis that occurs as a part of the healing process. Complex epigenetic mechanisms orchestrate expression of subsets of genes at various time points during disease progression resulting in tissue damage and healing. Bronchial asthma is a common allergic condition of small airways. Hyper responsive and hypertrophic airway smooth muscle cells and mucosal hypertrophy are the major pathologic findings. There is a marked increase in HAT activity in both tissues and alveolar macrophages isolated from bronchoalveolar lavage in patients [125]. A small decrease in HDAC activity due to reduced HDAC1 is seen albeit no changes in HDAC2 and 3 [126]. Interestingly, an increase in SIRT1 (class III HDAC) levels and activity is found to be responsible for OVA (Ovalbumin) induced bronchial hyper reactivity in mouse models necessitating further investigation into the role of other histone modifying enzymes [127]. COPD (Chronic Obstructive Pulmonary Disease), associated invariably with smoking occurs as a destruction of lung alveoli. It has been correlated to reduced HDAC levels in peripheral lung especially HDAC2 with a lesser extent of HDAC5 and HDAC8 reduction [128]. Reduction in HDAC2 follows severity of disease, with 95% decrease in Stage 4 disease. Reduction in HDAC2 levels also directly correlates with airway inflammation as evidenced by an increase in IL-8 and the number of inflammatory cells in small airway. Thus a decrease in the deacetylation status implies a constant level of acetylation within

10 10 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx tissues that leads to prolonged expression of inflammation associated genes resulting in chronic and widespread lung damage. Inhibition of HAT could in theory be able to limit inflammatory gene expression and thus reduce the damage Role of lysine methylation in inflammation associated diseases Apart from cancer, altered methylation patterns are also observed in diabetes and other inflammatory diseases. Glucose induced insulin transcription is regulated by PDX1 which recruits SET9 methyltransferase to the insulin promoter. Several PDX1 mutations associated with diabetes that lead to epigenetic changes are known [129]. Hyperglycemia also leads to changes in epigenetic patterns on the genes involved in vascular inflammation. H3K4 methyltransferase SET7/9 influences recruitment of NF-κB p65 to promoters and thereby regulate expression of pro-inflammatory genes [130]. Vascular smooth muscle cells of db/db mice show increased H3K4 dimethylation and decreased H3K9 dimethylation at the promoters of inflammatory genes. Interestingly, overexpression of SUV39H1 in vascular smooth muscle cells reverses diabetic phenotype, whereas silencing this protein in normal smooth muscle cells increases the expression of inflammatory genes [131]. In type I diabetes, IL-1β and NF-κB promoters show increase in H3K9 dimethylation occupancy in lymphocytes. SET7 is known to be involved in regulating epigenetic changes at NF-κB promoter in response to transient glycemia. Recently, SET7/9 was shown to play an important role in TNF-α expression in monocytes implicating a possible role in manifestation of diabetes and inflammatory disorders where TNF-α is over expressed [130] Role of arginine methylation in inflammatory disorders Arginine methylation has not been directly implicated with diabetes manifestation; however, important members in the glucose metabolism are methylated by PRMTs. Further investigation in this context might reveal new roles for the PRMTs in diabetes too. Both PRMT1 and PRMT4 are known transcriptional co-activators of NF-κB [132] Chromatin modifications in cardiovascular disease conditions Similar to the inflammatory disorders, the information available about acetylation and cardiovascular disease is much more than the other two modifications. Described below are the compelling evidences of these modifications on different cardiac disease conditions Acetylation and cardiovascular disorders Cardiac hypertrophy is associated with increase in cardiac muscle size, re-induction of fetal cardiac gene program (α to β isoforms of Myosin Heavy Chain), increased protein synthesis and sarcomere reorganisation resulting in decreased cardiac performance. The role of several signaling pathways including the chromatin modifying enzymes, has been implicated in cardiac hypertrophy. The acetyltransferase p300 acetylates GATA-4 and induces expression of genes like ANF, ET-1, and α-mhc [133]. Acetylated GATA-4 has increased DNA binding ability and also increased expression of GATA-4 regulated genes as mentioned above [134]. Cardiac overexpression of p300 results in increased mortality [135]. p300 along with MEF2D is known to activate cardiac genes in differentiated cardiac myocytes [136]. p300 mediated acetylation of MEF2C affects MEF2C DNA binding and transactivation function of MEF2C [137]. MEF2 basal activity is very low and tightly regulated by deacetylation mediated by HDAC5 and 9. Even a small increase in myocardial p300 can switch MEF2 from a basal deacetylated state to an active acetylated state. Recent studies argue whether MEF2 or GATA-4 mediated activation is important 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 [ ]. Recent studies with p300 inhibitors such as anacardic acid and curcumin have been shown to prevent the induction of hypertrophy in isolated neonatal rat cardiomyocytes, which will be discussed in the subsequent section [141,142]. Interestingly, HDACi treatment has also been shown to repress cardiac hypertrophy, but through an acetylation independent mechanism, possibly by blocking the HAT and MEF2C interactions [143]. HDACs may also directly or indirectly modulate hypertrophic signal transduction cascades like the HAT- GATA4-hypertrophy axis [144]. The delicate balance between HATs and HDACs in normal physiology implicates a careful investigation of the role of each of these components in the disease manifestation Lysine methylation and cardiovascular disorders There is an active effort towards identifying the possible alteration in lysine methylation associated changes and cardiovascular disease onset, but very less success has been achieved in this field. One of the notable studies done on a rat model of cardiac disease signifies the alteration of H3K4 methylation across the stages of disease progression. Another mark, H3K9 methylation that was also analyzed showed very minimal changes across the different disease states. The exact role of lysine methylation on these disorders also remains to be elucidated, but the fact that inflammation and diabetic complications show the involvement in these disorders indicates a possible role of lysine methylation in cardiovascular disorders [145] Arginine methylation and cardiovascular diseases All the class I PRMTs are involved in the formation of asymmetric dimethyl arginine (ADMA), which is a competitive inhibitor of nitric oxide synthase (NOS), and thus decreases nitric oxide (NO) availability in the cells [146]. Decreased NO levels are a hallmark of cardiovascular diseases. Thus, ADMA levels play an important role in the cardiovascular diseases as well as conditions related to oxidative stress like hyperlipidemia, diabetes, arterial hypertension, hyperhomocysteinemia and heart failure. ADMA levels are also considered to be indicative of atherosclerosis [147]. Thus, all the class I enzymes are responsible for the oxidative stress induced disease manifestation via the ADMA intermediate [148] Chromatin modifications in retroviral diseases Retroviruses are RNA viruses which use the reverse transcriptase to form DNA and get integrated into the host genome upon infection. These have also been shown to form chromatinized templates with properly positioned nucleosomes as in the case of HIV. One of the major retroviral diseases of global concern is AIDS caused by HIV, wherein the epigenetic mechanisms regulating several steps of viral integration and replication have been well studied. Other retroviral diseases are indirect causes of retroviral infections as in the case of retroviral induced cancer. Rous sarcoma virus (RSV) and mouse mammary tumor viruses are two such viruses wherein the src gene is a (RSV) viral oncogene Role of acetylation in retroviral infection The major challenge in the field of retroviral infections is due to the integration of the viral genome into the host genome resulting in viral latency [149]. An actively replicating virus can be targeted by the enzyme inhibitors such as protease and reverse transcriptase inhibitors; however, the latent viruses are difficult to eradicate. Hence, various strategies have been devised to break this latency; one of which is increasing the acetylation status. HDAC inhibitors have been tried for this purpose since these can lead to indirect activation of the viral genome and once the virus is active, it can be targeted by the inhibitors [150]. This indicates that acetylation plays an important role in the viral gene expression. The HATs p300 and PCAF acetyltransferases have

11 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx 11 been shown to be absolutely essential for the transcription of the viral genome especially in the case of HIV. The nucleosome organization of the integrated HIV genome has been well characterized. PCAF has been shown to be one of the initial acetyltransferases involved for the activation [151]. The transactivation protein Tat is a p300 substrate and is activated in an acetylation dependent manner [152]. The multiple acetylation events are known to bring about remodeling events which leads to the formation of the TAR (transactivation response element), which is bound by the Tat and p-tefb complex. These together facilitate the recruitment of CDK9 to mediate RNA polymerase II phosphorylation thereby initiating transcription [153]. Thus, targeting the Tat acetylation and the subsequent acetylation events are attractive therapeutic strategies which will be discussed in the last section Role of lysine methylation in retroviral infection Histone lysine methylation is also known to play a role in modulation of gene expression upon viral infection. Recent reports suggest that integration site selection by HIV depends on the chromatin environment of the site. Integration is particularly favored in regions having transcription associated histone modifications such as H3K4 trimethylation but disfavored in regions associated with transcription-inhibitory marks such as H3K27 trimethylation [154]. One of the major hurdles in the treatment of HIV is its latency which prevents its complete eradication. One of the mechanisms responsible for HIV latency involves SUV39H1, HP1 and H3K9 trimethylation which are reversibly associated with HIV-1 and help in the maintenance of latency [155]. Apart from silencing, Menin, a component of MLL1 complex, H3K4 methyltransferase is shown to be required for HIV-1 tat transactivation [156]. These observations suggest that targeting lysine methyltransferases to combat HIV can provide a hope for treatment of HIV Role of arginine methylation in retroviral pathogenesis Arginine methylation has both positive and negative modulatory effects on viral pathogenesis. PRMT1 methylates the adenoviral 100 k protein which is essential for the synthesis of late proteins and the establishment of the viral infection [157]. PRMT1 also methylates the Epstein Barr viral EBNA1 protein which is required for the replication and transcriptional activation of the viral genes [158]. Yet another viral 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 negative regulator of viral pathogenesis [160]. PRMT6 is the only arginine methyltransferase that methylates HIV-Tat protein and the HIV nucleocapsid. Tat methylation leads to its decreased binding to TAR and therefore the viral transactivation is impaired. The methylation of nucleocapsid reduces the initiation of reverse transcription [161] Chromatin modifications in neurodegenerative diseases Another important area of research that has an epigenetic link to disease is the neurodegenerative disorders. Although epigenetic regulation of gene expression in the brain has been linked to neural development and differentiation, circadian rhythmicity, memory formation, and synaptic plasticity, its role in neurologic disorders like seizures and psychiatric disorders like depression and schizophrenia has been recently identified [162]. There are enough convincing evidences which suggest that chromatin modifying machineries have a role in the CNS disorders. Acetylation of histones mediated by CBP is known to be vital in neuronal cell survival [163].A fine balance between acetylation and deacetylation of histones is expected to be pivotal in post mitotic neuron physiology [164]. The absence of acetylated histones is conspicuous in neurodegenerative states like Alzheimer's, Parkinson's and Huntington's diseases. Multiple factors that affect normal function of histone acetyltransferases (especially CBP) like decreased expression, degradation, and sequestration of CBP into compartments that do not allow histone acetylation finally lead to hypoacetylation of histones inducing neuronal apoptosis. Inhibition of histone deacetylases increases the pool of acetylated histones and hence, is an attractive approach for treatment of neurodegenerative disorders. However, the pleotropic effects inherent to these inhibitors, that include apoptosis induction in neurons (primarily due to lack of specificity to any one class of HDACs), are a cause of concern [165]. Activation of enzyme functions of CBP and p300 in the neurons could be a more specific approach and a potential alternative to the problem, thereby reducing pleotropic effects associated with indiscriminate hyperacetylation associated with HDACi. In addition to diseased states, importance of histone acetylation is also recognized now in the process of synaptic plasticity and has been extrapolated to learning and memory [162]. Yet again, CBP mediated histone acetylation is implicated but it is also known that p300 and PCAF HATs are essential for establishment of long term memory [166]. Recent studies report specific increase in H2B and H4 acetylation in the rat dorsal hippocampus, while spatial memory was being consolidated, which was associated with a global increase in HAT activity as well as increase of CBP, p300, and PCAF expression [166]. The role of acetylation in neuronal physiology and pathophysiology has been discussed extensively in another article in this same issue (Selvi BR et al.). Other modifications of histones like methylation and phosphorylation have also been identified to be important for various neuronal functions. It was recently reported that misregulation of the H3K4 tridemethylase SMCX, that acts as a transcriptional repressor on sodium channel type 2A (SCN2A) and synapsin1 promoters, results in X- linked mental retardation [167]. Another independent study has also shown that postnatal neuron-specific deficiency of the complex composed by the H3-K9 dimethyltransferases G9a/GLP results in derepression of non-neuronal (e.g. Serpinb1b, Serpinb5) and early neuronal progenitor (e.g. Dach2) genes [168]. This decrease in H3K9 methylation reflects on complex behavioural abnormalities, such as defects 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 of chromatin modifications in transcription and gene expression. It also illustrates the role of altered modifications in abnormal states, i.e. diseased conditions. These altered modifications are a consequence of altered enzyme activity of the chromatin modifying machineries. Hence, if these activities could be modulated, the alterations could also be minimized. There are several ways of modulating an enzyme function but the successful examples so far implicate the use of small molecules which can inhibit or activate the enzymes. The field of epigenetic modifications also is abound with such examples but the monopoly is of the DNA methylation and histone deacetylation inhibitors [169]. However, of late, several histone acetylation inhibitors have also been identified. Furthermore, protein methylation inhibitors have also been identified which will be briefly discussed below. These inhibitors and their targets are represented in Fig HAT inhibitors Synthetic inhibitors. The very first class of inhibitors identified for the HATs, p300 and PCAF were the bi-substrate inhibitors which showed remarkable selectivity. Lys-CoA was specific to p300 while H3-CoA was specific for PCAF [170]. The major drawback of these inhibitors was their lack of cell permeability. Hence, a coenzyme A analogue conjugated to a cell permeabilizing oligo-arginine peptide

12 12 B.R. Selvi et al. / Biochimica et Biophysica Acta xxx (2010) xxx xxx Fig. 2. Histone modification inhibitors and their targets: the figure represents the histone modifications on the core histone substrate with the different histone acetyltransferases and the methyltransferases which bring about the histone acetylation, histone lysine methylation and arginine methylation. Since the inhibitors enlisted are for p300, PCAF, and GCN5, these are represented in green. The available inhibitors of these enzymes and the inhibited modification residue are indicated alongside. The acetylation inhibitors are enlisted in 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 and chaeotocin inhibit H3K9 methylation. H3K27 methylation is inhibited by BIX and DZNep. The latter also inhibits H4K20 methylation. The well characterized arginine methylation 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 cellular histone acetylation and transcription using p300 reporter assays. GCN5 specific inhibitors, γ-butyrolactones have been synthesized based on the catalytic site [171]. High throughput screening has led to the identification of PCAF specific inhibitors, isothiazolones [172], which inhibit acetylation of histones in the cells in a time and dosedependent manner. Many analogues for isothiazolones have been synthesized raising hope for more better and specific inhibitors. The isothiazolone functional group can easily be modified with a variety of substitutions that will enhance binding to the enzyme active site. Importantly, isothiazolone core structure has been used as a starting point to explore PCAF inhibition [173]. HAT inhibition in vitro has been initially reported with 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 a thioglycolic acid linkage. Each of the two isomeric forms of Spd-CoA, linking the N1 or N8 atom of spermidine to CoA, respectively, has subsequently been shown to be HAT inhibitors in vitro. Negativelycharged CoA moiety is considered to be a hindrance to cellular permeability. Though there have been doubts about this issue, banking on the fact that polyamines such as spermidine are efficiently transported across cellular membranes, it has been hypothesized that Spd-CoA could also be internalized into whole cells and that internalization would lead to inhibition of HATs. In line with the hypothesis, bi-substrate HAT inhibitor, Spd(N1)-CoA, acts on cells bringing about inhibition of histone acetylation [175]. Bi-substrate inhibitors of MYST family enzymes like Esa1 and TIP60 have also been synthesized [176]. An elegant study from Cole P.A.'s group, reported a commercially available pyrazolone-containing small molecule; C646 as a potent p300 HAT inhibitor. Incidentally, C646 is a competitive p300 inhibitor with low micromolar inhibitory constant [177]. However, natural products having HAT inhibitory properties have drawn attention and raised expectations of having better cell permeability and more selectivity for HATs, which are summarized below Natural products. Anacardic acid (AA) is a simple salicylic acid derivative (nonadecyl salicylic acid), purified from cashew nut shell liquid. It is a potent inhibitor of p300 and p300/cbp-associated factor histone acetyltransferase activities [178]. Anacardic acid non-specifically inhibits both p300 and PCAF with an IC 50 less than 10 μm. Since anacardic acid is a non-specific inhibitor, the search for specific inhibitors led to the identification of the polyphenolic compound from Curcuma longa rhizome, curcumin, as a p300 specific HAT inhibitor and does not inhibit PCAF [179]. However, curcumin has its limitations in possessing pleotropic effects [180]. Recently, a water soluble curcumin derivative, CTK7A has been synthesized which has a broad spectrum HAT inhibitory property as compared to curcumin [88]. A naturally occurring polyisoprenylated benzophenone, garcinol, isolated from Garcinia indica or kokum fruit ( indigenous to southern parts of India) as a potent inhibitor of both PCAF and p300 [181]. IC 50 of garcinol for p300 acetyltransferase activity was 7 μm while it was 5 μm for PCAF. However, by virtue of its non-specificity, garcinol is found to be immensely cytotoxic when treated to HeLa cells. It caused apoptosis of HeLa cells and caused downregulation of majority of genes. The potential use of garcinol to target p300 for understanding the physiological role of HATs or to develop a therapeutic molecule is less