J Biosci (2020)45:135 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-020-00099-2 (0123456789().,-volV)(0123456789().,-volV) Review

Evaluation of post-translational modifications in histone proteins: A review on histone modification defects in developmental and neurological disorders

1 1 2 SHAHIN RAMAZI ,ABDOLLAH ALLAHVERDI * and JAVAD ZAHIRI 1Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, P.O. Box: 14115-111, Tehran, Iran 2Bioinformatics and Computational Omics Lab (BioCOOL), Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, P.O. Box: 14115-111, Tehran, Iran

*Corresponding author (Email, [email protected])

MS received 2 March 2020; accepted 14 September 2020

Post-translational modification (PTM) in histone proteins is a covalent modification which mainly consists of methylation, phosphorylation, acetylation, ubiquitylation, SUMOylation, glycosylation, and ADP-ribosylation. PTMs have fundamental roles in chromatin structure and function. Histone modifications have also been known as epigenetic markers. The PTMs that have taken place in histone proteins can affect gene expression by altering chromatin structure. Histone modifications act in varied biological processes such as transcriptional activation/inactivation, chromosome packaging, mitosis, meiosis, apoptosis, and DNA damage/repair. Defects in the PTMs pathway have been associated with the occurrence and progression of various human diseases, such as cancer, heart failure, autoimmune diseases, and neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. Histone modifications are reversible and used as potential targets for cancer therapy and prevention. Recent different histone PTMs have key roles in cancer cells since it has been shown that histone PTMs markers in cancers are acetylation, methylation, phospho- rylation, and ubiquitylation. In this review, we have summarized the six most studied histone modifications and have examined the role of these modifications in the development of cancer.

Keywords. Chromatin; disease; histone proteins; histone modifications; post-translational modifications Abbreviations: ADP, adenosine diphosphate; ADP-ribose, adenosine diphosphate ribose; ART, ADP- ribosyltransferases; ARTD1/ PARP1, poly [ADP-ribose] polymerase 1; ATM/ATR, ATM/ATR serine/threonine kinase; ATP, adenosine triphosphate; BRCA1, breast cancer type 1 susceptibility protein; CARM1, coactivator- associated arginine methyltransferase 1; ChIP, combines chromatin immunoprecipitation; ChIP-seq, ChIP- sequencing; CRC, colorectal cancer; DMFS, distant metastasis-free survival; DNase I, deoxyribonuclease I; DSBs, DNA double-strand breaks; E1, ubiquitin/sumo activating; E2, ubiquitin/sumo conjugating; E3, ubiquitin/sumo ligase; EZH2, Zeste homolog 2; HATs, histone acetyltransferases; HDAC, histone deacetylase; HMTs, histone methyltransferases; HP1, heterochromatin protein; LSD1, lysine-specific histone demethyla1; MAP kinase, mitogen-activated protein kinase; MARTs, mono-ADP-ribosyltransferases; MLL gene fusion, MOZ/MORF (MYST family) gene fusion; NAD, nicotinamide adenine dinucleotide; NATs, N-terminal acetyltransferases; OS, overall survival; PAR, polymeric ADP-ribose; PARP, poly-ADP ribose synthetase; PcG proteins, polycomb group (PcG) proteins; PO4, phosphate; PRC2, polycomb repressive complex 2; PRMTs, protein arginine methyltransferases; PTMs, post-translational modifications; RING, really interesting new gene; RNF20, ring finger protein 20; SET domain, Su(var)3-9, enhancer-of-Zeste and Trithorax (suppressor of variegation, Enhancer of Zeste, and Trithorax); SUMO, small ubiquitin related modifier; TSSs, transcription start sites; USP22, ubiquitin-specific peptidase 22. http://www.ias.ac.in/jbiosci 135 Page 2 of 29 S Ramazi, A Allahverdi and J Zahiri

1. Introduction

associate together, and then with two H2A/H2B 1.1 Histone proteins dimers make the histone octamer. In physiological salt conditions, the H3/H4 tetramer is the core of histone DNA in eukaryotic organisms is organized in a octamer, and the H2A/H2B is symmetrically placed nucleoprotein complex called chromatin (Jenuwein and on each side of the tetramer. Approximately 75% of Allis 2001). In recent years it has been shown that the core histone protein mass comprises the ‘histone chromatin has a key role in the regulation of gene fold’ domains which form the spool onto which the expression. Understanding chromatin structure and nucleosome DNA is wrapped. The core histones are dynamics is of fundamental importance for life science highly conserved proteins even from yeast to human, and contributes decisively to our knowledge of cell and this property is very important for the mainte- function, organism development, and treatment of nance of genomic material. The remaining *25–30% diseases. In the eukaryotic nucleus, the proteins which of the mass of the core histones consists of the largely bind to DNA to form chromosomes are divided into structurally undefined but highly evolutionarily con- two groups: the histone and the non-histone proteins. served ‘tail’ domains. X-ray crystal structures of the The complex of both groups of protein with the nuclear nucleosome core particle show that the tail domains DNA is known as chromatin. The total histone mass in follow the minor grooves of DNA to the exterior of chromatin is approximately equal to the DNA (Bow- the nucleosome. man and Poirier 2014; Bulger 2005; Crane-Robinson The tails of histone H3 and H2B follow aligned et al. 1997; Olins and Olins 2003; Xhemalce et al. minor grooves between adjacent DNA super-helical 2011; Zhang and Dent 2005). Histone proteins are gyres, while those of H2A and H4 follow minor responsible for the first and most basic level of chro- grooves over or under the top/bottom super-helical mosome organization, consisting of the nucleosome, turns such that the bulk of the tail domains exit to the which was discovered in 1884 by Albrecht Kossel exterior of the nucleosome and cannot be structurally (Chen et al. 2014). located in the NCP crystal (Bulger 2005; Luger et al. Most of the chromatin in the cell is in the form of a 1997). These histone tails do not contribute to estab- 30-nm filament throughout most of the cell cycle, and lishing the structure of individual nucleosomes signif- at interphase, appears to be looped onto the nuclear icantly; however, they play an essential role in matrix. At mitosis, the 30-nm filament is subjected to regulating the condensation degree of chromatin into further levels of folding to give the highly dense higher-order structure (Peterson and Laniel 2004; metaphase chromosome in which the overall length Sawan and Herceg 2010). In vitro, removal of the compaction of the DNA is about 10,000-fold. The histone tails results in nucleosomal arrays that cannot 30-nm filament is not a suitable template for tran- scription by the large eukaryotic RNA polymerases and clearly has to be unfolded first; formation of an ‘open’ (‘transcriptional competent’) chromatin state is an essential prerequisite for transcription (Sawan and Herceg 2010). The nucleosome is the fundamental repeating unit of chromatin and is the first level of compaction of DNA in eukaryotes (Maleszewska et al. 2016;Mu¨ller and Muir 2014; Shogren-Knaak et al. 2006). Each nucleosome consists of a 145–147 bps DNA molecule wrapped around a histone octamer which comprises two copies of four histone proteins, H2A, H2B, H3 and H4 (Cao and Yan 2012; Mal- eszewska et al. 2016; Rogakou et al. 1998; Stu¨tzer et al. 2016) (figure 1). The nucleosomes are joined by linker DNA and histone H1 to form chromatin (Crane-Robinson et al. 1997). All histones have a Figure 1. X-Ray Structure of the nucleosome core particle; similar structure of the globular domain and unstruc- pdb code (1kx5). DNA is wrapped around two copies each tured N-terminal ‘tail’. Two dimers of H3/H4 of H2A, H2B, H3, and H4. Evaluation of post-translational modifications in histone proteins Page 3 of 29 135 Table 1. Different types of PTMs and the amino acids modified in histone proteins

Residues Degree of Modification modified modification Abbreviation Functions Regulated Acetylation Lysine (K) Mono ac Transcription, replication, condensation, gene silencing, DNA repair, and cell – cycle progression (Vendone et al. 2005) Methylation Lysine (K) Mono me1 Transcription, repair (Lohrum et al. 2007;Ng Di me2 et al. 2009) Tri me3 heterochromatin formation, X-chromosome inactivation, transcriptional regulation and regulation of gene expression (Martin and Zhang 2005; Shi et al. 2012) Methylation Arginine (R) Mono me1 DNA repair, and transcription, signal transduction Di- me2s and transcriptional regulation, protein symmetric mea2s translocation, ribosomal biosynthesis and all Di- aspects of RNA metabolism (Litt et al. 2009; asymmetric Murn and Shi 2017; Wesche et al. 2017) Phosphorylation Serine (S) Mono ph Transcriptional regulation, apoptosis, cell cycle threonine (T) progression, DNA repair, chromosome tyrosine (Y) condensation, developmental gene regulation, and heat shock-induced pathways (Banerjee and Chakravarti 2011;Loet al. 2005) Ubiquitylation Lysine (K) Mono ub1 Repair, transcriptional activation and silencing (Cao and Yan 2012; Sawan and Herceg 2010) SUMOylation Lysine (K) Mono sum Replication, meiosis and mitosis, cell division, transcriptional regulation and enhancement of transcriptional activity, nuclear and protein trafficking, and the DNA damage response (Gareau and Lima 2010; Munk et al. 2017) ADP_ribosylation Asparagine (N), Mono ar 1 RNA synthesis, cellular differentiation, heat shock Glutamic acid(E), poly ar n response, or nuclear matrix structure (Boulikas and Arginine (R) 1989) DNA replication, repair and recombination, transcriptional regulation, the unfolded protein response, insulin secretion and immunity, cell cycle regulation, and mitosis (Feijs et al. 2013; Messner and Hottiger 2011)

further condense to 30-nm fiber. Although the highly reported by Stedman and Stedman in 1951 and recently positively charged histone tails are generally interact- in the biochemistry research was shown that histones ing with DNA- binding regions, their essential roles in do block DNA function and lead to suppression of the tail-mediated chromatin folding might involve in RNA synthesis (Allfrey et al. 1964). histone–histone interactions and subsequently inter- Additionally, multiple non-canonical histone iso- nucleosomal aggregation (Banerjee and Chakravarti forms have been identified. Table 1 is referred for the 2011; Sawan and Herceg 2010). Three different groups various histone isoforms. The N- and C-terminal from proteins are involved in regulating chromatin histone tails exit from the nucleosome core and have activity: (1) ATP-dependent chromatin remodeling the potential to interact with adjoining nucleosomes proteins for organizing the histone proteins within and the linker DNA (Xhemalce et al. 2011). Consis- chromatin; (2) histone chaperone proteins for assem- tent with this observation, it is clear that chromatin bling, disassemble or exchange different histones has a crucial role in many nuclear events, such as within chromatin; (3) post-translational modification transcription, DNA damage repair, and replication enzymes for add or remove functional groups from the apoptosis, and cell-cycle regulation (Cao and Yan histone proteins (Hodawadekar and Marmorstein 2012; Chen et al. 2014; Kouzarides 2007; Xhemalce 2007). The inhibitory role of histones was originally et al. 2011). 135 Page 4 of 29 S Ramazi, A Allahverdi and J Zahiri

Figure 2. Schematic representation of PTM in histone tails. The location of each modification is shown in black and the amino acid modified at each position is also shown (K = lysine, R = arginine, S = serine, T = threonine, Y= tyrosine).

1.2 Post -translational modifications (PTMs) condensation during mitosis and meiosis to gene transcription, DNA damage response (Banerjee and In eukaryotes, transcription works in the chromatin Chakravarti 2011; Kaimori et al. 2016; Khan et al. context. Some genes are active in all cell types, 2015), cell cycle control, protein–protein interactions, whereas others are active only in specific cell types. and protein functions (Arnaudo and Garcia 2013; Active genes in higher eukaryotes have a chromatin Duan and Walther 2015; Minguez et al. 2012; structure characterized by a greater sensitivity (7- to Shortreed et al. 2015; Wang et al. 2015a, b) (fig- 10-fold) to the endonuclease DNase I than the bulk of ure 2). Moreover, different types of PTMs like the chromatin in the nucleus. This state precedes active acetylation, methylation, phosphorylation and Citrul- transcription and marks ‘transcriptional competence’; it lination or deimination of the histone core usually is the first step in the control of the transcriptional affect three types of interactions: histone–DNA activity. DNase I sensitivity is probably due to (partial) interactions, histone–histone interactions, and histone– relaxation of the 30-nm filament toward the 10-nm chaperone interactions (Tessarz and Kouzarides 2014). filament state, which is probably facilitated by partial In this review, we focus on the importance of histone loss of linker histones and acetylation of the core his- modifications in biological events. As we will describe tone N-terminal tails. Histone proteins undergo a large below, covalent modifications in histone proteins have number of post-translational modifications (PTMs), essential roles in chromatin structure and controlling such as acetylation, methylation, phosphorylation, the transcription of eukaryotic genes and the disruption ubiquitylation, SUMOylation, and ADP-ribosylation of of these processes lead to differences in human diseases lysine (K), arginine (R), serine (S), and threonine (Labrador and Corces 2003; Nowak and Corces 2004). (T) (Sawan and Herceg 2010). In most cases, PTMs For example, methylation in H3-K10, H3-K28, and have been seen in the N-terminal tails, but recently H4-K21 has been related with gene silencing, while some modifications were found in the histone-core H3-K5, H3-K37, and H3-K80 associate with actively domain as well (Sawan and Herceg 2010). Histone transcribed genes (Wood et al. 2009). Therefore, these modification is likely to control the function of the modifications can regulate chromatin structure directly chromatin fiber, with different modifications yielding and frequently act as binding sites for the recruitment distinct functional consequences (Sawan and Herceg of other non-histone proteins to chromatin (Romanoski 2010). et al. 2015). Acetylation and methylation have hap- According to a growing number of studies within pened on different lysine and arginine residues in his- the last decade, it was discovered that these modifi- tones H3 and H4 and have important roles in cations in the chromatin play a critical role in regu- transcriptionally active or transcriptionally repressed lating many aspects of cell function including gene states of gene expression (Eberharter and Becker 2002; expression, chromosome dynamics, DNA replication, Goto et al. 1999; Kaimori et al. 2016) while phos- DNA repair, chromatin packaging, and DNA phorylation of histone H3 was primarily related to Evaluation of post-translational modifications in histone proteins Page 5 of 29 135

Figure 3. The prevalent modifications to lysine and arginine residues. (a) In methylation, lysine contains one, two or three methyl groups (left) that do not effect positive charge in lysine residue; In acetylation modification, lysine can connect to a single acetate group (middle) and lead to neutralizing the positive charge of lysine; or lysine residue can form an isopeptide bond with the C-terminal glycine of ubiquitin or SUMO (right). (b) Arginine in the methylation process can be modified with a single methyl group (left), or with two methyl groups that can be arranged symmetrically or asymmetrically. chromosome condensation during mitosis (Preuss et al. covalent addition to the N-terminal and C-terminal of 2003; Zhiteneva et al. 2017). histone tails and to examples of modifications within the globular domain (Sawan and Herceg 2010). His- tone PTMs are key regulatory factors of the nucleo- 1.3 Histones post-translational modifications some structural changes, nonetheless; the molecular mechanisms related to PTMs function remain not In the early 1960s Vincent Allfrey’s determined that understood (Simon et al. 2011). In this article, the histones contain PTMs (Bannister and Kouzarides different types of modifications in histone proteins 2011). Histone modifications have been identified as a have been investigated. Structurally, histones can be 135 Page 6 of 29 S Ramazi, A Allahverdi and J Zahiri

Figure 4. Each histone molecule has a long and rich tail in lysine residues (K) which are the sites of enzymatic modification, such as acetylation. Acetylation has a key role in the charge of the molecule and leading to DNA unwinding. Thus, gene activation and repression are regulated by the acetylation of core histones and enable the transcriptional machinery to access gene promoters. This particular modification is carried out by enzymes called histone acetyltransferases (HATs) and histone deacetylase (HDAC). divided into the core domain (two each of H2A, H2B, PTMs involves the addition or removal as small as just H3, and H4) and the protein (Schiza et al. 2013) a few atoms, such as acetyl (Lys), methyl (Lys, Arg, (figure 3). Gln), phosphoryl groups (Ser, Thr), or as large as an Generally, epigenomics refers to study mechanisms entire protein in the case of ubiquitin or SUMO (Lys) without a change in the primary DNA sequence lead to by specific enzymes (figure 4) (Bulger 2005; Hoda- regulating gene expression in a cell (Romanoski et al. wadekar and Marmorstein 2007; Huang et al. 2013; 2015; Wood et al. 2009). Epigenetic regulation of gene Sawan and Herceg 2010; Wood et al. 2009). More than expression involves three major mechanisms, DNA 60 residues of histones have been detected which methylation at the CpG islands of gene promoters, include different modifications (Sawan and Herceg chromatin remodeling, and histone modifications 2010). Generally, the histones contain different types of (Jaenisch and Bird 2003; Rotili and Mai 2011). PTMs, for example, lysine acetylation, arginine and Although these systems are carried out by different lysine methylation (mono-, di-, and tri-methylation), chemical reactions and need dissimilar sets of enzymes, phosphorylation, proline isomerization, and ubiquiti- there seems to be a biological relationship between nation by specific modifying enzymes. But other DNA methylation and histone modification which modifications have been reported, such as SUMOyla- plays a part in modulating gene repression program- tion, deamination, ADP-ribosylation, arginine citrulli- ming in the organism (Cedar and Bergman 2009). nation, N-formylation, crotonylation, propionylation, For the first, epigenetics was discovered by CH and butyrylation, as well as proline and aspartic acid Waddington as ‘the causal interactions between genes isomerization, and biotinylation (Hassan and Zempleni and their products, which creates the phenotype into 2008; Sadakierska-Chudy and Filip 2015;Tanet al. being’. After a while, it became clear that epigenetics 2011; Wood et al. 2009). involved in many biological processes such as DNA It has been observed that directly acetylation, phos- methylation, noncoding RNA, histone variants, and phorylation, and ubiquitylation modifications have histone PTMs. Hence, the new definition is, ‘the study affected the structure of chromatin. However, acetyla- of heritable changes in gene expression which happen tion and phosphorylation led to a reduction in the net independently of changes in the primary DNA positive charge of histones, and thus, weaken electro- sequence’ (Khan et al. 2015). DNA methylation and statically interactions with the negatively charged DNA. histone modifications are the mechanisms that collec- PTMs effects on the biophysical properties of the target tively define epigenetics (Lohrum et al. 2007). Histone protein and create a docking site for specific interaction Evaluation of post-translational modifications in histone proteins Page 7 of 29 135 partners, interfere with binding events of other factors, 1.4.1 Histone acetylation: Histone acetylation was or act through a combination of these mechanisms discovered by Allfrey in 1964 (Crane-Robinson et al. (Mu¨ller and Muir 2014). Often, this modification occurs 1997; Kingston and Narlikar 1999; Kouzarides 2000; in histones H3 and H4 that are highly correlated with Sawan and Herceg 2010; Yang and Seto 2007), and is gene expression (Sawan and Herceg 2010; Zentner and the first studied type of histone modification. It was Henikoff 2013). For example, acetylation and methy- initially linked to transcriptional activation and subse- lation of histones H3 and H4 have been related to quently linked to such diverse processes as gene transcriptional activation or repression of certain genes silencing, DNA repair, and cell- cycle progression (Kaimori et al. 2016), while phosphorylation of these (Vendone et al. 2005). This particular modification is histones during mitosis and meiosis have been entailing carried out by enzymes called histone acetyltrans- in chromosome condensation, while the same modifi- ferases (HATs), which catalyze the transfer of an acetyl cation in interphase cells regulates gene transcription group from acetyl Co-A to the lysine e-amino groups (Banerjee and Chakravarti 2011). For instance, histone on the N-terminal tails of histones (Vendone et al. acetylation in H3 at K9, 14, or 27 plays an important 2005). The enzymes that act to reverse this activity are role in transcription start sites (TSSs) and enhancers of known as histone deacetylase (HDAC). Histone transcriptionally active genes and DNA repair, and acetylation is a reversible process and involves in the DNA replication. Histone H4 acetylated in K5, 8, 12, regulation of many cellular processes such as chro- and 16 is associated with highly active genes, but matin dynamics and transcription, gene silencing, cell acetylation in K16 has a key role in cellular senescence cycle progression, apoptosis, differentiation, DNA (Kaimori et al. 2016) and is selective for interphase replication, DNA repair, nuclear import, and neuronal histones (Zhiteneva et al. 2017). repression (Kuo and Allis 1998;Liet al. 2017; Xhe- The histone proteins can contain a couple of PTMs, malce et al. 2011;Xuet al. 2005). Furthermore, this and the modified sites are often on the histone N-ter- modify regulates many varied functions, counting minal tails (Maleszewska et al. 2016). Moreover, the DNA recognition, protein–protein interaction and pro- C-terminal tails and the non-tail regions contain PTMs; tein stability (Kouzarides 2000). Histone acetylation however, they are few in number (Wood et al. 2009). typically occurs within the histone N-terminal tails that The histone tails are readily accessible to various include adding enzymatically an acetyl group from modifying enzymes; it means the tails can add or acetyl-coenzyme A to the epsilon-amino group of remove covalent modifications that are called ‘writers’ lysine residues in histones by ‘histone acetyltrans- and ‘erasers’ respectively (Fan et al. 2015; Sabari et al. ferases’ (HATs). (Li et al. 2017), but lysine acetylation 2017; Sadakierska-Chudy and Filip 2015). The N-ter- at the histone fold region and C-terminal tails are also minal and C-terminal tails of histones are affected by reported (Simon et al. 2011). N-terminal tails of his- the reversible PTMs that change their interaction with tones are involved in acetylation process at the posi- DNA and are used as a docking station for nuclear tions of H2A-K5, H2B-K2, 12, 15 and 20, H3-K9, proteins (Sadakierska-Chudy and Filip 2015). Cur- K14, 18 and 23, 27, and H4-K5, 8, 12, and 16 (Yang rently it has been discovered that histone modification 2004; Zentner and Henikoff 2013). Acetylation in these crosstalk can occur between different histones, and the regions has essential roles in interactions with DNA greatest number of modifications on the H3 histones and therefore regulates transcription and repression and then in the H4, H2B, and H2A histones have been (Nadal et al. 2018; Simon et al. 2011). observed (Lohrum et al. 2007). It is not only the core However, acetylation can occur at the alpha-amino histones that are containing to PTMs, but on the other group in the N-termini of histone proteins and cat- hand, the linker histone H1 can also be modified such alyzed by N-terminal acetyltransferases (NATs). This that H1 plays significant roles in the stabilization of modification is one of the major acetylations in higher-order chromatin structure (Starkova et al. 2017; eukaryote proteins and acts as a protein degradation Wood et al. 2009). signal, an inhibitor of endoplasmic reticulum (ER) translocation, and a mediator of protein complex for- mation (Starheim et al. 2012). This modification is 1.4 Types of post-translational modifications more abundant in histones H2A and H4 and has a in histone proteins range of molecular and biological roles, including regulation of protein degradation, protein translocation, In the following subsections the six most studied his- protein complex formation, membrane attachment, tone PTMs have been described in greater detail. apoptosis and cellular metabolism (Schiza et al. 2013). 135 Page 8 of 29 S Ramazi, A Allahverdi and J Zahiri Acetylation of lysine is performed by the opposite 1.4.1.1 .1 Role of histone acetylation in transcrip- action of two families of enzymes, histone acetyl- tion The correlation between histone acetylation and transferases (HATs) and histone deacetylases (HDACs) transcriptional activity was proposed 40 years ago by (Bannister and Kouzarides 2011; Bulger 2005). Iden- Allfrey et al. (1968). In addition, transcriptional acti- tification of the first histone acetyltransferases (HATs) vation is generally correlated with histone acetylation, and histone deacetylases (HDACs) were in the mid- and repression is often associated with histone 1990s (Sawan and Herceg 2010; Yang and Seto 2007). deacetylation. Several studies have proven that this The HATs use acetyl CoA as a cofactor and catalyze correlation is not exclusive. Still, histone acetylation the transfer of an acetyl group (COCH3) to the e-amino emerges as a central switch that allows inter-conversion group of lysine side chains, resulting in chromatin between permissive and repressive chromatin struc- opening and gene activation (Han et al. 2016), whereas tures and domains (Vendone et al. 2005). One major HDACs decreased acetylation and is associated with point which is still not clear is related to the role of repression of gene expression (Kingston and Narlikar histone acetylation in controlling gene expression 1999). (Vendone et al. 2005). There are two possible (not HATs play a critical role in controlling histone H3 mutually exclusive) explanations for the molecular and H4 acetylation and these families of enzymes are mechanism underlying the effects exerted by this par- the two main classes (type-A and type-B) (Ho- ticular modification: histone acetylation changes the dawadekar and Marmorstein 2007). More than 20 structure of the nucleosome and chromatin or histone HATs have been identified which can be classified into acetylation provides a signal for protein binding four families: N-acetyltransferases (GNAT1), p300/ (Vendone et al. 2005). According to the first explana- CBP, MYST, and TF-related family (Han et al. 2016; tion, changes in the structure of the nucleosome, the Hodawadekar and Marmorstein 2007; Kouzarides addition of an acetyl group to the lysine e-amino 2000; Kuo and Allis 1998; Yang and Seto 2007). In groups of histone N- terminal tails neutralizes the contrast, HDAC enzymes functionally are opposed to positive charge of the lysine side chain. This modifi- the HATs enzymes and reverse lysine acetylation (Yang cation may affect the interaction between the lysine and Seto 2007). HDAC enzymes create the DNA residue and DNA, leading to weaker contact with the tightly wrapped around the histone cores, leading to the negatively charged DNA and subsequently losing suppression of gene transcription (Bannister and Kou- chromatin compaction (Vendone et al. 2005). Accord- zarides 2011; Kouzarides 2007). This restores the ing to the second mechanism, the addition of an acetyl positive charge of the lysine. HDACs are two families; group to lysine residue creates a new surface for pro- the classical and silent information regulator 2 (Sir2)- tein association, thus exerting its effects through gain related protein (Sirtuin) families and HDAC families of of function mechanisms. Lysine acetylation creates enzymes are four classes (Hodawadekar and Mar- docking sites for a protein module known as bromod- morstein 2007; Yang and Seto 2007). There are 18 omain, which has been found to be present in many HDAC enzymes in humans that are classified based on transcription and chromatin regulators. For example in their homology into three main classes (Marks and S. cerevisae there are 10 bromodomain proteins; in Breslow 2007; Seto and Yoshida 2014). Generally, it Drosophila about 16; and in humans, more than 30 was reported that acetylation in histone H3K56 is bromodomains are recognized (Vendone et al. 2005). essential for DNA replication, repair, and transcrip- Several studies have led to the identification of at least tional activation (Tessarz and Kouzarides 2014;Ke 5 functions for bromodomains. However, whether all Zhang and Dent 2005) and histone H4-K20 is a major bromodomains are able to recognize acetyl-lysine is methylation site, can also be acetylated that is associ- currently unknown. In addition, evidence has been ated with gene repression (Kaimori et al. 2016; Simon reported of a bromodomain-containing protein, P300, et al. 2011). This modified in H4-K5, 12 is essential for that binds to histone in an acetylation-independent the deposition of newly synthesized core histones and manner. One of the functions of bromodomains in gene chromatin assembly (Yang 2004) and is associated in regulation is chromatin acetylation by HATs. In S. H3K4me1(enhancers) and H3K27 acetylation cerevisiae the bromodomain of the acetyltransferase (H3K27ac; enhancer/promoter activation) (Gjoneska Gcn5 is required for the coordination of nucleosome et al. 2015). But, table 2 completely refers to the sites remodeling. Another function of bromodomains is to of acetylation and other five PTMs in the histone link the activity of ATP-dependent chromatin remod- proteins. elers to the acetylation of specific lysine. Selective Evaluation of post-translational modifications in histone proteins Page 9 of 29 135 Table 2. Sites of histone post-translational modifications and their known functions

Histone Modification Role References

H1 H1-S/T ph Mitosis; Transcription Rossetto et al. (2012) H2A H2A-S1 ph Mitosis: chromatin assembly Barber et al. (2004) H2A-S16 ph EGF signaling Rossetto et al. (2012) H2A-S122ph DNA repair; mitosis; meiosis Rossetto et al. (2012) (Sc) H2A-S129ph DNA repair Rossetto et al. (2012) (Sc) H2AX-S139ph DNA repair Rossetto et al. (2012) (Hs) H2A-T120ph DNA repair; mitosis; meiosis Rossetto et al. (2012) (Hs) H2AX-Y142ph Apoptosis; DNA repair Rossetto et al. (2012) H2A-K4/5ac Transcriptional activation Fusauchi and Iwai (1984) H2A-K7ac Transcriptional activation Suka et al. (2001) H2A-K119ph Spermatogenesis Baarends et al. (2007) H2A-K119ub Transcriptional activation Wang et al. (2004) H2B H2B-S10ph Apoptosis; meiosis Cheung et al. (2003) H2B- S14ph Apoptosis; meiosis Rossetto et al. (2012) H2B-S32ph EGF signaling Rossetto et al. (2012) H2B-S33ph Transcriptional activation Maile et al. (2004) H2B-S36ph Transcription Rossetto et al. (2012) H2B-K5ac Transcriptional activation Golebiowski and Kasprzak (2005) H2B-K11/12ac Transcriptional activation Suka et al. (2001) H2B-K15/16ac Transcriptional activation Suka et al. (2001) H2B-K20ac Transcriptional activation Golebiowski and Kasprzak (2005) H2B-K120ub Spermatogenesis/meiosis Baarends et al. (2007) H2B-K123ub Transcriptional activation Robzyk et al. (2000) (Sc) H2B-Y40 (Sc)/ Transcription Rossetto et al. (2012) Y37 (Hs)ph H3 H3K4-me2 Permissive euchromatin Santos-Rosa et al. (2002) H3K4-me3 Transcriptional elongation; active euchromatin Rossetto et al. (2012), Strahl et al. (1999), Zhang et al. (2002b) H3K9-me Transcriptional activation Khan et al. (2015) H3K9-me2 Transcription initiation and repression Khan et al. (2015) H3K9-me3 Transcriptional repression; imprinting; DNA methylation; gene Barski et al. (2007), Rea et al. (2000), Wood and silencing; heterochromatin formation Shilatifard (2004) and Zhang et al. 2002a H3R17-me Transcriptional activation Bauer et al. (2002) and Chen et al. (1999) H3K27-me3 Transcriptional silencing; X-inactivation; bivalent genes/gene Zhang et al. (2002b) and Barski et al. (2007) poising H3K36-me3 Transcriptional elongation Zhang et al. 2002b H3K4-ac Transcriptional activation Strahl et al. (1999) H3K9-ac Histone deposition; Transcriptional activation; Transcription Khan et al. (2015) and Suka et al. (2001) initiation H3K14-ac Transcriptional activation; DNA repair Tan et al. (2011) H3K18-ac Transcriptional activation; DNA repair; DNA replication Tan et al. (2011) H3K23-ac Transcriptional activation; DNA repair Tan et al. (2011) H3K27-ac Transcriptional activation Tan et al. (2011) H3T3-ph Mitosis Zhiteneva et al. (2017) H3T6-ph Transcription Rossetto et al. (2012) H3T11-ph Meiosis (Sc); Mitosis (Hs) Transcription DNA damage response Rossetto et al. (2012) H3S10-ph Mitosis; meiosis; Transcriptional activation Sawan and Herceg (2010) H3S28-ph Mitosis; Transcriptional activation Rea et al. (2000) H3Y41-ph Transcription Rossetto et al. (2012) H3Y45-ph Apoptosis Rossetto et al. (2012) H4 H4R3-me Transcriptional activation Schiza et al. (2013) and Wang et al. (2001) H4K20-me1 Transcriptional silencing Nishioka et al. (2002) H4K20-me3 Heterochromatin Kaimori et al. (2016) H4K5-ac Histone deposition; Transcriptional activation; DNA repair Zhang et al. (2002b) H4K8-ac Transcriptional activation; DNA repair; Transcriptional Zhang et al. (2002b) elongation H4K12-ac Histone deposition; telomeric; silencing; Transcriptional Zhang et al. (2002b) activation; DNA repair H4K16-ac Transcriptional activation; DNA repair Shogren-Knaak et al. (2006) H4K20-ac Transcription repression; gene repression Kaimori et al. (2016) H4S1-ph Mitosis Rossetto et al. (2012) H4S47-ph (H3.3-H4) deposition Rossetto et al. (2012) 135 Page 10 of 29 S Ramazi, A Allahverdi and J Zahiri recognition of acetylated histones by bromodomain- model (model 3) involves the recruitment of a histone- containing proteins have been recently established in modifying activity to particular promoters by sequence- the intact nuclei of a living cell by FRET analysis specific, DNA-binding proteins (figure 5). (Vendone et al. 2005). The best studied for such gene-specific targeting is transcriptional repression by Sin3-HDAC/Rpd3 histone deacetylase complexes (Struhl 1998). This repression 1.4.1.2 Model for how histone acetylase and deacety- mechanism appears to be conserved from yeast to lase selectivity affect gene expression Although his- mammals, and the supporting arguments for this tones are associated with virtually all eukaryotic mechanism derive and benefit from complementary genomic DNA sequences, histone acetylases and experimental approaches and observations. First, the deacetylases do not universally affect the transcription yeast and mammalian histone deacetylase complexes of all genes. In yeast cell, Gcn5, and Rpd3 are specifically interact with DNA-binding repressor pro- important for the transcription of a small subset of teins (e.g. Mad, YY1, Ume6) or their associated core- genes; expression of most genes is unaffected in Gcn5- pressors (NCor, SMRT). Second, studies from or Rpd3- deletion strains (Struhl 1998). These obser- mutational analysis indicate that the ability of these vations are in accord with the fact that histone acety- proteins to interact physically with histone deacetylase lation patterns are not uniform in eukaryotic complexes is correlated to repress transcription target chromosomes. There are three basic models to account genes in vivo. Third, artificial recruitment of histone for how histone-modifying activities can affect tran- deacetylase complexes to promoters via heterologous scriptional activity in a gene-specific manner (figure 5). DNA-binding domains are sufficient to mediate tran- In the untargeted model (model 1) the histone-modi- scriptional repression; in mammalian cells, this fying activities operate on a genome-wide basis with repression is sensitive to histone deacetylase inhibitors. minimal specificity for particular chromosomal regions. Fourth, yeast Rpd3 deacetylases histone in vivo and In this case, selective gene expression would arise from histone deacetylase activity is important for repression. promoter-specific responses to a generally altered chromatin structure. For example, promoters whose 1.4.2 Histone phosphorylation: Protein phosphoryla- DNA sequences inherently cause nucleosomes to be tion was first reported in 1906 by Phoebus Levene at tightly packed or preferentially positioned over binding the Rockefeller Institute for Medical Research with the sites for transcription factors might be particularly discovery of phosphorylated vitellin (phosvitin) sensitive to acetylation status (Struhl 1998). Thus, (Levene and Alsberg 1906) and in 1932 phosphoserine histone acetylases that function by general promoter in casein protein was discovered by Fritz Lipmann targeting might be part of the mechanisms that account (Lipmann and Levene 1932). However, it took another for the long-standing and the general correlation 20 years for Eugene P Kennedy to describe the first between an active chromatin structure and gene ‘enzymatic phosphorylation of proteins’ (Burnett and transcription. Kennedy 1954; Choudhury 2016; Pawson and Scott For each model, sites of acetylation (green Ac) are 2005). ‘Histone phosphorylation’ was first observed in indicated with respect to five promoters (arrows) that the late-1960s and the first kinase responsible for either is (blue) or not (green) affected by acetylation. In phosphorylating of histone tail was detected to be an model 1, histone acetylases are not targeted, and his- AMP-dependent kinase (Gutierrez and Hnilica 1967). tone acetylation occurs at promoter regions. In model The phosphorylation of histones is highly dynamic and 2, histone acetylases are generally targeted to promot- the majority of histone phosphorylation sites are within ers (arrows), because of their association with a general the N-terminal tails and give a negative charge to its component of the Pol II transcription machinery. For modified residue (Zentner and Henikoff 2013). The both model 1 and 2 selective effects on transcription DNA backbone has a negative charged due to the are attributable to inherent differences in the promoters presence of phosphate, and addition phosphates in with respect to the state of histone acetylation. In model histones would loosen the association of DNA with 3, histone acetylase is targeted to specific promoters by histones. In fact, phosphorylated histones are effective gene-specific activator proteins, leading to selective for un-phosphorylated histones at inhibiting DNase I effects on transcription. In model 2, the histone-modi- digestion of chromatin (Zentner and Henikoff 2013). fying activity is selectively targeted to promoter Phosphorylation is a reversible PTMs and the levels regions, in a manner that is relatively nonspecific for of this modification are controlled by kinases and individual genes (figure 5). The gene-specific targeting phosphatases and usually take place on serine, Evaluation of post-translational modifications in histone proteins Page 11 of 29 135

Figure 5. Models for how histone acetylation selectively affects transcriptional activity. threonine, and tyrosine residues that add and remove the site can increase acetylation histone H3 at K14 and the phosphate (PO4) group from adenosine triphos- prevent methylation of histone H3 at K9 (Greer and Shi phate (ATP) to receptor residues and thus create ade- 2012). Also, methylation of histone H3 at K9 interferes nosine diphosphate (ADP) (Bannister and Kouzarides with phosphorylation of histone H3 at S10 (Goto et al. 2011). In addition, this modifies in arginine and his- 2002; Nowak and Corces 2004; Prigent and Dimitrov tidine residues have been detected; however, their 2003; Sawan and Herceg 2010). However, phospho- biochemistry is not known due to the chemical insta- rylation in H3 at T3 is a target of Haspin kinase in bility (Mu¨ller and Muir 2014; Nguyen et al. 2015). mitosis, and H3 at T11 is phosphorylated in response to Histone phosphorylation has been associated with a gene activation and is not cell-cycle regulated (Zhite- variety of cellular processes, containing transcriptional neva et al. 2017). Histone H2B amino-terminal tail regulation, apoptosis, cell cycle progression, DNA contains phosphorylation sites in S10, 14, 32, 33, 36 repair, chromosome condensation, developmental gene that is associated with dramatic alterations in higher- regulation, and heat shock induced pathways (Banerjee order chromatin structure during meiosis, chromatin and Chakravarti 2011;Loet al. 2005). Often, post- condensation, response to DNA double-strand breaks, translational phosphorylation occurs in histone H3 and apoptosis, meiosis, and transcription activation events then in histone H2B, H2A, H4, H1. Recently, evidence (Oki et al. 2007; Rossetto et al. 2012). Moreover, has shown N-terminal phosphorylation of H3 at S10 phosphorylation affects the level of ubiquitination of and S28 are vital for chromosome condensation and H2B-K120 and tri-methylation of H3-K4 and H3-K79, cell-cycle progression during mitosis and meiosis. which is a marker for active gene expression (Sueoka Furthermore, it has been detected that H3 S10 phos- et al. 2017). However, in H2A this reaction carries out phorylation has an important role in the transcriptional in S1, 122, 129 and T 57, 119, 126 (Oki et al. 2007; activation of eukaryotic genes in various organisms and Sueoka et al. 2017). Phosphorylation in C-terminal at is associated with activation of transcription at inter- H2A T 119 has been related to the regulation of chro- phase (Banerjee and Chakravarti 2011; Goto et al. matin structure and function during mitosis and this 2002; Labrador and Corces 2003; Prigent and Dimitrov modification in H2A-T57 regulates the stability of the 2003; Rogakou et al. 1998). Phosphorylation of H3 histone H2A–H2B dimer and leads to the regulation of S10,28 has been linked with the activation of tran- transcriptional elongation that inhibits de-ubiquitinase scription in yeast, mammals, and Drosophila. Based on activity in H2B-K120 (Sueoka et al. 2017). In mam- current studies, there exists an interaction between malian, histone type of H2AX is quickly phosphory- phosphorylation of histone H3 at S10 and acetylation lated at S139 upon exposure to DNA damaging agents. or methylation of histone H3 which phosphorylation in It is also proposed that H2AX functions as a tumor 135 Page 12 of 29 S Ramazi, A Allahverdi and J Zahiri suppressor gene, playing a major role in cancer. Thus, associated with active transcription, it has not been it can be said that phosphorylation in histone proteins clear whether they occurred on the same N-terminal tail mediates a variation of chromatin structure, facilitates of histone H3 or if they occurred in H3 molecules in DNA repair and maintains genome integrity (Oki et al. different nucleosomes. Using specific antibodies gen- 2007). In yeast, the H2AX variant does not exist and erated against phosphorylated and acetylated histone this modification occurs on the H2A-S129 in all phases H3 tails has shown that both phosphorylated and of the cell cycle. Furthermore, serine 122 of histone acetylated histone H3 can be observed in vivo. More- H2A is phosphorylated and is together with the nearby over, di-modified H3 isoform was observed in both H2AS129 so that their modification plays a key role in in vivo and in vitro. Further experiments revealed that response to DNA damage (Rossetto et al. 2012). the yeast GCN5 histone acetyltransferase displayed a Phosphorylation of histone H4 at S1 as well as histone preference for binding to a portion of the histone H3 H2A at S1 are evolutionarily conserved modifications tail that is pre-phosphorylated at the Ser-10 position that have separate roles during the mitosis and S-phase in (Yang 2004). Taken together, these results suggest a the cell cycle. For example, in S. cerevisae, phosphory- synergistic mechanism of the addition of each modifi- lation of histone H4 at S1 is associated with DNA dou- cation to the N-terminal tails of histone H3 in the ble-strand breaks and acetylation in this site by the NuA4 nucleosome particles at these promoters, whereby complex has an important role for normal gene expres- MAP-kinase signaling results in the phosphorylation of sion and DNA repair (Oki et al. 2007; Rossetto et al. histone H3 at Ser-10 (Yang 2004). This relationship 2012). Phosphorylation in H1 histone happens via two between acetylation and phosphorylation is shown in major enzymes called CDK1 and CDK2 (Oki et al. (figure 6). 2007), which are associated with cell growth and divi- sion (Zhang and Dent 2005). This modification is 1.4.3 Histone methylation: Methylation research dates increased in dividing cells and decreased in non-prolif- back to 1939 and is one of the PTMs which performed erating or quiescent cells. During the cell cycle, phos- reversibly on the proteins and often occurs in the cell phorylation begins in the late G1 phase and enhances nucleus and on the nuclear proteins such as histone throughout the S and G2 phases. Thus, phosphorylation proteins (Bannister and Kouzarides 2005). Histone in histone’s tails during the cell cycle have reflected the methylation is a transfer of methyl group to lysine, effect of H1 on modulates chromatin condensation and arginine, or histidine residues of histones that result in decondensation and in chromatin folding (Talasz et al. transcriptional activation or silencing. Lysine can be 1996). Furthermore, post-translational phosphorylation methylated with one, two, or three methyl group of this histone tail plays a significant role in regulation of (Bannister and Kouzarides 2005; Martin and Zhang ATP-dependent chromatin remodeling enzyme and 2005) So far, histone lysine methylation has been transcription of some specific genes, including oncoge- found to occurs at six major sites, including histone nes (Oki et al. 2007). H3-K4, H3-K9, H3-K27, H3-K36, H3-K79, and H4- K20 (Barski et al. 2007; Lan and Shi 2009;Nget al. 2009) and can be found near active or poised tran- 1.4.2.1 Correlation between phosphorylation and his- scriptional units. Methylation in histone H3-K9, K20, tone H3 acetylation The Ser-10 amino acid in the K27 plays a vital role in silenced or heterochromatic N-terminal tail of histone H3 has been located in the regions (Ng et al. 2009; Zhang et al. 2015) and H3-K4, region that is subject to other post-translational modi- K3-K36, H3-K79 methylation with actively transcribed fications as well. Moreover, Lys-9 and Lys-14 acety- genes (Lohrum et al. 2007;Nget al. 2009; Zhang et al. lation have been linked with transcriptional activation, 2015). Recently it was reported that H4 K20me plays while methylation of Lys-9 amino acid leads to the an important role in transcriptional activation via being silencing genome and formation of heterochromatin. enriched in active promoters or in transcribed regions Thus, these kinds of modifications may affect the (Barski et al. 2007). However, methylations histone H3 phosphorylation of Ser-10. Recent studies from in vitro at K4, K9 is the best-studied example of histone and in vivo experiments agree with this conclusion methylation-mediated transcriptional regulation (An (Yang 2004). In addition, a recent investigation sup- 2007). Tri-methylation in H3K27 is identified by ported a relationship between acetylation and phos- polycomb group (PcG) proteins, which maintain epi- phorylation in histone H3 by a wealth of experimental genetic silencing (LeRoy et al. 2013). Mono-, di- and data. Although acetylation and phosphorylation act as tri-methylation level in histone H3-K4 were increased two modifications which had been found to be in the surrounding transcription starts sites (TSSs) on Evaluation of post-translational modifications in histone proteins Page 13 of 29 135

Figure 6. A schematic representation of the two pathways of histone H3 N-terminal modification during gene transcription activation. (a) The synergistic effect between histone H3 phosphorylation and acetylation for gene induction. It was found that Lys-9 in histone H3 is acetylated via GCN5 histone acetyltransferase (HAT) activity before phosphorylation at the Ser-10 position. (i) The result of MAPK signaling in the phosphorylation of histone H3 at Ser-10. (ii) Phosphorylation of Ser-10 in histone H3 N-terminal tail increases binding affinity for HAT activity of GCN5, which results in acetylation of H3 at the lysine 14 amino acid. (iii) The acetylation of H3 at lysine 14 position led to enhance in the transcription of the associated genes. (b) The parallel independent pathway of histone modification. (i) Acetylation of histone H3 at lysine 9 and 14 is regulated by an equilibrium between the endogenous HAT and histone deacetylase (HDAC) activity. (ii) Kinase activity coming from either MAPK-signaling or gene induction by phosphorylation of pre-acetylated lysine 9 and 14 in histone H3 tail. (iii) Transcription from the genes associated with nucleosomes having di-acetylated, Ser-10 phosphorylated histone H3 N-terminal tail. Abbreviations: K, lysine; MAPK, mitogen-activated protein–threonine kinase; Ser, serine. identified genes. H3K4me3 is correlated primarily with lysine methyltransferases (Peterson and Laniel 2004). active promoters and gene expression, and H3K4me1 The closely related SET domain proteins Suv 39h1 and with enhancers, while methylation of H3K9 has been Suv 39h2 were shown to be essential for normal his- involved in heterochromatin formation and gene tone methylation within heterochromatin; deletion of silencing and is present in pericentromeric and telom- Suv 39 h1 and Suv 39h2 genes reduced net methylation eric regions (Barski et al. 2007; Maleszewska et al. of histone H3 lysine (H3-K9) by approximately one- 2016). Morover, it was discovered on the inactive X half, and in wild-type cells Suv 39h1 was seen to chromosome and at silenced promoters (Egger et al. colocalize with the polycomb group protein HP1 at 2004). heterochromatic regions of interphase nuclei (Banerjee Rea et al. found that many (not all) mammalian and Chakravarti 2011). The phenotype of mice that proteins that contain a SET (suppressor of variegation, lack a second H3-K9 methyltransferase acts in the Enhancer of Zeste, and Trithorax) domain are histone euchromatic compartment. Loss of this histone 135 Page 14 of 29 S Ramazi, A Allahverdi and J Zahiri methyltransferase (G9a) reduced total levels of H3-K9 different HMTs (Wood and Shilatifard 2004). This methylation to approximately one-eighth of wild-type covalent modification has important functions in many and residual H3-K9 methylation was localized to biological processes that include heterochromatin for- pericentric heterochromatin, where histones are under- mation, X-chromosome inactivation, transcriptional methylated in the Suv 39h null mutant. G9a-deficient regulation, and regulation of gene expression (Martin mice showed defects in Lys9 methylation at euchro- and Zhang 2005; Shi et al. 2012) and has important matic sites and suffered a more severe phenotype than roles in cell-cycle regulation, DNA damage and stress did suv39h-null mutants. In Drosophila, H3-K9 response, development, and differentiation (Greer and methylation demonstrates an intimate association both Shi 2012). However, downregulation of a SET-domain with cytological heterochromatin and the heterochro- function can be causal of many types of cancer (Sch- matin binding protein HP1. It has been shown recently neider et al. 2002; Zhang and Dent 2005). that the HP1 chromo-domain specially binds histone In recent studies, it has been exemplified that argi- H3 N-terminal tails when those tails are methylated at nine in histone H3R2, R8, R17 and R26, H4R3 and H3 Lys9 (Luger et al. 1997). It has been suggested H3R42 are methylated and has roles in defining both previously that two distinct modification states were active and repressed chromatin states (Barski et al. interchanged by means of a switch between H3-K9 2007;Nget al. 2009; Tessarz and Kouzarides 2014). methylation and H3 Ser10 phosphorylation. It may be Protein arginine methyltransferases (PRMTs) are that H3 Lys9 methylation mediates transcriptional enzymes for catalyzing arginine methylation and target modulation in euchromatin regions, whereas its role in multiple arginine residues on the N-terminal tails of heterochromatin is related to chromosome structure. histone H3 and H4 (Ng et al. 2009; Santos-Rosa et al. An interesting question that stems from this distinction 2002). There are two types of PRMTs: the type I in function is how two different H3 Lys 9 methylation PRMTs (PRMT1, PRMT3, CARM1/PRMT4, and events might be distinguished (Banerjee and Chakra- Rmt1/Hmt1) catalyzes mono- and asymmetric di- varti 2011)? methylation of arginine, and the type II methyl trans- It is worth mentioning that unlike acetylation and ferases (JBP1/PRMT5) catalyzes mono- and symmetric phosphorylation, histone methylation does not alter the di-methylation of arginine. PRMTs exist in fungi, charge of the histone protein and is stable (Bannister plants, Caenorhabditis elegans, Drosophila, and ver- and Kouzarides 2011; Sadakierska-Chudy and Filip tebrate animals (Litt et al. 2009), and so far, several 2015; Wood and Shilatifard 2004; Xhemalce et al. PRMTs are known for histone proteins. Saccharomyces 2011; Xie et al. 2007). In addition, it is possible lysine Rmt1, as well as in the mammalian PRMT1, PRMT5, mono-, di- or tri-methylated and each methylation state JBP1, and CARM1, have histone methyl transferase may confer a distinct biological impact, while arginine activity (Wood and Shilatifard 2004). In mammals, may be mono-methylated symmetrically or asymmet- methyl transferases (PRMT1 and CARM1) produced rically di-methylated (Bedford and Clarke 2009;Mu¨ller asymmetric dimethyl-arginine which involves in gene and Muir 2014; Zentner and Henikoff 2013). In this activation. Whereas methyl transferase (PRMT5) has modification, methyl groups transferred from the ability to form symmetric dimethyl-arginine which S-adenosyl-L-methionine to lysine or arginine residues associate in gene repression (Wysocka et al. 2006). of histone proteins by histone lysine methyltransferases Arginine methylation have pivotal roles in functions (HMTs), and deletion of a methyl group by lysine such as DNA repair and transcription, signal trans- demethylases enzyme occurs (Greer and Shi 2012; duction and transcriptional regulation, protein translo- Sadakierska-Chudy and Filip 2015; Schiza et al. 2013; cation, ribosomal biosynthesis and all aspects of RNA Wesche et al. 2017). The first histone methyltransferase metabolism, including mRNA transcription, splicing, (HMTs) on lysine was discovered in 2000 and so far, transport, and translation (Litt et al. 2009; Murn and have been identified as an extended family of histone Shi 2017; Wesche et al. 2017); in recent research it has lysine methyltransferases. The majority of HMTs share been suggested the arginine methylation can play a key a SET domain as a catalytic core in the N-terminal tail role in the expression genes involved in tumor sup- of histones (Ng et al. 2009). HMTs in SET-domain pression (Litt et al. 2009; Martin and Zhang 2005; include approximately 130 amino acids in length. Singh et al. 2004). Some areas of the sequence in SET-domain are highly In histone proteins, methylation occurs more fre- conserved between species; others are much more quently in H3 and than in H4. Methylation on lysines varied. The upstream and downstream sequence and may have extremely diverse consequences and be structure related to SET-domain can differ between recognized as a marker of transcriptionally active Evaluation of post-translational modifications in histone proteins Page 15 of 29 135 ‘euchromatin’ or transcriptionally repressed ‘hete- histone antibody processes and they can be arranged rochromatin’ (Greer and Shi 2012; Sawan and Herceg symmetrically or asymmetrically. For instance, in his- 2010). For instance, methylation in histone H3-K9 and tone H3 at R2, 17, 26 residues and H2A-R3 are asso- H4 at K20, K27 in the form of me2/me3 connected ciated with dimethylation asymmetrically, and with heterochromatin formation, and transcriptional dimethylation symmetrically in histone H3-R8 and repression (Barski et al. 2007; Bulger 2005; Egger H2A-R3 was recognized (Di Lorenzo and Bedford et al. 2004; Jones and Baylin 2007; Lohrum et al. 2011; Greer and Shi 2012; Strahl et al. 1999). 2007; Sadakierska-Chudy and Filip 2015). On the contrary, histone H3-K4, K36, and K79 methylation 1.4.3.1 Lysine methylation and acetylation of his- correlate with euchromatin (Bedford and Clarke 2009). tones The studies showed that enzymatic acetylation Additionally, it has been found that histone of the e-amino group in conserved lysine amino acids monomethylation at H3-K27, K9, K20, K79, and H2B in the N- terminal tail of H3 and H4 histones reduce the K5 related to gene activation, whereas tri-methylation positive charge. Moreover, the recent discoveries sug- of H3-K27, K9, and K79 are correlated to repression gest that histone acetylation may change the structure (Barski et al. 2007). This process occurred on histone of histone tails via increasing their alpha-helical con- tail in H3 K4me1, me2 and me3, and H3-K36me3, and tent (Luger et al. 1997). In contrast, methylation does was detected in promoters of active genes and sur- not alter the overall charge of the histone tails; how- rounding transcription start sites (Barski et al. 2007; ever, the addition of methyl group (mono-, di- or tri-) Egger et al. 2004), and H3-K4-me1 is a suitable marker increases its basicity and hydrophobicity (figure 7) for best predicts enhancer regions, whereas there exists (Luger et al. 1997). In contrast to acetylation, it has a difference between the di- and tri- methylated state in been found that methylation has an irreversible effect the H3. Di-methylation occurs at both inactive and on chromatin structure and function. Some investiga- active euchromatic genes, while tri-methylation is tions showed that histone methylation to be biochem- present only at active genes; consequently, the exis- ically stable and irreversible. In other words, tence of a tri-methylated at K4 describes an active state methylation could be a dead-end modification (Allfrey of gene expression (Howe and Gamble 2016; Santos- et al. 1964; Hodawadekar and Marmorstein 2007). In Rosa et al. 2002; Wood and Shilatifard 2004). In some organisms, LSD1 was reported as a nuclear mammals and yeast, di-methylation of H3R2 is pre- homolog of amine oxidases functions as a histone vented by H3-K4me3; conversely, H3R2me2 prevents demethylase (Chen et al. 2014). However, in recent H3-K4 methylation (Greer and Shi 2012). Furthermore, studies, it has been observed as a mechanism for methylation H3-K79 catalyzed by Dot (disruptor of methylation (similar to acetylation) that this process telomeric silencing 1) is important for silencing at the occurs at specific residues at specific promoters (Luger telomeres that leads to restricts Sir protein binding to et al. 1997). the telomeres (Wood and Shilatifard 2004). Conserved lysine residues located in N-terminal of Methylation in histone H4 arginine 3 (H4R3) can be histone proteins H3 and H4 are the major targets for catalyzed by the protein arginine methyltransferases PTMs in both acetylation and methylation; these PRMT1 and PRMT5, which are involved in nuclear modifications can happen on the H2A and H2B tails. receptor-mediated transcriptional activation (Barski Acetylation of specific lysine amino acid in a desired et al. 2007). Methylation asymmetrically in histone H4 residue is only catalyzing by specific histone acetyl arginine 3 (H4R3) is a lesser extent arginine 3 in his- transferase (HAT). Histone methylation is a non-ran- tone H2A, which is associated with active transcription dom event in vivo, which is similar to acetylation. In in mammals and is catalyzed by the PRMT1enzyme this process specific lysines are methylated by spe- (Sawan and Herceg 2010; Schiza et al. 2013; Strahl cialized histone methyl transferase (HMT). Histone et al. 1999; Wang et al. 2001; Wysocka et al. 2006). methyl transferases are similar to HATs, and have their Histone modifications are often investigated through own kinetic parameters, catalytic site, as well as histone antibody-based techniques, such as Western blot anal- and lysine as substrate (Luger et al. 1997). ysis (Huang et al. 2013). Methyl-specific antibodies Histone acetylation takes place in the whole of the have been known in the arginine methylation sites on cell cycle; however, histone methylation is observed histone tails and these antibodies have been utilized in mostly in the G2 phase. This phenomenon is due to several studies, including traditional ChIP, ChIP-chip, DNA replication and histone synthesis in S phase that and ChIP-seq experiments (Fuchs and Strahl 2011). chromatin required to be more open. An example of the There are several effective sites of methylation in synergistic effect of histones acetylation and 135 Page 16 of 29 S Ramazi, A Allahverdi and J Zahiri

Figure 7. Histone methylation versus histone acetylation (Luger et al. 1997). (a) The molecular structure of lysine, acetyl- lysine, and methyl lysine. Conserved lysine amino acid within histone N-terminal can be modified by acetylation or methylation. Acetylation is catalyzed by HAT and the removing of an acetyl group histone tail residue is catalyzed and regulated by histone deacetylase (HDACs), as long as the binding of acetyl to the e-amino group of lysine results in a more acidic, hydrophobic residue. The addition, methyl group is catalyzed by HMTs; however, the demethylase enzymes which are responsible for removing of the methyl group have not been completely identified. (b) The acetyl and methyl PTMs of lysine residues on N-terminal tails of human histone H3 and H4. Here, the conserved lysines are indicated in bold and their amino- acid position in the histone tail indicated by numbers. The in vivo positioning sites for lysine acetylation and methylation are shown. methylation on chromatin unfolding is that hyper- attachment of ubiquitin molecule (ubiquitin is a acetylated H4 is a preferential target of H3 methylation, 76-amino-acid protein) to proteins through the forma- which suggests these modifications may act synergis- tion of an isopeptide bond between the ubiquitin tically to increase transcription level in an unknown C-terminus and the side chain lysine of target proteins. mechanism. Therefore, it can be summarized that In target, proteins can occur mono- or poly-ubiquiti- combinations of acetyl and methyl modifications of nation, and this process involves an enzyme complex lysine in histone tail residue may have synergistic or with three different enzymes: E1 (activating), E2 antagonistic effects (Luger et al. 1997). For example, (conjugating), and E3 (ligase) enzymes (Bhogaraju and dimethylation and trimethylation of H3-K4, often in Dikic 2016; Popovic et al. 2014; Radivojac et al. 2010; combination with H3-K14 acetylation have been an Varshavsky 2017). Until now, its functions were less important role in transcriptional activation, and to the well understood than other histone modifications such contrary, dimethylation and trimethylation in H3-K9 as methylation and acetylation (Cao and Yan 2012). have been associated with chromatin condensation for This reversible reaction in histones have a crucial role transcriptional repression (An 2007). Usually, in pro- in stem cell preservation and differentiation by regu- moter regions of active genes, high levels of acetylation lation of the pluripotency and differentiation genes and methylation in H3-K4 have been identified (Barski expression (Cao and Yan 2012). This process plays et al. 2007). important roles in many various activities of cells like as in the proliferation and differentiation, gene tran- 1.4.4 Histone ubiquitination: Ubiquitination was dis- scription, and regulation of transcription, DNA repair, covered in the late 1970s and is involves the and replication, intracellular trafficking and virus Evaluation of post-translational modifications in histone proteins Page 17 of 29 135

Figure 8. Schematic representation of different types of modifications in histone (H1, H2A, H2B, H3, H4). Most phosphorylation occurs in histone H1. H2A, H2B histone contain more modifications such as phosphorylation, acetylation, and ubiquitination, and in H3, H4 histone occurs methylation, acetylation, and phosphorylation, in which these existing modification in a histone octamer have vital roles in many cellular processes. budding, the control of signal transduction, the impli- remodeling via recruiting chromatin remodeling factors cation of endocytosis and sorting, degradation of the (Cao and Yan 2012). In the recent studies, it was protein, and innate immune signaling (Alonso and observed that H2Bub has several basic roles outside of Friedman 2013; Haglund and Dikic 2005; Micel et al. chromatin and is required for chromosome segregation. 2013; Wagner et al. 2011). The dominant form of ubiquitination in histones are Histone ubiquitination has been linked with both mono-ubiquitination in H2A (H2Aub) and H2B transcriptional activation and silencing. The ubiquiti- (H2Bub) at positions H2AK119 and H2AK123 in yeast nation process was identified for the first in Histone as well as H2BK120 in vertebrate. In addition to H2A H2A by ubiquitin in cells. However, ubiquitination is and H2B, ubiquitination has been reported in core the most common reaction in H2A and H2B histones histones H3, H4, and linker histone H1 (Cao and Yan and has an important role in the inhibition of tran- 2012). Ubiquitination in H2B K120 is selective for scription and in transcriptional activation (Cao and Yan interphase histones and is a mark linked to chromatin 2012; Sawan and Herceg 2010). In female mammals, de-compaction (Zhiteneva et al. 2017). It has been RING1B and H2Aub have a key role in the initiation of shown that in Saccharomyces cerevisiae Rad6 (DNA imprinted and random X chromosome inactivation. repair gene) is involved in the attachment of ubiquitin H2Bub is needed for chromatin boundary integrity that to H2B-K123 which is required as a signal for decreasing the activity causes changes in other histones methylation of histone H3 at K4 and K79 (Greer and PTMs and increasing of H2Bub are associated with Shi 2012; Wood and Shilatifard 2004). chromatin compaction and lead to a closed chromatin structure. Thus, H2Bub has a significant role in 1.4.5 Histone SUMOylation: SUMOylated protein was homologous recombination through chromatin primarily discovered in 1996 (Feligioni and Nistico 135 Page 18 of 29 S Ramazi, A Allahverdi and J Zahiri 2013). In the process of SUMOylation, SUMO proteins 1.4.6 Histone ADP_ribosylation: ADP-ribosylation covalently and reversibly are conjugated to specific was discovered by P Chambon and colleagues (Hassa lysine residues in the target proteins (Matic et al. et al. 2006). This modification is known as one of the 2010). SUMO is a small protein with 100 amino acids reversible types of PTMs in core histones and as well and resembles the three-dimensional structure of as linker histone H1 (Burzio et al. 1979; Messner and ubiquitin that is conserved in all eukaryotes (Droescher Hottiger 2011; van der Heden et al. 2010; Zentner and et al. 2013). SUMO family has four isoforms in Henikoff 2013). Mono-, oligo- and poly-modified humans called SUMO-1 (also known as UBL1, GMP1, forms have been seen in H1 and core histones that have SENTRIN, and SMT3C), SUMO-2 (SMT3A), SUMO- to serve a specific function (Messner and Hottiger 3 (SMT3B), and SUMO-4, and one isoform in yeast 2011). ADP-ribosylation occurs widely at nucleophilic (Saccharomyces cerevisiae) that is known as Smt3p side chains of amino acid residues, such as asparagine, and eight isoforms in plants (Filosa et al. 2013; Ramazi glutamic acid, lysine, arginine, cysteine, and phospho- et al. 2016). SUMO attaches to proteins via an enzy- serine residues, and ADP ribosylation imparts a nega- matic cascade and shows a high degree of similarity tive charge to its modified residues (Burzio et al. 1979; with the ubiquitination pathway. This modification is Messner and Hottiger 2011; Van der Heden van Noort involving three enzymes: an activating enzyme (E1), a et al. 2010; Zentner and Henikoff 2013). ADP-ribo- conjugating enzyme (E2), and a ligating enzyme (E3), sylated histone has been described in amino-terminal and finally, SUMO is detached by a sumo-specific regions in glutamate at second amino acid in histone protease (Gong et al. 2016; McManus et al.2016). H2B and in amino acids 2, 14 and 116 in H1. More- SUMOylation has vital roles in numerous biological over, this modification exists in the carboxyl-terminal processes in the cells such as replication, cell-cycle COOH group of the lysine residue at position 213 in progression, protein transport, meiosis and mitosis, cell H1 (Sadakierska-Chudy and Filip 2015). Until now it division, transcriptional regulation and enhancement of has not been discovered which enzymes catalyze this transcriptional activity, nuclear and protein trafficking, modification in glutamic acid residues (Messner and apoptosis, and the DNA damage response (Gareau and Hottiger 2011). ADP-ribosylations have been divided Lima 2010; Munk et al. 2017). Moreover, SUMOyla- into four major groups: mono-ADP-ribosylation tion induces different effects such as changes in cellular (MARylating), poly-ADP-ribosylation (PARylating), localization or stability, modulation of protein–protein ADP-ribose cyclization, and formation of O-acetyl or protein–DNA interactions (Nathan et al. 2006). ADP-ribose. Mono-ADP-ribosylation of proteins has Thus, SUMOylation is necessary for the conservation been seen both in prokaryotes and eukaryotes. ADP- of genomic totality, regulation of gene expression, ribosyl cyclase activities happen only in unicellular and and intracellular signaling. Defects in the SUMOy- multicellular eukaryotes. The poly-ADP-ribosylation lation pathway have been associated with the process occurs in multicellular eukaryotes and is per- occurrence and progression of various diseases, such haps present in unicellular eukaryotes (Hassa et al. as cancer, heart failure, and neurodegeneration 2006). However, most of ADP-ribosylation carried out (Gong et al. 2016). in cells is ‘mono’ ADP-ribosylations mediated by oli- By using Western blot analysis with anti-Flag and gomeric or polymeric ADP-ribose (PAR) chains and anti-SUMO antibodies, it has been specified that have been predominantly found on proteins outside of SUMOylation is occurred in all four core histones the nucleus. Poly-ADP-ribosylation have been detected in vivo, while target sites on H3 have not yet been more in histones, especially the nucleosomal core his- known. In general, histone SUMOylation happens in a tones by adding short oligomers rather than long small percentage of total histone (Nathan et al. 2006). polymers (Messner and Hottiger 2011). Histone SUMOylation has a repressive role in gene ADP-ribosylation is catalyzed by two different types expression, whereas it helps to retain a low basal level of enzymes that are responsible for transferring of of transcription (Yang et al. 2017). Also, based on adenosine diphosphate ribose (ADP-ribose) groups present evidence it is suggested this modification plays from cofactor NAD? to the side chain of amino acid a key role in transcriptional repression through residues in the target protein, followed by releasing recruitment of histone deacetylases and heterochro- nicotinamide (Boulikas 1989; Moyle and Muir 2010; matin protein 1 (HP1). SUMOylation on histone H4 is Van der Heden van Noort et al. 2010). In MARylating, likely to be related to transcriptional repression, transfer of only one ADP-ribose molecule to the resi- whereas a relatively lower degree is observed for H2A, dues in target proteins is catalyzed by mono-ADP-ri- H2B, and H3 (Shiio and Eisenman 2003). bosyltransferases (MARTs) and PARylating is Evaluation of post-translational modifications in histone proteins Page 19 of 29 135 catalyzed by poly-ADP ribose synthetase enzyme Campbell 2016; LeRoy et al. 2013; Lohrum et al. (PARP) (Boulikas 1989; Hassa et al. 2006). ADP-ri- 2007; Polak et al. 2015; Rotili and Mai 2011). It is bosylation involves in numerous nuclear processes proved that any change in the histone modification such as DNA strand breaks in replication, repair, or profile could be the initiator of cancers (Lohrum et al. recombination (Feijs et al. 2013), transcriptional reg- 2007; Rotili and Mai 2011). Until now, the mechanism ulation, unfolded protein responses, insulin secretion, of interaction of histone modification to the cancer immunity (Feijs et al. 2013; Messner and Hottiger pathophysiological characteristics such as cellular 2011), inter- and intracellular signaling, cell cycle transformation, angiogenesis, and metastasis has not regulation, mitosis, necrosis, and apoptosis(Hassa et al. been specified (Khan et al. 2015). Post-translational 2006). methylation and acetylation create signals in many ADP-ribosylation reversibly occurs in 1% of all transcriptions and chromatin regulators, and their reg- histone proteins in lysine residues (Messner and Hot- ulatory enzymes have important roles in gene regula- tiger 2011) and is catalyzed by a number of NAD? tion, and the disruption of the regulatory signals lead to dependent poly(ADP-ribose) polymerases (PARPs) a variety of wide-range diseases in human such as such as poly(ADP-ribose) polymerase 1 (ARTD1/ cancer, leukemia, lymphomas, mental retardation PARP1) and more by ADP-ribosyltransferases (ART) (Angelman syndrome), or diabetes mellitus as well as which are ADP-ribosylate histone and non-histone pathogen infection and neurodegenerative disorders proteins. PARP1 is a nuclear chromatin-linked the such as Parkinson’s disease, Alzheimer’s disease, and multifunctional enzyme that exists in most eukaryotes Huntington’s disease (Chen et al. 2014; Peleg et al. apart from yeast (Hassa et al. 2006) and is the best- 2016; Robertson 2005; Sadri-Vakili and Cha 2006; Shi characterized histone ADP-ribosylating enzyme in the et al. 2012). amino-terminal on all core histones in environmental Moreover, studies suggest imbalances of histone stresses. In recent studies it has been shown that ADP- acetylation/deacetylation in promoter regions of the ribosylate occurs in the amino-terminal tails of all core gene cause deregulation of gene expression and asso- histones, specifically at H2A-K13, H2B-K30, H3-K27, ciated with tumor development (Lohrum et al. 2007), and -K37 as well as H4-K16 and acetylation in this site and aberrant histone methylation plays a role in neu- prevent of ADP-ribosylation by PARP1 (Audia and rodevelopmental, neurodegenerative, behavioral disor- Campbell 2016; Messner et al. 2010; Messner and ders, and cancers (table 3) (Greer and Shi 2012). Hottiger 2011). However, identification of the modifi- Therefore, identification of methylation and acetylation cations in histones H3 and H4 are important, since sites can be very useful for understanding their modi- ADP-ribosylation on the lysine residues are competing fication dynamics and molecular mechanism, as well as with phosphorylation and acetylation and lead to drug design for various related diseases. Histone crosstalk. For example, acetylation of histone H4 K16 modifications are reversible and they can be used as inhibits the ADP-ribosylation of H4 (Messner and potential targets for cancer therapy and prevention Hottiger 2011). Table 2 summarizes different types of (Sawan and Herceg 2010). HDAC inhibitors through PTMs histone and the histone residues currently known increased acetylation of histones can lead to increased and as well as the cellular processes implicated with transcription of silenced genes, and also, it has a role in chromatin remodeling and gene expression. Mostly, promoting growth arrest by inducing the expression of different types of PTMs histone occur in Saccha- tumor suppressor genes. Therefore, HDACi is utilized romyces cerevisiae (SC) and Homo sapiens (HS) as anticancer drugs (Sadri-Vakili and Cha 2006). (table 2). Figure 8 is a schematic representation of Interestingly, many histone-modifying enzymes have different types of modifications (table 2) that has been been identified as oncogene or tumor suppressors such drawn by Cytoscape software. as BRCA1, RNF20 and RNF40, and USP22 in the ubiquitylation process. The BRCA1 protein is a RING- finger-domain-containing E3 and H2A and H2B, which 2. Correlation between histone modifications have been discovered as substructures in BRCA1. The and diseases complex of BRCA1 and protein BAP1(BRCA1/BAP1) is a tumor suppressor in multiple cancers, such as lung Cancerous cells are created in effect of genetic alter- cancer, breast cancer, uveal melanoma, and mesothe- ations and the genomic instability in several classes of lioma. RNF20 is the chief H2B-specific E3 ubiquitin genes, such as oncogenes, tumor suppressor genes, ligase in mammalian cells and known to be a tumor apoptotic genes, and DNA repair genes (Audia and suppressor. USP22 is a ubiquitin hydrolase and has a 135 Page 20 of 29 S Ramazi, A Allahverdi and J Zahiri major role in the removal of ubiquitin from glioblastoma multiform, cutaneous nodular melanoma, monoubiquitinated histones H2A and H2B which are cutaneous melanoma, breast cancer, esophageal squa- known as a putative cancer stem cell marker and are mous cell carcinoma, gastric cancer, melanoma, and expressed in malignant tumor samples (Cao and Yan nasopharyngeal carcinoma (Khan et al. 2015). More- 2012). over, monoubiquitination of histones H2A, H2B, and In 2005, it was detected which defects in histone H2AX occurs with poly-ubiquitination of histones at acetylation at H4-K16 and trimethylation H4-K20 are the sites of DNA damage. However, respectively related to human cancers (Fraga et al. 2005; Khan et al. monoubiquitination is catalyzed in H2A/H2AX K119 2015). Moreover, monomethylation in H3-K4, and H2B K120 by using the RING1B/BMI1 and dimethylation in H3-K9, trimethylation in H3-K9, RNF20/RNF40 in DSB site (Cao and Yan 2012). acetylation in H3-K18 and in H4-K12 were reduced in It is proved that mutation and/or altered expression prostate cancer compared to nonmalignant prostate of histone acetyl and methyl modifiers, as well as tissue. The decrease in dimethylation H3-K4, -K9 has methyl-binding protein profiles, lead to increased been found as the dictator in various neoplastic tissues incidence of various different cancers (Schneider et al. like pancreatic adenocarcinomas, lung, breast, and 2002; Zhang and Dent 2005). For example, the kidney cancers, and in contrast, further elevation of enhancer of Zeste homolog 2 (EZH2) is a histone- H3K9ac levels was described in liver cancer (Ellinger lysine N-methyltransferase enzyme and is one of the et al. 2010). Lung cancer cells, compared to normal catalytic components of the polycomb repressive lung epithelium, have an aberrant pattern of histone H4 complex 2 (PRC2) and is involved in methylation H3 modifications along with hyperacetylation in histones K27me3 which has the repressive role in gene tran- H4-K5, -K8, as well as hypoacetylation in histones H4- scription. EZH2 is upregulated in a number of cancers, K12, -K16 which been considered as biomarkers of including prostate cancer, breast cancer, and lym- non-small-cell lung carcinoma (Van Den Broeck et al. phomas. Some mutations have been identified in EZH2 2008). Moreover, studies show an increase in that cause loss of methyltransferase activity in EZH2 as H3K4me3 levels along with a reduction on the levels a result of myelodysplastic syndromes. Thus, this of H3K27me3 in cells from patients with rheumatoid enzyme functions has a key role as a tumor suppressor arthritis leads to development of the disease. Moreover, myelodysplastic cancers (Cross 2012; LeRoy et al. it is specified that specific genes in the systemic lupus 2013). erythematosus (SLE) change the H3K4me3 levels. Furthermore, some severe diseases are caused by Thus, recent studies show an association between his- suppression of HATs, HDACs,HMTs, and HDMCs tone modifications and epigenetics in causing neu- (Audia and Campbell 2016; Zhang and Dent 2005). rodegenerative disease such as amyotrophic lateral Among the various types of HATs known, p300/CBP sclerosis, frontotemporal dementia, and Parkinson’s and PCAF have been identified in primary human disease (Cobos et al. 2019). tumor development and are the key player in cell dif- In mammalian cells in response to DNA double- ferentiation, growth, transformation, apoptosis, and strand breaks (DSBs), phosphorylation at c position somatic mutations (An 2007; Zhang and Dent 2005). (cH2AX) is induced along chromatin tracks flanking Disruptions and alteration in HATs enzymes such as DSBs by ATM, ATR, and DNA-PK(Cao and Yan mutation, MLL gene fusion, MOZ/MORF (MYST 2012; Gjoneska et al. 2015). If DNA double-strand family) gene fusion, deletion/point mutation, translo- breaks (DSBs), do not repair, or repair incorrectly, it cation, nonsense, missense amplification lead to human can cause cell death, mutations, and chromosomal diseases (colon, lung, and stomach cancer, leukemia, translocations and can lead to cancer (Oki et al. 2007). Rubinstein-Taybi syndrome, breast cancer, epithelial For instance, poor overall survival was observed in cancer, colorectal, glioblastoma, and uterine leiomy- breast tumors that have a high level of cH2AX, which omata, ovarian) (An 2007). In GNAT family (GCN5 usually comes with shorter telomere length. Moreover, and PCAF) disturbances like missense, frameshift, high cH2AX expression level, in CRC tissues in col- deletion, and amplification lead to diseases as breast, orectal cancer, is associated with tumor stage and colorectal, prostate, lung, and kidney sarcoma (Hassa perineurial invasion. In addition, high cH2AX et al. 2006). HDACs are part of a vast family of expression level is correlated with poor distant metas- enzymes and they have vital roles in numerous bio- tasis-free survival (DMFS) and longer overall survival logical processes. HDACs play a repressive role in (OS). Elevation of phosphorylation in H3S10 is cor- transcription. HDACs are classified into four families related with poor prognosis in several cancers such as (class I, IIa, IIb, and IV). These family enzymes are Evaluation of post-translational modifications in histone proteins Page 21 of 29 135 Table 3. Correlation of aberrant histone methylation marks with neurodevelopmental, neurodegenerative, behavioral dis- orders and cancers

Target residue Diseases Enzyme and modification Enzyme References Weaver syndrome EZH2 (KMT6) / H3K27me3 Cohen et al. (2016) methylation Sotos syndrome NSD1 (KMT3B) / H4K20 and H3K36 Berdasco et al. (2009) methylation Mental retardation, autism KDM5C (JARID1C)/ H3K4 Santos-Rebouc¸as et al. (2011) demethylation Mental retardation and facial PHF8 /demethylation H3K9 Laumonnier et al. (2005) deformity Deletion syndrome EHMT1/ methylation H3K9 Kleefstra et al. (2006) Friedreich ataxia SETDB1/ methylation H3K9 Sandi et al. (2011) Schizophrenia MLL1 / methylation H3K4 me3 Huang et al. (2007) Addiction G9a / methylation H3K9 me2 Covington III et al. (2011) Huntington’s disease SETDB1/ methylation H3K9 me3 Lee et al. (2013) Parkinson’s disease Jmjd3 / demethylation H3K27me3 Wang et al. (2015a, b) Ataxia-telangiectasia EZH2 (KMT6)/ H3K27me3 Li et al. (2013) methylation Several cancers EZH1/2 (KMT6B / H3K27me3 Ezhkova et al. (2011) and Nakagawa KMT6) / methylation and Kitabayashi (2018) Gastric carcinoma, prostate, breast SUV39H1 / methylation H3K9me3 Cai et al. (2014) and Yokoyama et al. and colorectal cancers (2013) X-linked mental retardation, Breast PHF8 / demethylation H4K20me1, Qi et al. (2010) and Shao et al. cancers H3K9me1 and (2016) me2 Breast, ovarian and gastric cancer PRMT8 / methylation H4R3me1 Hernandez et al. (2017) Breast cancer SETD7 (SET7/9) / H3K4me1 Huang et al. (2017) and Song et al. methylation (2016)

different in structure, enzymatic function, subcellular (Chen et al. 2014). Disruption of a limited number of localization, and expression patterns. There is evidence histone modifications disrupts the pattern of gene that levels of HDACs are altered in Alzheimer’s dis- expression. Over the last decade, epigenetic alterations ease, specifically HDAC2 and HDAC6 protein levels and specific histone modifications such as phosphory- (Coneys and Wood 2020). There is in mammalian lation, acetylation, ubiquitination, ADP-ribosylation, genomes encoded another group of HDACs called the deamination, and other histone modifications have Sirtuins (Haberland et al. 2009). Sirtuins are a class III been reported in the development of cell death (apop- of HDACs and for their activity use NAD? as a totic and non-apoptotic cell death) and the most cofactor. Impairment of the Sirtuins family has key prevalent human autoimmune diseases (Dieker and roles in different diseases such as cancers, Alzheimer’s Muller 2010), such as systemic lupus erythematosus disease, insulin resistance, aging, diabetes, and (SLE) (Liu et al. 2012), rheumatoid arthritis (RA), inflammation, but their role in cardiovascular diseases systemic sclerosis (SSc), Sjogren’s syndrome (SS), (CVDs) is being studied. Therefore, it can be said that autoimmune thyroid diseases (AITD), and type 1 dia- Sirtuins have been considered as a therapeutic target for betes (T1D) (Mazzone et al. 2019). For example, various cancers, aging, and neurodegenerative disor- phosphorylation of histones in N-terminal tails, at ders (Aggarwal et al. 2020). Ser10 and Ser28 of H3, Ser1 of H4, and Ser14 of H2B, Recently studies have suggested that epigenetic are repeated targets for autoantibodies in SLE and other pathways and histone PTMs have key roles in the connective tissue diseases. Moreover, in SLE disease, function of the immune system and in the development N-terminal tails in histones H4 and H2B exist epitopes and regulation of various cell lines and in the modu- targeted by lupus autoantibodies and have been prone lation of immune tolerance and autoimmune disorders to acetylate. Monoubiquitination of the Ro/SSA protein 135 Page 22 of 29 S Ramazi, A Allahverdi and J Zahiri and histones in H2A and H2B at Lys119 of H2A and Dysregulation of DNA methylation, histone hypo- Lys120 in H2B are common targets in SLE and SS acetylation due to decreased HAT activity or increased (Dieker and Muller 2010). HDAC activity, has been reported in neurodegenerative Modifications of histone in immune cells are diseases (Mirabella et al. 2016). Current evidence important during SLE development, and modified shows that in Alzheimer’s disease (AD) and Hunting- histones, such as H3K4me3, H3K27me3, H4K8ac, ton’s disease (HD) there exist alterations in histone H4K16ac, and H2BK12ac, are identified as autoanti- methylation (Rowe et al. 2019). In terms of PD, it was gens related to SLE that lead to trigger NETosis (Don˜as recently discovered that a-synuclein is methylated and et al. 2019). In the NETosis phenomenon, neutrophils reduction in methylation is effective in the accumula- can continue to phagocytose and kill microbes (Yang tion of the protein a-synuclein and progression of PD. et al. 2016). Evidence indicates that histone modifica- Interestingly, in animal models of PD, it has been tions and histone methyltransferase Ezh2 (important in detected that HDACi inhibits a-synuclein toxicity in autoimmune responses of CD4? T cells) have a key the dopamine neuron. PD patients have hypomethyla- role in SLE (Don˜as et al. 2019). In SLE patients, we tion of the tumor necrosis factor (TNF) gene in the have observed hypoacetylation of H3/4 and cortex and higher levels of TNF-a cytokine in the hypomethylation of H3K9 in CD4? T cells and it is cerebrospinal fluid (Wen et al. 2016). TNF-a is a associated with a loss of HDAC2, HDAC7, and lysine complex linker between inflammation and cancer via methyltransferase KMT1B and KMT6 expression intermediary the process of apoptosis and cell-mediated compared to healthy controls. Furthermore, studies immunity (Liu et al. 2016). TNF-a has a key role in represent an inverse correlation between H3 acetyla- pathogenesis AD patients. AD patients have a DNA tion levels and SLE disease activity (Mehta and Jef- methylation state of duplicate elements (Wen et al. frey 2016). Moreover, it can be said that HDAC 2016). inhibitors (HDACi) lead to increase the transcription of various genes, containing those involved in autoimmunity and the effect on the influence immune 3. Discussion and inflammatory processes in T cells (Mazzone et al. 2019). Different types of modifications in histone proteins in a With increasing population age, neurodegenerative coordinated and orderly fashion affect chromatin disorders are one of many serious health problems in structure and function. Consequently, histone modifi- modern societies across the world. Numerous of neu- cations have key roles in regulating cellular processes rodegenerative diseases including Alzheimer’s disease such as gene transcription, DNA replication, and DNA (AD) and Parkinson’s disease (PD) and Huntington’s repair. Studies on histone modifications in chromatin disease (HD) may be associated with dysfunctions of research have increased our knowledge about epige- methylation and histone modifications such as chro- netic mechanisms in controlling cellular processes. matin acetylation, deacetylation of histone proteins, Thus, research on histone modifications is a new and hypoacetylation, and transcriptional dysfunction. These rapidly expanding field that leads to advance our diseases are created by continuous degeneration and knowledge about the key processes underlying disease death of nerve cells, and after overtaking cancer, they development such as different cancers. A review of will be considered as the second leading cause of death recent studies indicates the existence of interdepen- by 2050. In the recent decade, HDACi conferring dence between histone markers including acetylation, neuroprotective effects in Huntington’s disease has methylation, and phosphorylation. According to critical been reported, and its cause is re-stabilization of gene roles of histone modifications in essential cellular transcription, owing to a shift of histone acetylation processes, evidence has suggested which deregulation equilibrium towards increased acetylation of histones, of histone marking is exactly related to a number of relaxation of DNA-chromatin complexes, and pursuant human diseases. Hence, a better understanding of the increase of gene transcription (Lovrecˇic´ et al. 2013). mechanisms controlling the establishment and mainte- Thus, it can be said that HDACi reduce the symptoms nance of histone markers and how the disruption of observed in Huntington’s disease in a range of neuro- these mechanisms, will facilitate the development of logic diseases, from monogenic neurologic diseases to novel strategies to prevent, diagnose, and treat human common neurodegenerative disorders, such as Parkin- diseases. Histone modifications are one type of epige- son’s and Alzheimer’s disease (Lovrecˇic´ et al. 2013; netic alterations which in contrast to genetic changes is Sadri-Vakili and Cha 2006). a reversible process and may provide the basis for the Evaluation of post-translational modifications in histone proteins Page 23 of 29 135 development of new therapeutic and preventive Esteller M 2009 Epigenetic inactivation of the Sotos approaches targeting aberrant histone modifications. overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma. PNAS. 106 21830–21835 Bhogaraju S and Dikic I 2016 Cell biology: Ubiquitination References without E1 and E2 enzymes. Nature. 533 43–44 Boulikas T 1989 DNA strand breaks alter histone ADP- Aggarwal S, Banerjee SK, Talukdar NC and Yadav AK 2020 ribosylation. 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