**Chromatin Remodelling During Host-Bacterial Pathogen Interaction**

Yong Zhong Xu, Cynthia Kanagaratham and Danuta Radzioch

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55977

#### **1. Introduction**

Eukaryotic DNA is tightly packaged into nucleosome repeats, which form the basic unit of cellular chromatin. The nucleosome consists of an octamer core wrapped with a segment of 146 base pairs of double stranded DNA. Each octamer core is composed of two molecules of each core histone proteins H2A, H2B, H3 and H4 (Figure 1). A fifth histone protein, linker H1, binds to the nucleosomal core particle and assists in further compaction of the chromatin into higher-order structure(Lusser and Kadonaga, 2003;Roberts and Orkin, 2004). This compaction of genomic DNA into chromatin restricts access of a variety of DNA regulatory proteins to the DNA strand, which are involved in the processes of transcrip‐ tion, replication, DNA repair and recombination machinery. To overcome these barriers, eukaryotic cells possess a number of multi-protein complexes which can alter the chroma‐ tin structure and make DNA more accessible. These complexes can be divided into two groups, histone-modifying enzymes and ATP-dependent chromatin remodelling com‐ plexes. The histone-modifying enzymes post-translationally modify the N-terminal tails of histone proteins through acetylation, phosphorylation, ubiquitination, ADP-ribosylation and methylation. On the other hand, ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to disrupt the contact between DNA and histones, move nucleosomes along DNA, and remove or exchange nucleosomes(Kallin and Zhang, 2004;Lusser and Kadonaga, 2003;Roberts and Orkin, 2004). The importance of chromatin structure and its functional role in genome regulation and development is becoming increasingly evident, especially in diseases such as cancer.

© 2013 Xu et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

to manipulate the host cellular function through histone modification and subversion of host

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Histone acetylation/deacetylation is a key epigenetic regulator of chromatin structure and gene expression, in combination with other posttranslational modifications. These patterns of histone modification are maintained by histone modifying enzymes such as histone acetyl‐ transferases (HATs) and histone deacetylases (HDACs). While HATs acetylate histones, conferring an ''open" chromatin structure that allows transcriptional activation, HDACs have the opposite effect resulting in transcriptional repression by closing chromatin structure. Global HDAC-mediated transcriptional changes can have a concomitant effect on cell function – an epigenetic mechanism often exploited by viruses to promote infection (Punga and Akusjarvi, 2000;Radkov *et al.*, 1999;Valls *et al.*, 2007). Recent reports also show that intracellular bacteria manipulate host cell epigenetics to facilitate infection (Arbibe *et al.*, 2007;Hamon *et al.*, 2007;Hamon and Cossart, 2008). Disruption of HDAC activity with inhibitors or by siRNA affects gene expression profilling in different cell types (Glaser *et al.*, 2003a;Glaser *et al.*, 2003b;Lee *et al.*, 2004;Zupkovitz *et al.*, 2006). The potential of HDAC inhibitors in treatment of

In this chapter, the chromatin modifications in host cells induced by bacterial pathogens and their effects on host gene expression and infection will be reviewed. Furthermore, the potential role of HDAC inhibitors, as a therapeutic immunomodulator, in treatment of infections will

The packaging of DNA into chromatin does not only simply facilitate the compaction of eukaryotic DNA genomes into the cell nucleus but also plays a profound and ubiquitous roles in almost all DNA-related cellular processes such as DNA replication, repair, recombination and transcription (Clapier and Cairns, 2009;Li *et al.*, 2007a). Chromatin structure is not a simple static unit. It possesses dynamic properties that are orchestrated by ATP-dependent chroma‐ tin-remodeling complexes and histone-modifying enzymes. In conjunction with other coregulators, these chromatin remodelers modify histone-DNA interaction and regulate

Histone sequences are highly conserved. A core histone protein typically consists of an unstructured N-terminal tail, a globular core including a central histone-fold domain, and a conformationally mobile C-terminal tail (Garcia *et al.*, 2007b;Mersfelder and Parthun, 2006). Both N-terminal tails and globular domains are subject to a variety of posttranslational modifications (Kouzarides T, Cell, 2007, 128:693-705) (Figure 1). At least fourteen different types of posttranslational (or covalent) modifications involving more than 60 different residues on histones have been reported to date including acetylation, methylation, phosphorylation, ubiquitination, poly-ADP ribosylation, sumoylation, butyrylation, formylation, deimination,

innate immune responses for their survival or to infect the host.

**2. Chromatin structure in transcription regulation**

infection has being studied.

transcription at specific genomic loci.

**2.1. Histone modifications and transcription**

also be discussed.

**Figure 1. Schematic representation of a nucleosome (A) and major histone modifications (B).** Modifications on histones are described in text. The major modifications shown include acetylation (A), methylation (M), phosphoryla‐ tion (P) and ubiquitination (U). Histone modifications mainly occur on the N-terminal tails of histones but also on the C-terminal tails and globular domains, for example, ubiquitination of the C-terminal tails of H2A and H2B and acetyla‐ tion and methylation of the globular domain of H3 at K56 and K79, respectively.

Intracellular pathogens, through a long-standing coexistence with host cells, have evolved mechanisms that provide pathogens with the amazing capacity to adapt and survive in the variable and often hostile environments of their hosts (Galan and Cossart, 2005). The concept of chromatin modification as a mechanism by which pathogens affect host immune responses to facilitate infection has emerged in recent years. For example, listeriolysin O (LLO), secreted by *Listeria monocytogenes*, induces a dramatic dephosphorylation of histone H3 at serine 10 and deacetylation of histone H4, and these modifications are associated with changes in host gene expression during early stages of infection (Hamon *et al.*, 2007). Arbibe and colleagues also indicate that *Shigella flexneri* effector OspF dephosphorylates ERK and p38 mitogen-activated protein kinase (MAPK) in the nucleus; this subsequently prevents histone H3 phosphorylation at Ser10 at the promoters of a specific subset of genes, which blocks the activation of nuclear factor –κB (NF-κB)- responsive genes leading to a compromised inflammation in the infected tissue(Arbibe *et al.*, 2007). These results suggest a strategy developed by microbial pathogens to manipulate the host cellular function through histone modification and subversion of host innate immune responses for their survival or to infect the host.

Histone acetylation/deacetylation is a key epigenetic regulator of chromatin structure and gene expression, in combination with other posttranslational modifications. These patterns of histone modification are maintained by histone modifying enzymes such as histone acetyl‐ transferases (HATs) and histone deacetylases (HDACs). While HATs acetylate histones, conferring an ''open" chromatin structure that allows transcriptional activation, HDACs have the opposite effect resulting in transcriptional repression by closing chromatin structure. Global HDAC-mediated transcriptional changes can have a concomitant effect on cell function – an epigenetic mechanism often exploited by viruses to promote infection (Punga and Akusjarvi, 2000;Radkov *et al.*, 1999;Valls *et al.*, 2007). Recent reports also show that intracellular bacteria manipulate host cell epigenetics to facilitate infection (Arbibe *et al.*, 2007;Hamon *et al.*, 2007;Hamon and Cossart, 2008). Disruption of HDAC activity with inhibitors or by siRNA affects gene expression profilling in different cell types (Glaser *et al.*, 2003a;Glaser *et al.*, 2003b;Lee *et al.*, 2004;Zupkovitz *et al.*, 2006). The potential of HDAC inhibitors in treatment of infection has being studied.

In this chapter, the chromatin modifications in host cells induced by bacterial pathogens and their effects on host gene expression and infection will be reviewed. Furthermore, the potential role of HDAC inhibitors, as a therapeutic immunomodulator, in treatment of infections will also be discussed.

#### **2. Chromatin structure in transcription regulation**

The packaging of DNA into chromatin does not only simply facilitate the compaction of eukaryotic DNA genomes into the cell nucleus but also plays a profound and ubiquitous roles in almost all DNA-related cellular processes such as DNA replication, repair, recombination and transcription (Clapier and Cairns, 2009;Li *et al.*, 2007a). Chromatin structure is not a simple static unit. It possesses dynamic properties that are orchestrated by ATP-dependent chroma‐ tin-remodeling complexes and histone-modifying enzymes. In conjunction with other coregulators, these chromatin remodelers modify histone-DNA interaction and regulate transcription at specific genomic loci.

#### **2.1. Histone modifications and transcription**

**Figure 1. Schematic representation of a nucleosome (A) and major histone modifications (B).** Modifications on histones are described in text. The major modifications shown include acetylation (A), methylation (M), phosphoryla‐ tion (P) and ubiquitination (U). Histone modifications mainly occur on the N-terminal tails of histones but also on the C-terminal tails and globular domains, for example, ubiquitination of the C-terminal tails of H2A and H2B and acetyla‐

Intracellular pathogens, through a long-standing coexistence with host cells, have evolved mechanisms that provide pathogens with the amazing capacity to adapt and survive in the variable and often hostile environments of their hosts (Galan and Cossart, 2005). The concept of chromatin modification as a mechanism by which pathogens affect host immune responses to facilitate infection has emerged in recent years. For example, listeriolysin O (LLO), secreted by *Listeria monocytogenes*, induces a dramatic dephosphorylation of histone H3 at serine 10 and deacetylation of histone H4, and these modifications are associated with changes in host gene expression during early stages of infection (Hamon *et al.*, 2007). Arbibe and colleagues also indicate that *Shigella flexneri* effector OspF dephosphorylates ERK and p38 mitogen-activated protein kinase (MAPK) in the nucleus; this subsequently prevents histone H3 phosphorylation at Ser10 at the promoters of a specific subset of genes, which blocks the activation of nuclear factor –κB (NF-κB)- responsive genes leading to a compromised inflammation in the infected tissue(Arbibe *et al.*, 2007). These results suggest a strategy developed by microbial pathogens

tion and methylation of the globular domain of H3 at K56 and K79, respectively.

174 Chromatin Remodelling

Histone sequences are highly conserved. A core histone protein typically consists of an unstructured N-terminal tail, a globular core including a central histone-fold domain, and a conformationally mobile C-terminal tail (Garcia *et al.*, 2007b;Mersfelder and Parthun, 2006). Both N-terminal tails and globular domains are subject to a variety of posttranslational modifications (Kouzarides T, Cell, 2007, 128:693-705) (Figure 1). At least fourteen different types of posttranslational (or covalent) modifications involving more than 60 different residues on histones have been reported to date including acetylation, methylation, phosphorylation, ubiquitination, poly-ADP ribosylation, sumoylation, butyrylation, formylation, deimination, citrullination, isomerisation, O-GlcNAcylation, crotonylation and hydroxylation (Martin and Zhang, 2007;Ruthenburg *et al.*, 2007;Sakabe *et al.*, 2010;Tan *et al.*, 2011). The majority of known histone modifications are located within the N-terminal tails of core histones. These modifi‐ cations play an important role in the control of chromatin dynamics and its availability for transcription (Kouzarides, 2007). It has been suggested that all these modifications are combinatorial and interdependent and therefore may constitute a ``histone code`` (Jenuwein and Allis, 2001;Strahl and Allis, 2000). According to this hypothesis, the "histone code" is read by effector proteins (readers) which recognize and bind to modifications via specific domains and result in distinct and consistent cellular processes, such as replication, transcription, DNA repair and chromosome condensation (Kouzarides, 2007;Shi and Whetstine, 2007). Specific histone modifications are essential for partitioning the genome into functional domains, such as transcriptionally silent heterochromatin and transcriptionally active euchromatin (Martin and Zhang, 2005).

either a lysine or an arginine residue, is catalyzed by three different classes of methyltransfer‐ ases: SET domain-containing histone methyltransferases (HMTs), non-SET domain-containing lysine methyltransferases as well as protein arginine methyltransferase (PRMT). Methylation is implicated in both activation and repression of transcription depending on the methylation site and the type of methyltransferase involved (Shilatifard, 2006;Wysocka *et al.*, 2006a). For example, methylation of lysine 4, 36 or 79 of H3 correlates with activation of transcription whereas methylation of lysine 9, 27 of H3 or lysine 20 of H4 is usually linked to transcriptional repression (Pawlak and Deckert, 2007). Type I PRMT, such as CARM1 (cofactor associated arginine methyltransferase 1), PRMT1 and PRMT2, catalyze the formation of monomethyland asymmetric dimethyl-arginine derivatives and is involved in transcriptional activation. Type II PRMT, such as PRMT5, catalyzes the formation of monomethyl- and symmetric dimethyl-arginine derivatives and is involved in transcriptional repression. In addition, a lysine can be mono-, di- or trimethylated with different effect on gene transcription (Santos-Rosa *et al.*, 2002;Schneider *et al.*, 2005). Both lysine and arginine methylations can be reversed by histone demethylases, which had been discovered many years after the discovery of HMTs. LSD1 was the first histone demethylase discovered in 2004 and was shown to demethylate H3K4 and to repress transcription (Shi *et al.*, 2004). However, LSD1 was also shown to demethylate H3K9 and activate transcription when present in a complex with the androgen receptor (Metzger *et al.*, 2005). Following the discovery of LSD1, a number of other related enzymes were subsequently discovered. Among them, Jumonji domain–containing 6 protein (JMJD6) is the only direct arginine demethylase reported to date shown to demethylate H3 at arginine 2 and H4 at arginine 3 (Chang *et al.*, 2007). In addition, human peptidylarginine deiminase 4 protein (Pad4) can regulate histone arginine methylation by converting monomethylated arginine into citrulline via demethylimination or deimination (Cuthbert *et al.*, 2004;Wang *et al.*, 2004). Histone methylation may affect the binding of other histone-modifying enzymes to the chromatin, which then mediates other posttranscriptional modifications, such as histone phosphorylation and DNA methylation (Mosammaparast and Shi, 2010;Pedersen

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Acetylation, another well-characterized modification, occurs on lysine residues mainly in the N-terminal tail of core histones. However, a lysine 56 within the globular domain of H3 (H3K56) has been found to be acetylated in yeast. Yeast protein SPT10, a putative histone acetyltransferase (HAT), was shown to mediate the H3K56 acetylation of histone genes at their promoter regions. H3K56 acetylation allows the recruitment of Snf5, an essential component of SWI/SNF chromatin remodeling complex and subsequently regulating transcription (Xu *et al.*, 2005). Compared with the SPT10, the Rtt109 acetyltransferase mediates H3K56 acetylation more globally (Driscoll *et al.*, 2007;Han *et al.*, 2007;Schneider *et al.*, 2006). The acetylation level correlates with transcriptional activation (Davie, 2003;Legube and Trouche, 2003). The level of acetylation is balanced by HATs and HDACs. Generally, increased levels of histone acetylation by HATs enhance chromatin decondensation and DNA accessibility for transcription factors to activate gene expression. In contrast to acetylation, deacetylation of histones catalyzed by HDACs leads to chromatin condensa‐ tion and gene silencing (Berger, 2007;Li *et al.*, 2007a). The relationship between histone

and Helin, 2010).

There are two major mechanisms underlying the function of histone modifications (Kouzar‐ ides, 2007;Ruthenburg *et al.*, 2007). The first is the modulation of chromatin structure either by altering DNA-nucleosome interaction or by altering nucleosome-nucleosome interactions via changing the histone charges or by addition of physical entities. For example, histone acety‐ lation, a modification associated with transcriptional activation, has been proposed to unfold chromatin structure via neutralization of the basic charges of lysines (Kouzarides, 2007). Indeed, *in vitro* studies using recombinant nucleosomal arrays have demonstrated that acetylation of H4K16 restricts the formation of a 30-nanometer fiber and the generation of higher-order structures (Shogren-Knaak *et al.*, 2006;Shogren-Knaak and Peterson, 2006). Secondly, histone modifications provide docking sites for the recruitment of specific binding proteins, which recognize and interact with modified histones via specialized domains such as bromo-, chromo- and PHD (plant homeodomain) domains, thereby influence chromatin dynamics and function (Wysocka *et al.*, 2005;Wysocka *et al.*, 2006b;Zeng and Zhou, 2002). A number of proteins have been identified that are recruited to specific modifications. For example, methylation of H3K4, H3K9 and H3K27 can be recognized by inhibitor of growth (ING) proteins, heterochromatin protein 1 (HP1) and polycomb proteins, respectively. It has been shown that histone modification binding proteins can tether, directly or indirectly, an enzyme to chromatin. The activity of this recruited enzyme can be regulated (Pena *et al.*, 2006;Shi *et al.*, 2006;Wysocka *et al.*, 2006b). BPTF, a component of the NURF chromatin remodelling complex, binds to H3K4me3 via a PHD domain and tethers the SNF2L ATPase to H0XC8 gene and activates the expression of the latter (Wysocka *et al.*, 2006b). JMJD2A and CHD1, two other H3K4me-binding proteins, possess enzymatic activities themselves and can directly deliver enzymatic activities to chromatin when recruited (Huang *et al.*, 2006;Pray-Grant *et al.*, 2005;Sims, III *et al.*, 2005).

The link between histone modifications and transcriptional regulation has been widely studied. It has been found that a specific modification can be associated with transcriptional activation or repression. Among the histone modifications, methylation and acetylation of H3 and H4 play a major role in the regulation of transcriptional activity (Berger, 2007;Jenuwein and Allis, 2001;Li *et al.*, 2007a;Shahbazian and Grunstein, 2007). Methylation, which occurs on either a lysine or an arginine residue, is catalyzed by three different classes of methyltransfer‐ ases: SET domain-containing histone methyltransferases (HMTs), non-SET domain-containing lysine methyltransferases as well as protein arginine methyltransferase (PRMT). Methylation is implicated in both activation and repression of transcription depending on the methylation site and the type of methyltransferase involved (Shilatifard, 2006;Wysocka *et al.*, 2006a). For example, methylation of lysine 4, 36 or 79 of H3 correlates with activation of transcription whereas methylation of lysine 9, 27 of H3 or lysine 20 of H4 is usually linked to transcriptional repression (Pawlak and Deckert, 2007). Type I PRMT, such as CARM1 (cofactor associated arginine methyltransferase 1), PRMT1 and PRMT2, catalyze the formation of monomethyland asymmetric dimethyl-arginine derivatives and is involved in transcriptional activation. Type II PRMT, such as PRMT5, catalyzes the formation of monomethyl- and symmetric dimethyl-arginine derivatives and is involved in transcriptional repression. In addition, a lysine can be mono-, di- or trimethylated with different effect on gene transcription (Santos-Rosa *et al.*, 2002;Schneider *et al.*, 2005). Both lysine and arginine methylations can be reversed by histone demethylases, which had been discovered many years after the discovery of HMTs. LSD1 was the first histone demethylase discovered in 2004 and was shown to demethylate H3K4 and to repress transcription (Shi *et al.*, 2004). However, LSD1 was also shown to demethylate H3K9 and activate transcription when present in a complex with the androgen receptor (Metzger *et al.*, 2005). Following the discovery of LSD1, a number of other related enzymes were subsequently discovered. Among them, Jumonji domain–containing 6 protein (JMJD6) is the only direct arginine demethylase reported to date shown to demethylate H3 at arginine 2 and H4 at arginine 3 (Chang *et al.*, 2007). In addition, human peptidylarginine deiminase 4 protein (Pad4) can regulate histone arginine methylation by converting monomethylated arginine into citrulline via demethylimination or deimination (Cuthbert *et al.*, 2004;Wang *et al.*, 2004). Histone methylation may affect the binding of other histone-modifying enzymes to the chromatin, which then mediates other posttranscriptional modifications, such as histone phosphorylation and DNA methylation (Mosammaparast and Shi, 2010;Pedersen and Helin, 2010).

citrullination, isomerisation, O-GlcNAcylation, crotonylation and hydroxylation (Martin and Zhang, 2007;Ruthenburg *et al.*, 2007;Sakabe *et al.*, 2010;Tan *et al.*, 2011). The majority of known histone modifications are located within the N-terminal tails of core histones. These modifi‐ cations play an important role in the control of chromatin dynamics and its availability for transcription (Kouzarides, 2007). It has been suggested that all these modifications are combinatorial and interdependent and therefore may constitute a ``histone code`` (Jenuwein and Allis, 2001;Strahl and Allis, 2000). According to this hypothesis, the "histone code" is read by effector proteins (readers) which recognize and bind to modifications via specific domains and result in distinct and consistent cellular processes, such as replication, transcription, DNA repair and chromosome condensation (Kouzarides, 2007;Shi and Whetstine, 2007). Specific histone modifications are essential for partitioning the genome into functional domains, such as transcriptionally silent heterochromatin and transcriptionally active euchromatin (Martin

There are two major mechanisms underlying the function of histone modifications (Kouzar‐ ides, 2007;Ruthenburg *et al.*, 2007). The first is the modulation of chromatin structure either by altering DNA-nucleosome interaction or by altering nucleosome-nucleosome interactions via changing the histone charges or by addition of physical entities. For example, histone acety‐ lation, a modification associated with transcriptional activation, has been proposed to unfold chromatin structure via neutralization of the basic charges of lysines (Kouzarides, 2007). Indeed, *in vitro* studies using recombinant nucleosomal arrays have demonstrated that acetylation of H4K16 restricts the formation of a 30-nanometer fiber and the generation of higher-order structures (Shogren-Knaak *et al.*, 2006;Shogren-Knaak and Peterson, 2006). Secondly, histone modifications provide docking sites for the recruitment of specific binding proteins, which recognize and interact with modified histones via specialized domains such as bromo-, chromo- and PHD (plant homeodomain) domains, thereby influence chromatin dynamics and function (Wysocka *et al.*, 2005;Wysocka *et al.*, 2006b;Zeng and Zhou, 2002). A number of proteins have been identified that are recruited to specific modifications. For example, methylation of H3K4, H3K9 and H3K27 can be recognized by inhibitor of growth (ING) proteins, heterochromatin protein 1 (HP1) and polycomb proteins, respectively. It has been shown that histone modification binding proteins can tether, directly or indirectly, an enzyme to chromatin. The activity of this recruited enzyme can be regulated (Pena *et al.*, 2006;Shi *et al.*, 2006;Wysocka *et al.*, 2006b). BPTF, a component of the NURF chromatin remodelling complex, binds to H3K4me3 via a PHD domain and tethers the SNF2L ATPase to H0XC8 gene and activates the expression of the latter (Wysocka *et al.*, 2006b). JMJD2A and CHD1, two other H3K4me-binding proteins, possess enzymatic activities themselves and can directly deliver enzymatic activities to chromatin when recruited (Huang *et al.*, 2006;Pray-

The link between histone modifications and transcriptional regulation has been widely studied. It has been found that a specific modification can be associated with transcriptional activation or repression. Among the histone modifications, methylation and acetylation of H3 and H4 play a major role in the regulation of transcriptional activity (Berger, 2007;Jenuwein and Allis, 2001;Li *et al.*, 2007a;Shahbazian and Grunstein, 2007). Methylation, which occurs on

and Zhang, 2005).

176 Chromatin Remodelling

Grant *et al.*, 2005;Sims, III *et al.*, 2005).

Acetylation, another well-characterized modification, occurs on lysine residues mainly in the N-terminal tail of core histones. However, a lysine 56 within the globular domain of H3 (H3K56) has been found to be acetylated in yeast. Yeast protein SPT10, a putative histone acetyltransferase (HAT), was shown to mediate the H3K56 acetylation of histone genes at their promoter regions. H3K56 acetylation allows the recruitment of Snf5, an essential component of SWI/SNF chromatin remodeling complex and subsequently regulating transcription (Xu *et al.*, 2005). Compared with the SPT10, the Rtt109 acetyltransferase mediates H3K56 acetylation more globally (Driscoll *et al.*, 2007;Han *et al.*, 2007;Schneider *et al.*, 2006). The acetylation level correlates with transcriptional activation (Davie, 2003;Legube and Trouche, 2003). The level of acetylation is balanced by HATs and HDACs. Generally, increased levels of histone acetylation by HATs enhance chromatin decondensation and DNA accessibility for transcription factors to activate gene expression. In contrast to acetylation, deacetylation of histones catalyzed by HDACs leads to chromatin condensa‐ tion and gene silencing (Berger, 2007;Li *et al.*, 2007a). The relationship between histone acetylation and gene expression has been well documented (Verdone *et al.*, 2006). HATs can also acetylate non-histone proteins, such as transcription factors and nuclear recep‐ tors to facilitate gene expression (Bannister and Miska, 2000;Masumi, 2011)

Other histone modifications, such as phosphorylation, ubiquitylation and sumoylation, have also been shown to be involved in transcriptional regulation. For example, H3S10 phosphor‐ ylation has been demonstrated to be involved in the activation of NF-κB-regulated genes as well as "immediate early" genes, such as c-fos and c-jun (Macdonald *et al.*, 2005). Ubiquitina‐ tion of H2AK119 and H2BK120 are associated with transcriptional repression and activation, respectively (Wang *et al.*, 2006;Zhu *et al.*, 2005).

> **Figure 2. ATPase subunits of the four main families of ATP-dependent chromatin remodeling complexes.** The ATPase subunit of each ATP-dependent chromatin-remodeling complex belongs to the SNF2 ATPase superfamily, whose ATPase domain comprises an N-terminal DExx and a C-terminal HELICc subdomain, separated by an insert re‐ gion. The SWI/SNF family contains an HSA domain for actin binding, and a bromodomain which recognizes and binds to the acetylated histone tails. The ISWI family contains the SANT and SLIDE domains, important for histone binding. The CHD/NURD/Mi-2 family is characterized by the presence of two N-terminal chromodomains that is involved in the remodeling of chromatin structure and the transcriptional regulation of genes. The INO80 family, like the SWI/SNF family, also contains an HSA domain, however the insert region between the DExx and the HELICc subdomains is three

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**Figure 3. Mechanisms of ATP-dependent chromatin remodeling activity to alter the accessibility of nucleoso‐ mal DNA.** Upon utilization of the energy from ATP hydrolysis, the nucleosomal structure is altered to make protected region of chromatin available to DNA binding protein complexes, such as transcription factors, which involves mobili‐ zation of nucleosome position(sliding), dissociation of DNA-histone contact (unwrapping), and eviction of histones (histone eviction). In some cases ATP dependent remodeling complexes can use the energy from ATP hydrolysis to in‐ troduce histone variants into the nucleosome (exchange of histone variants), such as H2A–H2B or H2A variants

times longer than that of other three families.

(H2Avar)–H2B dimers.

#### **2.2. Chromatin remodelling complex and transcription**

The second major class of chromatin-modifying factors are the protein complexes that use energy from ATP hydrolysis to alter nucleosomal structure and DNA accessibility and hence are generally referred to as chromatin remodeling complex (Flaus and Owen-Hughes, 2004;Saha *et al.*, 2006). Each ATP-dependent chromatin-remodeling complex characterized to date contains a highly conserved ATPase subunit that belongs to the SNF2 ATPase superfamily (Marfella CGA, Mutate Res, 2007). Based on the similarities of their ATPase subunits and the presence of other conserved domains, these complexes can be classified into at least four different families (Figure 2): the SWI/SNF (mating type switching /sucrose non-fermenting) family; the ISWI (imitation switch) family; the NuRD/Mi-2/CHD (chromodomain helicase DNA-binding) family and INO80 (inositol requiring 80) family (Farrants, 2008;Saha *et al.*, 2006). The ATPase subunits of the SWI/SNF family members, including yeast Snf2 and Sth1, *Drosophila melangaster* brahma (BRM) and mammalian BRM and BRG1 (brahma-related gene 1), contain an C-terminal bromodomains which recognize and binds to acetylated histone tails (Hassan *et al.*, 2002;Marfella and Imbalzano, 2007). The members of ISWI family, such as yeast homologues ISW1 and ISW2, and mammalian homologues SNF2H and SNF2L, each contains an ATPase subunit with homology to *Drosophila* ISWI protein and has nucleosome-stimulated ATPase activity. These enzymes are characterized by the presence of a SANT (SWI3-ADA2- NCoR-TFIIIB) domain, which functions as a histone-tail-binding module (Boyer *et al.*, 2004;de la Serna *et al.*, 2006). SANT domain has been found in a number of transcriptional regulatory proteins and is therefore thought to play a role in transcriptional regulation (Aasland *et al.*, 1996;Boyer *et al.*, 2002). The NuRD/Mi-2/CHD family members include a number of proteins that are highly conserved from yeast to humans and are characterized by the presence of two N-terminal chromodomains involved in the remodeling of chromatin structure and regulation of transcription (Brehm *et al.*, 2004;Eissenberg, 2001;Jones *et al.*, 2000). The INO80 family contains the INO80 remodeling complex (INO80.com) and the SWR1 remodeling complex (SWR1.com), which are distinguished by the split ATPase domains and the presence of two RuvB-like proteins, Rvb1 and Rvb2 (Bao and Shen, 2007).

ATP-dependent chromatin remodelers can reposition (slide, twist, or loop) nucleosomes along the DNA, evict histones from DNA or facilitate exchange of histone variants, and thus creating nucleosome-free regions for gene activation (Figure 3) (Wang *et al.*, 2007).

acetylation and gene expression has been well documented (Verdone *et al.*, 2006). HATs can also acetylate non-histone proteins, such as transcription factors and nuclear recep‐

Other histone modifications, such as phosphorylation, ubiquitylation and sumoylation, have also been shown to be involved in transcriptional regulation. For example, H3S10 phosphor‐ ylation has been demonstrated to be involved in the activation of NF-κB-regulated genes as well as "immediate early" genes, such as c-fos and c-jun (Macdonald *et al.*, 2005). Ubiquitina‐ tion of H2AK119 and H2BK120 are associated with transcriptional repression and activation,

The second major class of chromatin-modifying factors are the protein complexes that use energy from ATP hydrolysis to alter nucleosomal structure and DNA accessibility and hence are generally referred to as chromatin remodeling complex (Flaus and Owen-Hughes, 2004;Saha *et al.*, 2006). Each ATP-dependent chromatin-remodeling complex characterized to date contains a highly conserved ATPase subunit that belongs to the SNF2 ATPase superfamily (Marfella CGA, Mutate Res, 2007). Based on the similarities of their ATPase subunits and the presence of other conserved domains, these complexes can be classified into at least four different families (Figure 2): the SWI/SNF (mating type switching /sucrose non-fermenting) family; the ISWI (imitation switch) family; the NuRD/Mi-2/CHD (chromodomain helicase DNA-binding) family and INO80 (inositol requiring 80) family (Farrants, 2008;Saha *et al.*, 2006). The ATPase subunits of the SWI/SNF family members, including yeast Snf2 and Sth1, *Drosophila melangaster* brahma (BRM) and mammalian BRM and BRG1 (brahma-related gene 1), contain an C-terminal bromodomains which recognize and binds to acetylated histone tails (Hassan *et al.*, 2002;Marfella and Imbalzano, 2007). The members of ISWI family, such as yeast homologues ISW1 and ISW2, and mammalian homologues SNF2H and SNF2L, each contains an ATPase subunit with homology to *Drosophila* ISWI protein and has nucleosome-stimulated ATPase activity. These enzymes are characterized by the presence of a SANT (SWI3-ADA2- NCoR-TFIIIB) domain, which functions as a histone-tail-binding module (Boyer *et al.*, 2004;de la Serna *et al.*, 2006). SANT domain has been found in a number of transcriptional regulatory proteins and is therefore thought to play a role in transcriptional regulation (Aasland *et al.*, 1996;Boyer *et al.*, 2002). The NuRD/Mi-2/CHD family members include a number of proteins that are highly conserved from yeast to humans and are characterized by the presence of two N-terminal chromodomains involved in the remodeling of chromatin structure and regulation of transcription (Brehm *et al.*, 2004;Eissenberg, 2001;Jones *et al.*, 2000). The INO80 family contains the INO80 remodeling complex (INO80.com) and the SWR1 remodeling complex (SWR1.com), which are distinguished by the split ATPase domains and the presence of two

ATP-dependent chromatin remodelers can reposition (slide, twist, or loop) nucleosomes along the DNA, evict histones from DNA or facilitate exchange of histone variants, and thus creating

tors to facilitate gene expression (Bannister and Miska, 2000;Masumi, 2011)

respectively (Wang *et al.*, 2006;Zhu *et al.*, 2005).

178 Chromatin Remodelling

**2.2. Chromatin remodelling complex and transcription**

RuvB-like proteins, Rvb1 and Rvb2 (Bao and Shen, 2007).

nucleosome-free regions for gene activation (Figure 3) (Wang *et al.*, 2007).

**Figure 2. ATPase subunits of the four main families of ATP-dependent chromatin remodeling complexes.** The ATPase subunit of each ATP-dependent chromatin-remodeling complex belongs to the SNF2 ATPase superfamily, whose ATPase domain comprises an N-terminal DExx and a C-terminal HELICc subdomain, separated by an insert re‐ gion. The SWI/SNF family contains an HSA domain for actin binding, and a bromodomain which recognizes and binds to the acetylated histone tails. The ISWI family contains the SANT and SLIDE domains, important for histone binding. The CHD/NURD/Mi-2 family is characterized by the presence of two N-terminal chromodomains that is involved in the remodeling of chromatin structure and the transcriptional regulation of genes. The INO80 family, like the SWI/SNF family, also contains an HSA domain, however the insert region between the DExx and the HELICc subdomains is three times longer than that of other three families.

**Figure 3. Mechanisms of ATP-dependent chromatin remodeling activity to alter the accessibility of nucleoso‐ mal DNA.** Upon utilization of the energy from ATP hydrolysis, the nucleosomal structure is altered to make protected region of chromatin available to DNA binding protein complexes, such as transcription factors, which involves mobili‐ zation of nucleosome position(sliding), dissociation of DNA-histone contact (unwrapping), and eviction of histones (histone eviction). In some cases ATP dependent remodeling complexes can use the energy from ATP hydrolysis to in‐ troduce histone variants into the nucleosome (exchange of histone variants), such as H2A–H2B or H2A variants (H2Avar)–H2B dimers.

### **3. The role of chromatin remodelling in the regulation of inflammatory gene expression**

shock. Therefore, the host has readily available mechanisms in place which allow to dampen the response to LPS or even confer unresponsiveness to successive stimuli with LPS, a phenomenon named LPS or endotoxin tolerance (Cavaillon and Adib-Conquy, 2006;Cavaillon *et al.*, 2003). The mechanisms underlying endotoxin tolerance are not completely understood, but are characterized by impaired TLR-mediated activation of both NF-κB- and MAPKdependent genes (Adib-Conquy *et al.*, 2006;Adib-Conquy *et al.*, 2000). Endotoxin tolerance has been shown to be associated with chromatin remodeling in the promoter regions of several tolerizable genes (Chan *et al.*, 2005;El *et al.*, 2007). Chang and colleagues have demonstrated that chromatin remodeling and NF-κB p65 recruitment at the IL-1β gene promoter are altered in LPS-tolerant THP-1 cells, when compared to normal THP-1 cells (Chan *et al.*, 2005). Upon LPS treatment, increased phosphorylation of H3S10 and demethylation of H3K9 are observed in normal THP-1 cells, which represent an "open" chromatin state; however, these modifica‐ tions are impaired in LPS-tolerant cells. Concomitantly, recruitment of NF- κB p65 but not NFκB p50 to the IL-1 gene promoter is impaired in LPS-tolerant cells despite that the activation and nuclear accumulation of NF- κB is not changed. Similar histone modifications and NF- κB binding were also observed at the TNF-α promoter during endotoxin tolerance (El *et al.*, 2007). Interestingly, LPS tolerance negatively regulates expression of proinflammatory mediators without affecting antimicrobial effectors. Using microarrays and real-time PCR, Foster and colleagues (Foster *et al.*, 2007) identified two classes of genes based on their responsiveness to re-stimulation with LPS: so called tolerizable genes, which include proinflammatory media‐ tors, and non-tolerizable genes, which include antimicrobial effectors. Induction of tolerance to LPS inhibits expression of the proinflammatory genes, while the other group of genes remain inducible. Both classes of gene promoters show H4 acetylation and H3K4 tri-methylation, which mark an "open" chromatin state, upon initial stimulation with LPS; however, this kind of "open" chromatin state and recruitment of Brg1 are lost in tolerizable genes upon LPS restimulation. In contrast, these epigenetic marks are maintained in the genes that remain inducible. Aung and colleagues reported that HDACs are transiently repressed then induced to express in murine bone marrow-derived macrophages when treated with LPS. HDACs are

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181

recruited to different gene promoters to regulate the expression of the latter.

**3.2. Manipulation of host chromatin remodelling process by bacteria to facilitate infection**

Interestingly, intracellular pathogens, such as *Listeria monocytogenes*, *Shigella flexneri*, and *Helicobacter pylori*, affecting the expression of host defense gene via modulation of chromatin structure has also been reported in recent years. *Listeria monocytogenes* is a gram positive bacterium that causes listeriosis. Two different mechanisms have been reported to be used by *L. monocytogenes* to modify histones during the course of infection. In endothelial cells, *L. monocytogenes* has been shown to selectively induce serine 10 phosphorylation and lysine 14 acetylation of H3 and lysine 8 acetylation of H4 at the IL-8 but not the Interferon-γ (IFN-γ) gene promoter through the activation of p38 and ERK MAPK pathway. A subsequent study showed that activation of p38 MAPK signaling pathway and NF-κB by *L. monocytogenes* depends on nucleotide-binding oligomerization domain-containing protein 1 (NOD1 ). NOD1 is critical for *L. monocytogenes* induced secretion of IL-8. Interestingly, only invasive bacteria which can enter into the host cell cytoplasm induce IL-8 production in endothelial cells (Opitz

The inflammatory response is a defense mechanism developed in higher organisms to protect themselves from infection with pathogens. It demands rapid and coordinated regulation of expression of multiple inflammatory genes in immune cells, including macrophages. It has increasingly become clear that alterations of chromatin architecture orchestrated by histone modifications and ATP-dependent chromatin remodeling complexes play a key role in controlling of inflammatory response genes (Medzhitov and Horng, 2009;Smale, 2010).

#### **3.1. LPS-induced chromatin modification and target gene expression**

LPS, a large molecule consisting of a lipid and a polysaccharide joined by a covalent bond, is the major component of the outer membrane of gram-negative bacteria and is one of the bestcharacterized agonist of host inflammatory response. LPS is recognized by Toll-like receptor 4 (TLR4) and activates the downstream signaling pathways, including the NF-κB signaling cascades, MAPK cascades and interferon regulatory factor (IRF) signaling cascades and induce the transcription of proinflammatory cytokine genes such as interleukin-6 (IL-6), IL-12 and tumor necrosis factor (TNF) (Akira and Takeda, 2004;Takeda *et al.*, 2003). The first evidence of the involvement of chromatin remodeling in LPS-induced gene expression dates back to 1999, when it was observed that nucleosome remodeling appears to contribute to the rapid induction of p40 subunit of IL-12 (IL-12p40). Upon activation by LPS, a positioned nucleosome, which spans the IL-12p40 gene promoter, is rapidly and selectively repositioned prior to initiation of transcription process (Weinmann *et al.*, 1999). Further studies demonstrated that the nucleo‐ some remodeling by LPS requires TLR4 signaling but is independent of c-Rel, one of the NFκB subunits required for transcription of integrated Il-12p40 promoter (Weinmann *et al.*, 2001). In the year 2000, Saccani and colleagues (Saccani *et al.*, 2002) revealed that upon LPS stimulation, H3 phosphorylation at serine 10 (H3S10) occurs selectively on the IL-12p40 promoter as well as promoters of a subset of other NF-κB-responsive proinflammatory genes such as IL-6, IL-8, and CC-chemokine ligand 2 (CCL2) but not TNF-α, MIP-1α and CCL3. This phosphorylation event was shown to be dependent on the activation of p38 MAPK signaling pathway by LPS, and specific inhibition of p38 activation blocks H3S10 phosphorylation, recruitment of NF-κB to the selective promoters and gene expression (Saccani *et al.*, 2002). Therefore, it is postulated that phosphorylation of H3S10 via the p38 MAPK signaling pathway promotes the loosening of chromatin at certain selective promoters, thereby permitting accessibility to NF-κB and allowing transcription to occur. There are some evidence that link H3S10 mark with transcriptional activation. Serine to alanine substitution at position 10 of H3 or deletion of Snf1, a histone H3 kinase which phosphorylates the serine 10, abrogates transcriptional activation of LPS- inducible genes (Lo *et al.*, 2001;Lo *et al.*, 2000).

LPS activates TLR-dependent signaling to produce inflammatory cytokines and chemokines, which contribute to the efficient control and clearance of invading pathogens. However, production of these inflammatory mediators is tightly regulated because excessive production results in amplified inflammatory response and fatal illness characteristic of severe septic shock. Therefore, the host has readily available mechanisms in place which allow to dampen the response to LPS or even confer unresponsiveness to successive stimuli with LPS, a phenomenon named LPS or endotoxin tolerance (Cavaillon and Adib-Conquy, 2006;Cavaillon *et al.*, 2003). The mechanisms underlying endotoxin tolerance are not completely understood, but are characterized by impaired TLR-mediated activation of both NF-κB- and MAPKdependent genes (Adib-Conquy *et al.*, 2006;Adib-Conquy *et al.*, 2000). Endotoxin tolerance has been shown to be associated with chromatin remodeling in the promoter regions of several tolerizable genes (Chan *et al.*, 2005;El *et al.*, 2007). Chang and colleagues have demonstrated that chromatin remodeling and NF-κB p65 recruitment at the IL-1β gene promoter are altered in LPS-tolerant THP-1 cells, when compared to normal THP-1 cells (Chan *et al.*, 2005). Upon LPS treatment, increased phosphorylation of H3S10 and demethylation of H3K9 are observed in normal THP-1 cells, which represent an "open" chromatin state; however, these modifica‐ tions are impaired in LPS-tolerant cells. Concomitantly, recruitment of NF- κB p65 but not NFκB p50 to the IL-1 gene promoter is impaired in LPS-tolerant cells despite that the activation and nuclear accumulation of NF- κB is not changed. Similar histone modifications and NF- κB binding were also observed at the TNF-α promoter during endotoxin tolerance (El *et al.*, 2007). Interestingly, LPS tolerance negatively regulates expression of proinflammatory mediators without affecting antimicrobial effectors. Using microarrays and real-time PCR, Foster and colleagues (Foster *et al.*, 2007) identified two classes of genes based on their responsiveness to re-stimulation with LPS: so called tolerizable genes, which include proinflammatory media‐ tors, and non-tolerizable genes, which include antimicrobial effectors. Induction of tolerance to LPS inhibits expression of the proinflammatory genes, while the other group of genes remain inducible. Both classes of gene promoters show H4 acetylation and H3K4 tri-methylation, which mark an "open" chromatin state, upon initial stimulation with LPS; however, this kind of "open" chromatin state and recruitment of Brg1 are lost in tolerizable genes upon LPS restimulation. In contrast, these epigenetic marks are maintained in the genes that remain inducible. Aung and colleagues reported that HDACs are transiently repressed then induced to express in murine bone marrow-derived macrophages when treated with LPS. HDACs are recruited to different gene promoters to regulate the expression of the latter.

**3. The role of chromatin remodelling in the regulation of inflammatory**

The inflammatory response is a defense mechanism developed in higher organisms to protect themselves from infection with pathogens. It demands rapid and coordinated regulation of expression of multiple inflammatory genes in immune cells, including macrophages. It has increasingly become clear that alterations of chromatin architecture orchestrated by histone modifications and ATP-dependent chromatin remodeling complexes play a key role in controlling of inflammatory response genes (Medzhitov and Horng, 2009;Smale, 2010).

LPS, a large molecule consisting of a lipid and a polysaccharide joined by a covalent bond, is the major component of the outer membrane of gram-negative bacteria and is one of the bestcharacterized agonist of host inflammatory response. LPS is recognized by Toll-like receptor 4 (TLR4) and activates the downstream signaling pathways, including the NF-κB signaling cascades, MAPK cascades and interferon regulatory factor (IRF) signaling cascades and induce the transcription of proinflammatory cytokine genes such as interleukin-6 (IL-6), IL-12 and tumor necrosis factor (TNF) (Akira and Takeda, 2004;Takeda *et al.*, 2003). The first evidence of the involvement of chromatin remodeling in LPS-induced gene expression dates back to 1999, when it was observed that nucleosome remodeling appears to contribute to the rapid induction of p40 subunit of IL-12 (IL-12p40). Upon activation by LPS, a positioned nucleosome, which spans the IL-12p40 gene promoter, is rapidly and selectively repositioned prior to initiation of transcription process (Weinmann *et al.*, 1999). Further studies demonstrated that the nucleo‐ some remodeling by LPS requires TLR4 signaling but is independent of c-Rel, one of the NFκB subunits required for transcription of integrated Il-12p40 promoter (Weinmann *et al.*, 2001). In the year 2000, Saccani and colleagues (Saccani *et al.*, 2002) revealed that upon LPS stimulation, H3 phosphorylation at serine 10 (H3S10) occurs selectively on the IL-12p40 promoter as well as promoters of a subset of other NF-κB-responsive proinflammatory genes such as IL-6, IL-8, and CC-chemokine ligand 2 (CCL2) but not TNF-α, MIP-1α and CCL3. This phosphorylation event was shown to be dependent on the activation of p38 MAPK signaling pathway by LPS, and specific inhibition of p38 activation blocks H3S10 phosphorylation, recruitment of NF-κB to the selective promoters and gene expression (Saccani *et al.*, 2002). Therefore, it is postulated that phosphorylation of H3S10 via the p38 MAPK signaling pathway promotes the loosening of chromatin at certain selective promoters, thereby permitting accessibility to NF-κB and allowing transcription to occur. There are some evidence that link H3S10 mark with transcriptional activation. Serine to alanine substitution at position 10 of H3 or deletion of Snf1, a histone H3 kinase which phosphorylates the serine 10, abrogates

**3.1. LPS-induced chromatin modification and target gene expression**

transcriptional activation of LPS- inducible genes (Lo *et al.*, 2001;Lo *et al.*, 2000).

LPS activates TLR-dependent signaling to produce inflammatory cytokines and chemokines, which contribute to the efficient control and clearance of invading pathogens. However, production of these inflammatory mediators is tightly regulated because excessive production results in amplified inflammatory response and fatal illness characteristic of severe septic

**gene expression**

180 Chromatin Remodelling

#### **3.2. Manipulation of host chromatin remodelling process by bacteria to facilitate infection**

Interestingly, intracellular pathogens, such as *Listeria monocytogenes*, *Shigella flexneri*, and *Helicobacter pylori*, affecting the expression of host defense gene via modulation of chromatin structure has also been reported in recent years. *Listeria monocytogenes* is a gram positive bacterium that causes listeriosis. Two different mechanisms have been reported to be used by *L. monocytogenes* to modify histones during the course of infection. In endothelial cells, *L. monocytogenes* has been shown to selectively induce serine 10 phosphorylation and lysine 14 acetylation of H3 and lysine 8 acetylation of H4 at the IL-8 but not the Interferon-γ (IFN-γ) gene promoter through the activation of p38 and ERK MAPK pathway. A subsequent study showed that activation of p38 MAPK signaling pathway and NF-κB by *L. monocytogenes* depends on nucleotide-binding oligomerization domain-containing protein 1 (NOD1 ). NOD1 is critical for *L. monocytogenes* induced secretion of IL-8. Interestingly, only invasive bacteria which can enter into the host cell cytoplasm induce IL-8 production in endothelial cells (Opitz *et al.*, 2006). In another study*, L. monocytogenes* has been found to induce a dramatic H3 dephosphorylation at serine 10 (H3S10) as well as a deacetylation of H4 during early phase of infection (Hamon *et al.*, 2007). In contrast to the report described as above, entry of bacteria into the host cells is not required for these histone modifications. The LLO released by *L. monocytogenes* is a member of CDC (cholesterol-dependent cytolysin) toxin family, which is identified as a major effector sufficient for induction of H3S10 dephosphorylation and H4 deacetylation. LLO –induced H3S10 dephosphorylation specifically occurs in the case of genes whose expression is regulated by LLO, a number of which are involved in immunity. Inter‐ estingly, other members of the large family of CDC toxins, such as PFO and PLY secreted by *Clostridium perfringens* and *Streptococcus pneumonia*, respectively, dephosphorylate H3S10 through a mechanism analogous to that of LLO (Hamon *et al.*, 2007), suggesting that different bacteria may subvert immune response through a similar mechanism.

mouse macrophage, *H. pylori* peptidyl prolyl cis-, trans-isomerase (HP0175) has been shown to induce H3S10 phosphorylation at the IL-6 promoter resulting in increased IL-6 gene transcrip‐ tion and protein expression (Pathak *et al.*, 2006). HP0175-induced IL-6 gene transcription is dependent on the TLR4 –dependent activation of ERK and p38 MAPKs, which subsequently activate mitogen- and stress-activated protein kinase 1 (MSK1), a serine kinase responsible for H3S10 phosphorylation. This modification allows for recruitment of NF-κB to the IL-6 promot‐ er and activation of gene transactivation. Interestingly, *H. pylori* infection has also been shown to dephosphorylate H3S10 and deacetylate H3K23 in a time- and dose- dependent manner in gastric epithelial cells (Ding *et al.*, 2010). Therefore, the effect of a specific histone modification in host cells appears to be cell type specific and gene promoter specific. Further studies demon‐ strate that *cag*pathogenicity island(PAI)is responsible forthedephosphorylation of H3S10 and this modification is independent of ERK and p38 signaling pathways as well as IFN signaling. In addition, H3S10 dephosphorylation is associated with changes in the host gene expression, whichcontributes tobacterialinfectionandpathogenesis (Ding*et al.*, 2010).Treatment ofgastric epithelial cells withTSA, a generalinhibitor of HDACs whichnon-specifically increaseshistone H3 and H4 acetylation at multiple sites results in altered gene transcription pattern in both *IL-8* and *c-fos* genes upon *H. pylori* infection. TSA reduces IL-8 but increases c-fos gene transcrip‐ tion in the presence of *H. pylori* infection (Ding *et al.*, 2010). *H. pylori* has also been shown to regulate the cell cycle controlled protein p21(WAF), which is associated with the release of

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HDAC-1from the promoter and histone H4 acetylation (Xia *et al.*, 2008).

expression of MHC-II (Schroder *et al.*, 2004).

**4. Chromatin remodeling and IFN-γ-induced transcriptional response**

IFN-γ is a cytokine secreted by activated T cells and natural killer cells. IFN-γ can induce expression of the major histocompatibility complex class II (MHC-II) on the cell surface (Boehm *et al.*, 1997), which presents antigens to CD4+ T cells and plays a crucial role in normal immune response. IFN-γ activates gene expression mainly via the activation of JAK (Janus tyrosine kinase)/STATI (signal transducer and activator of transcription) signaling pathway, leading to the translocation of active STAT1 homodimers into the nucleus. The STAT1 homodimers then bind to the IFN-γ -activated sites (GAS) present in the promot‐ ers of IFN-γ -responsive genes thereby mediating the transcription of these genes, includ‐ ing class II transactivator (CIITA), which is necessary for both constitutive and inducible

Chromatin remodeling, mediated by ATP-dependent chromatin remodeling complex and or histone-modifying enzymes, has also been shown to be involved in the activation of IFNγ -responsive genes, such as CIITA and HLA-DR (Ni *et al.*, 2005;Pattenden *et al.*, 2002;Zika *et al.*, 2003). SWI/SNF complex often cooperates with histone-modifying enzymes to regulate transcription of genes, including those which are induced by IFN-γ (Chi, 2004;Wright and Ting, 2006). Studies have demonstrated that the SWI/SNF complex and CREB-binding protein (CBP), a transcriptional co-activator with histone acetyltransferase activity, are recruited to CIITA promoter in an IFN-γ-inducible fashion, leading to transcriptional

*Shigella flexneri* is a human intestinal pathogen, causing dysentery by invading the epithelium of the colon and is responsible, worldwide, for more than one million deaths per year. Arbibe and colleagues have shown that *S. flexneri* infection abrogates phosphorylation of H3S10 at the promoters of a specific subset of genes, such as IL-8 and CCL-20. The underlying mechanism is that the type III effector protein, OspF, secreted by *S. flexneri* enters into the nucleus and specifically dephosphorylates ERK and p38 MAPKs and then blocks MAPK-dependent phosphorylation of H3S10. This occurs in a gene-selective way, and renders selected gene promoter sites inaccessible to NF-κB, thereby reducing the expression of a subset of NF-κBresponsive genes, including IL8 (Arbibe *et al.*, 2007). This specificity might be a consequence of OspF's ability to inactivate MAPKs, thereby preventing them from entering into nucleus. Once activated, MAPKs translocate into nucleus and are recruited to the chromatin covering their target genes, where they regulate the phosphorylation of transcription factors, histones and chromatin-remodeling enzymes (Chow and Davis, 2006). It has been shown that OspF– induced down-regulation of inflammatory response is accomplished through the interaction of OspF with host retinoblastoma (Rb) protein, which has been linked to histone modification (Zurawski *et al.*, 2009). OspF also has the phosphothreonine lyase activity, a unique activity that has been found in a family of conserved effectors secreted by type III secretion system including OspF, SpvC from nontyphoid *Salmonella species*, and HopAI1 from the plant pathogen *Pseudomonas syringae* (Kramer *et al.*, 2007;Li *et al.*, 2007b;Zhang *et al.*, 2007). These effectors specifically inactivate their host MAPK pathway by carrying out a β elimination reaction to irreversibly remove the phosphate moiety from the phosphothreonine in phos‐ phorylated MAPKs. Inhibition of MAPK signaling by OspF attenuates the recruitment of polymorphonuclear leukocytes to *Shigella* infection sites by suppressing the activation of a portion of NF-κB-responsive genes in mice (Arbibe *et al.*, 2007), thereby contributing to the survival and persistent infection of the pathogens.

*Helicobacter pylori* is a Gram-negative bacterium that colonizes the human gastric mucosa. The chronic infectiongenerates a stateofinflammationwhichmaydeveloptowardchronicgastritis, peptic ulcers and gastric malignancies (Peek, Jr. and Crabtree, 2006). The virulence factors of *Helicobacter pylori* have been suggested to play a crucial role in the development of inflamma‐ tion and in affecting the host immune system (Gebert *et al.*, 2003;Lu *et al.*, 2005). For example, in mouse macrophage, *H. pylori* peptidyl prolyl cis-, trans-isomerase (HP0175) has been shown to induce H3S10 phosphorylation at the IL-6 promoter resulting in increased IL-6 gene transcrip‐ tion and protein expression (Pathak *et al.*, 2006). HP0175-induced IL-6 gene transcription is dependent on the TLR4 –dependent activation of ERK and p38 MAPKs, which subsequently activate mitogen- and stress-activated protein kinase 1 (MSK1), a serine kinase responsible for H3S10 phosphorylation. This modification allows for recruitment of NF-κB to the IL-6 promot‐ er and activation of gene transactivation. Interestingly, *H. pylori* infection has also been shown to dephosphorylate H3S10 and deacetylate H3K23 in a time- and dose- dependent manner in gastric epithelial cells (Ding *et al.*, 2010). Therefore, the effect of a specific histone modification in host cells appears to be cell type specific and gene promoter specific. Further studies demon‐ strate that *cag*pathogenicity island(PAI)is responsible forthedephosphorylation of H3S10 and this modification is independent of ERK and p38 signaling pathways as well as IFN signaling. In addition, H3S10 dephosphorylation is associated with changes in the host gene expression, whichcontributes tobacterialinfectionandpathogenesis (Ding*et al.*, 2010).Treatment ofgastric epithelial cells withTSA, a generalinhibitor of HDACs whichnon-specifically increaseshistone H3 and H4 acetylation at multiple sites results in altered gene transcription pattern in both *IL-8* and *c-fos* genes upon *H. pylori* infection. TSA reduces IL-8 but increases c-fos gene transcrip‐ tion in the presence of *H. pylori* infection (Ding *et al.*, 2010). *H. pylori* has also been shown to regulate the cell cycle controlled protein p21(WAF), which is associated with the release of HDAC-1from the promoter and histone H4 acetylation (Xia *et al.*, 2008).

*et al.*, 2006). In another study*, L. monocytogenes* has been found to induce a dramatic H3 dephosphorylation at serine 10 (H3S10) as well as a deacetylation of H4 during early phase of infection (Hamon *et al.*, 2007). In contrast to the report described as above, entry of bacteria into the host cells is not required for these histone modifications. The LLO released by *L. monocytogenes* is a member of CDC (cholesterol-dependent cytolysin) toxin family, which is identified as a major effector sufficient for induction of H3S10 dephosphorylation and H4 deacetylation. LLO –induced H3S10 dephosphorylation specifically occurs in the case of genes whose expression is regulated by LLO, a number of which are involved in immunity. Inter‐ estingly, other members of the large family of CDC toxins, such as PFO and PLY secreted by *Clostridium perfringens* and *Streptococcus pneumonia*, respectively, dephosphorylate H3S10 through a mechanism analogous to that of LLO (Hamon *et al.*, 2007), suggesting that different

*Shigella flexneri* is a human intestinal pathogen, causing dysentery by invading the epithelium of the colon and is responsible, worldwide, for more than one million deaths per year. Arbibe and colleagues have shown that *S. flexneri* infection abrogates phosphorylation of H3S10 at the promoters of a specific subset of genes, such as IL-8 and CCL-20. The underlying mechanism is that the type III effector protein, OspF, secreted by *S. flexneri* enters into the nucleus and specifically dephosphorylates ERK and p38 MAPKs and then blocks MAPK-dependent phosphorylation of H3S10. This occurs in a gene-selective way, and renders selected gene promoter sites inaccessible to NF-κB, thereby reducing the expression of a subset of NF-κBresponsive genes, including IL8 (Arbibe *et al.*, 2007). This specificity might be a consequence of OspF's ability to inactivate MAPKs, thereby preventing them from entering into nucleus. Once activated, MAPKs translocate into nucleus and are recruited to the chromatin covering their target genes, where they regulate the phosphorylation of transcription factors, histones and chromatin-remodeling enzymes (Chow and Davis, 2006). It has been shown that OspF– induced down-regulation of inflammatory response is accomplished through the interaction of OspF with host retinoblastoma (Rb) protein, which has been linked to histone modification (Zurawski *et al.*, 2009). OspF also has the phosphothreonine lyase activity, a unique activity that has been found in a family of conserved effectors secreted by type III secretion system including OspF, SpvC from nontyphoid *Salmonella species*, and HopAI1 from the plant pathogen *Pseudomonas syringae* (Kramer *et al.*, 2007;Li *et al.*, 2007b;Zhang *et al.*, 2007). These effectors specifically inactivate their host MAPK pathway by carrying out a β elimination reaction to irreversibly remove the phosphate moiety from the phosphothreonine in phos‐ phorylated MAPKs. Inhibition of MAPK signaling by OspF attenuates the recruitment of polymorphonuclear leukocytes to *Shigella* infection sites by suppressing the activation of a portion of NF-κB-responsive genes in mice (Arbibe *et al.*, 2007), thereby contributing to the

*Helicobacter pylori* is a Gram-negative bacterium that colonizes the human gastric mucosa. The chronic infectiongenerates a stateofinflammationwhichmaydeveloptowardchronicgastritis, peptic ulcers and gastric malignancies (Peek, Jr. and Crabtree, 2006). The virulence factors of *Helicobacter pylori* have been suggested to play a crucial role in the development of inflamma‐ tion and in affecting the host immune system (Gebert *et al.*, 2003;Lu *et al.*, 2005). For example, in

bacteria may subvert immune response through a similar mechanism.

182 Chromatin Remodelling

survival and persistent infection of the pathogens.

#### **4. Chromatin remodeling and IFN-γ-induced transcriptional response**

IFN-γ is a cytokine secreted by activated T cells and natural killer cells. IFN-γ can induce expression of the major histocompatibility complex class II (MHC-II) on the cell surface (Boehm *et al.*, 1997), which presents antigens to CD4+ T cells and plays a crucial role in normal immune response. IFN-γ activates gene expression mainly via the activation of JAK (Janus tyrosine kinase)/STATI (signal transducer and activator of transcription) signaling pathway, leading to the translocation of active STAT1 homodimers into the nucleus. The STAT1 homodimers then bind to the IFN-γ -activated sites (GAS) present in the promot‐ ers of IFN-γ -responsive genes thereby mediating the transcription of these genes, includ‐ ing class II transactivator (CIITA), which is necessary for both constitutive and inducible expression of MHC-II (Schroder *et al.*, 2004).

Chromatin remodeling, mediated by ATP-dependent chromatin remodeling complex and or histone-modifying enzymes, has also been shown to be involved in the activation of IFNγ -responsive genes, such as CIITA and HLA-DR (Ni *et al.*, 2005;Pattenden *et al.*, 2002;Zika *et al.*, 2003). SWI/SNF complex often cooperates with histone-modifying enzymes to regulate transcription of genes, including those which are induced by IFN-γ (Chi, 2004;Wright and Ting, 2006). Studies have demonstrated that the SWI/SNF complex and CREB-binding protein (CBP), a transcriptional co-activator with histone acetyltransferase activity, are recruited to CIITA promoter in an IFN-γ-inducible fashion, leading to transcriptional activation of CIITA (Kretsovali *et al.*, 1998;Pattenden *et al.*, 2002). HLA-DR is a MHC–II surface molecule whose transcriptional activation is tightly associated with CIITA. Howev‐ er, forced expression of CIITA in BRG1- and BRM-deficient SW13 cells cannot activate expression of the MHC-II genes (Mudhasani and Fontes, 2002). BRG1 or BRM represent the catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, suggesting that the SWI/SNF complex, which contains BRG1 might play additional roles in MHC-II expression. Further studies have indicated that BRG1 is recruited by CIITA to the MHC-II gene promoters and this recruitment is essential for activation of MHC-II gene expres‐ sion (Mudhasani and Fontes, 2002). Interestingly, CIITA itself has intrinsic HAT activity, which can bind not onlyBRG1 but also HATs, such as CBP and/or p300 (Ting and Trowsdale, 2002). Furthermore, CIITA is associated with increased acetylation modifications of H3 and H4 at MHC-II promoter mediated directly through its intrinsic HAT activity or by the recruitment of HATs, such as CBP (Beresford and Boss, 2001;Kretsovali *et al.*, 1998). IFN-γ induced transactivation of CIITA and expression of MHC-II is inhibited by HDACs/ mSin3A corepressor complex whereas enhanced by TSA, a general inhibitor of HDAC. Coimmunoprecipitation assay revealed that CIITA interacts strongly with HDAC1 and weakly with HDAC2 (Zika *et al.*, 2003). All these data suggest that CIITA may act as a modula‐ tor to coordinate functions of chromatin remodeling complex, HATs and HDACs.

activities rescues histone acetylation, suggesting a role of HDACs in the transcriptional repression induced by *M. tuberculosis* (Wang *et al.*, 2005). Indeed, Mycobacterial infection increases the expression of mSin3A (a co-repressor associated HDACs), enabling competi‐

Chromatin Remodelling During Host-Bacterial Pathogen Interaction

http://dx.doi.org/10.5772/55977

185

A recent study has demonstrated that infection with *Toxoplasma gondii* renders murine macrophages globally unresponsive to IFN-γ stimulation without affecting the nuclear translocation of STAT1 triggered by IFN-γ in infected macrophages. However, the binding of STAT1 to the STAT1-responsive promoters is aberrant. A number of genes, which were induced by IFN-γ in uninfected macrophages, were not induced in the *T. gondii*-infected cells. Among them, there are several genes previously shown to be repressed by *T. gondii*, such as CIITA, MHC class II molecule H2-Eα, and interferon- regulatory factor 1(IRF-1) (Lang *et al.*, 2012). By analyzing the underlying mechanism, the authors revealed that assembly of chro‐ matin remodeling complex and histone acetylation at the IFN-γ -responsive promoters are impaired upon infection with *T. gondii*. Treatment with HADC inhibitor restores the respon‐ siveness of *T. gondii*-infected macrophages to IFN-γ, leading to an increase in the expression

**5. The potential role of HDAC inhibitors in treatment of infection**

HDAC inhibitors have been developed clinically for cancer therapy due to their abilities to induce cell-cycle arrest and apoptosis (Adcock, 2007). Studies have demonstrated that HDAC inhibitors can exert anti-inflammatory effects via the suppression of cytokine and nitric oxide production (Blanchard and Chipoy, 2005;Dinarello *et al.*, 2011), suggesting their therapeutic potential in inflammatory diseases including infectious diseases. For example, HDAC inhibitors have been examined for the treatment of HIV infection and the current results are exciting and encouraging (Wightman *et al.*, 2012). Couple of other studies have demonstrated that HDAC inhibitors, TSA and apicidin, can inhibit the growth of *Plasmodi‐ um falciparum*, the main parasite causing malaria in humans (Colletti *et al.*, 2001a;Colletti *et al.*, 2001b). Similarly, azelaic bishydroxamic acid and suberohydroxamic acid, two other HDAC inhibitors, also show anti-malarial activity against *P. falciparum* (Andrews *et al.*, 2000). The potential of HDAC inhibitors as anti-bacterial agents has also been investigat‐

**5.1. Inhibition of infection by targeting histone modifying enzymes in the pathogen**

*Candida albicans* is an opportunistic pathogen that is normally found in the gut microflora of healthy individuals; however, *C. albicans* can cause severe and life-threatening diseases in immuosuppressed patients such as HIV infected, organ transplant and cancer chemotherapy patients (Tzung *et al.*, 2001). There is a very high rate of mortality from systemic candidiasis, ranging between 14 and 90% and averaging between 30 to 40%, depending on the disease group studied (Blot *et al.*, 2003). For patients with *Candida* infections, antifungal drug resistance

tion with CBP for binding to the HLA-DR promoter.

of IFN-γ-inducible genes, such as CIITA and H2-A/E.

ed; however, the results are contradictory.

In the context of host-pathogen interaction, intracellular pathogens have been shown to subvert the host immune response by affecting the macrophage responsiveness to IFN-γ but the underlying mechanism remains unclear. Intracellular pathogens may affect IFN-γ response via different ways. For example, *Leishamania donovani* inhibited IFN-γ response through down-regulation of IFN-γ receptor expression or interfering with the JAK/STAT1 signaling pathway (Nandan and Reiner, 1995;Ray *et al.*, 2000). By contrast, mycobacteria such as *Mycobacterium avium* and *Mycobacterium tuberculosis* impair IFN-γ response through inhibition of IFN-γ -responsive gene expression without interfering with the JAK/STAT1 signaling pathway (Kincaid EZ, J Immunol, 2003, 171:2042-2049). Interestingly, only a subset of IFN-γ responsive genes get affected, including CIITA, HLA-DR and CD64, while others remained unaffected (Pennini *et al.*, 2006;Wang *et al.*, 2005). Further studies showed that infection with *M. tuberculosis* affects the chromatin remodeling on CIITA gene since IFN-γ -induced histone acetylation and recruitment of BRG1 were both impaired (Pennini *et al.*, 2006). Additionally, LpqH, a mycobacterial cell wall protein, induces binding of the C/EBP transcriptional repressor to the CIITA promoter and inhibits IFN-γ -induced CIITA transcription (Pennini *et al.*, 2007). It has been shown that C/EBP can recruit HDAC-1 containing transcriptional repressor complex to the promoter of peroxisome proliferatoractivated receptor beta thereby inhibiting its transcription (Di-Poi *et al.*, 2005). Therefore, *M. tuberculosis* might induce the recruitment of C/EBP resulting in transcriptional repres‐ sion. The exact molecular mechanism by which *M. tuberculosis* inhibits IFN-γ -induced CIITA transcription remains to be elucidated. Similarly to CIITA, IFN-γ -induced histone acetylation gets impaired at the HLA-DR promoter and HLA-DR transcription becomes inhibited when the cells get infected with *M. tuberculosis*. Furthermore, inhibition of HDAC activities rescues histone acetylation, suggesting a role of HDACs in the transcriptional repression induced by *M. tuberculosis* (Wang *et al.*, 2005). Indeed, Mycobacterial infection increases the expression of mSin3A (a co-repressor associated HDACs), enabling competi‐ tion with CBP for binding to the HLA-DR promoter.

activation of CIITA (Kretsovali *et al.*, 1998;Pattenden *et al.*, 2002). HLA-DR is a MHC–II surface molecule whose transcriptional activation is tightly associated with CIITA. Howev‐ er, forced expression of CIITA in BRG1- and BRM-deficient SW13 cells cannot activate expression of the MHC-II genes (Mudhasani and Fontes, 2002). BRG1 or BRM represent the catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, suggesting that the SWI/SNF complex, which contains BRG1 might play additional roles in MHC-II expression. Further studies have indicated that BRG1 is recruited by CIITA to the MHC-II gene promoters and this recruitment is essential for activation of MHC-II gene expres‐ sion (Mudhasani and Fontes, 2002). Interestingly, CIITA itself has intrinsic HAT activity, which can bind not onlyBRG1 but also HATs, such as CBP and/or p300 (Ting and Trowsdale, 2002). Furthermore, CIITA is associated with increased acetylation modifications of H3 and H4 at MHC-II promoter mediated directly through its intrinsic HAT activity or by the recruitment of HATs, such as CBP (Beresford and Boss, 2001;Kretsovali *et al.*, 1998). IFN-γ induced transactivation of CIITA and expression of MHC-II is inhibited by HDACs/ mSin3A corepressor complex whereas enhanced by TSA, a general inhibitor of HDAC. Coimmunoprecipitation assay revealed that CIITA interacts strongly with HDAC1 and weakly with HDAC2 (Zika *et al.*, 2003). All these data suggest that CIITA may act as a modula‐

184 Chromatin Remodelling

tor to coordinate functions of chromatin remodeling complex, HATs and HDACs.

In the context of host-pathogen interaction, intracellular pathogens have been shown to subvert the host immune response by affecting the macrophage responsiveness to IFN-γ but the underlying mechanism remains unclear. Intracellular pathogens may affect IFN-γ response via different ways. For example, *Leishamania donovani* inhibited IFN-γ response through down-regulation of IFN-γ receptor expression or interfering with the JAK/STAT1 signaling pathway (Nandan and Reiner, 1995;Ray *et al.*, 2000). By contrast, mycobacteria such as *Mycobacterium avium* and *Mycobacterium tuberculosis* impair IFN-γ response through inhibition of IFN-γ -responsive gene expression without interfering with the JAK/STAT1 signaling pathway (Kincaid EZ, J Immunol, 2003, 171:2042-2049). Interestingly, only a subset of IFN-γ responsive genes get affected, including CIITA, HLA-DR and CD64, while others remained unaffected (Pennini *et al.*, 2006;Wang *et al.*, 2005). Further studies showed that infection with *M. tuberculosis* affects the chromatin remodeling on CIITA gene since IFN-γ -induced histone acetylation and recruitment of BRG1 were both impaired (Pennini *et al.*, 2006). Additionally, LpqH, a mycobacterial cell wall protein, induces binding of the C/EBP transcriptional repressor to the CIITA promoter and inhibits IFN-γ -induced CIITA transcription (Pennini *et al.*, 2007). It has been shown that C/EBP can recruit HDAC-1 containing transcriptional repressor complex to the promoter of peroxisome proliferatoractivated receptor beta thereby inhibiting its transcription (Di-Poi *et al.*, 2005). Therefore, *M. tuberculosis* might induce the recruitment of C/EBP resulting in transcriptional repres‐ sion. The exact molecular mechanism by which *M. tuberculosis* inhibits IFN-γ -induced CIITA transcription remains to be elucidated. Similarly to CIITA, IFN-γ -induced histone acetylation gets impaired at the HLA-DR promoter and HLA-DR transcription becomes inhibited when the cells get infected with *M. tuberculosis*. Furthermore, inhibition of HDAC A recent study has demonstrated that infection with *Toxoplasma gondii* renders murine macrophages globally unresponsive to IFN-γ stimulation without affecting the nuclear translocation of STAT1 triggered by IFN-γ in infected macrophages. However, the binding of STAT1 to the STAT1-responsive promoters is aberrant. A number of genes, which were induced by IFN-γ in uninfected macrophages, were not induced in the *T. gondii*-infected cells. Among them, there are several genes previously shown to be repressed by *T. gondii*, such as CIITA, MHC class II molecule H2-Eα, and interferon- regulatory factor 1(IRF-1) (Lang *et al.*, 2012). By analyzing the underlying mechanism, the authors revealed that assembly of chro‐ matin remodeling complex and histone acetylation at the IFN-γ -responsive promoters are impaired upon infection with *T. gondii*. Treatment with HADC inhibitor restores the respon‐ siveness of *T. gondii*-infected macrophages to IFN-γ, leading to an increase in the expression of IFN-γ-inducible genes, such as CIITA and H2-A/E.

#### **5. The potential role of HDAC inhibitors in treatment of infection**

HDAC inhibitors have been developed clinically for cancer therapy due to their abilities to induce cell-cycle arrest and apoptosis (Adcock, 2007). Studies have demonstrated that HDAC inhibitors can exert anti-inflammatory effects via the suppression of cytokine and nitric oxide production (Blanchard and Chipoy, 2005;Dinarello *et al.*, 2011), suggesting their therapeutic potential in inflammatory diseases including infectious diseases. For example, HDAC inhibitors have been examined for the treatment of HIV infection and the current results are exciting and encouraging (Wightman *et al.*, 2012). Couple of other studies have demonstrated that HDAC inhibitors, TSA and apicidin, can inhibit the growth of *Plasmodi‐ um falciparum*, the main parasite causing malaria in humans (Colletti *et al.*, 2001a;Colletti *et al.*, 2001b). Similarly, azelaic bishydroxamic acid and suberohydroxamic acid, two other HDAC inhibitors, also show anti-malarial activity against *P. falciparum* (Andrews *et al.*, 2000). The potential of HDAC inhibitors as anti-bacterial agents has also been investigat‐ ed; however, the results are contradictory.

#### **5.1. Inhibition of infection by targeting histone modifying enzymes in the pathogen**

*Candida albicans* is an opportunistic pathogen that is normally found in the gut microflora of healthy individuals; however, *C. albicans* can cause severe and life-threatening diseases in immuosuppressed patients such as HIV infected, organ transplant and cancer chemotherapy patients (Tzung *et al.*, 2001). There is a very high rate of mortality from systemic candidiasis, ranging between 14 and 90% and averaging between 30 to 40%, depending on the disease group studied (Blot *et al.*, 2003). For patients with *Candida* infections, antifungal drug resistance is a major clinical problem. H3K56 acetylation is mediated by HAT Rtt109 and seems to be much more abundant in yeasts than in mammals (Garcia *et al.*, 2007a;Xie *et al.*, 2009), and close homologues of Rtt109 have not yet been detected in mammals (Bazan, 2008). Therefore, it is expected that Rtt109 might be a unique target for antifungal therapeutics. Indeed, Wurtele and colleagues demonstrated that modulation of the acetylation of H3K56 exhibits potential as an anti-fungal therapy (Wurtele *et al.*, 2010). Interestingly, similar results have been found in a study by Lopes da Rosa *et al* (Lopes da *et al.*, 2010). Wurtele and colleagues showed that deleting Rtt109, an acetyltransferase of H3K56, leads to increased sensitivity to some anti-fungal drugs. Both teams also demonstrated that Rtt109 mutants are considerably less virulent in a mouse model infected with *C.albicans.* Wurtele and colleagues further investigated how the growth of *C. albicans* is affected by chemical modification of H3 *in vitro* and *in vivo*. They have observed that the growth of *C. albicans* is greatly inhibited when HST3, the H3 deacetylase acting on lysine 56, is inhibited by nicotinamide (a form of Vitamin B3 and product of the NAD+ dependent deacetylation reaction). Furthermore, modulation of H3K56 acetylation reduces the virulence of wild-type *C. albicans* in mice when nicotinamide was given in the drinking water of mice to repress HST3 (Wurtele *et al.*, 2010). These results, together with the study by Lopes da Rosa and colleagues, provide basis for targeting H3 modifying enzymes to fight fungal infections. Although important catalytic residues in Rtt109 are much different from those in mammalian homologues, it is still a challenge to find suitable fungal-specific inhibitors of H3 modifying enzymes in the future.

data reveal the complex effector mechanisms of HDAC inhibitors and suggest that more

Chromatin Remodelling During Host-Bacterial Pathogen Interaction

http://dx.doi.org/10.5772/55977

187

The activation and suppression of innate immunity are central principles of host-patho‐ gen interaction and need to be very well controlled. To establish persistent infection, intracellular pathogens must acquire efficient mechanisms to evade the host immune response. Interference with host posttranscriptional modifications by bacterial pathogens is a strategy widely used by the pathogens to promote survival and replication during the course of infection. MAPK, IFN-γ and transcription factor NF-κB signaling pathways are common targets for bacteria-induced posttranscriptional modifications (Ribet and Cossart, 2010). Interestingly, in the past few years, evidence has accumulated that targeting of histone modifications and chromatin remodeling, and subsequently subverting the host immune response, is a new and exciting field in the study of host-pathogen interaction. Phosphory‐ lation of H3 and acetylation of H3 and/or H4 at lysine residues are frequently associated with transactivation. Conversely, dephosphorylation and methylation of histones are more often associated with gene suppression (Berger, 2002;Kouzarides, 2007;Verdone *et al.*, 2006). Several strains of bacteria, including *L. monocytogenes*, *C. perfringens*, *S. pneumonia* and *H. pylori*, induce the same dephosphorylation of H3S10, while *S. flexneri* blocks phosphoryla‐ tion of H3S10; all of which lead to decreased phosphorylation of H3S10 and are associat‐

The molecular mechanisms by which bacterial infection induces histone modification and chromatin remodeling remain to be understood. For many pathogens, it is very difficult to hypothesize about the extent or the mechanics of epigenetic change they might induce. Currently available data largely provide snapshots of what is happening to the usual host genes studied in an infection model. More comprehensive global studies, such as ChIP-on– chip (chromatin immunoprecipitation coupled with expression microarray technology) for mapping global chromatin modifications, are now necessary and possible. This might provide fundamental clues to better understand the role and mechanism of chromatin regulation in the control of immune gene expression in inflammatory and infectious

studies are required to fully understand this complex process.

**6. Concluding remarks**

ed with altered host immune response.

Yong Zhong Xu, Cynthia Kanagaratham and Danuta Radzioch

Department of Medicine, McGill University, Montreal, Canada

\*Address all correspondence to: danuta.radzioch@mcgill.ca

diseases.

**Author details**

#### **5.2. Effects of HDAC inhibitors on host defense against bacterial infection**

In a mouse model of septic shock induced by LPS, administration of of suberoylanilide hydroxamic acid (SAHA) (50mg/kg intraperitoneally), improves long-term survival rates of mice whether given before or post a lethal dose of LPS, which may be due to the downregulation of MyD88-dependent pathway and decreased expression of proinflammatory mediators such as TNF-alpha, IL-1β, and IL-6 (Li *et al.*, 2010;Li *et al.*, 2009). Further studies demonstrated that treatment with SAHA increases anti-inflammatory IL-10 levels while decreasing proinflammatory IL-6 and MAP kinase production in the liver of septic shock mice (Finkelstein *et al.*, 2010). In contrast, it has also been shown that treatment with HDAC inhibitors lead to impaired host defense against bacterial infections. Studies have shown that HDAC inhibitors, TSA, SAHA, and VPA, can impair innate immune responses to TLR agonists by down-regulating the expression of genes involved in microbial sensing, such as C-type lectins and adhesion molecules, as well as genes involved in host defense, such as cytokines and chemokines, thereby increasing susceptibility to infection (Roger *et al.*, 2011). Interestingly, while LPS-induced IFN-β production is enhanced by HDAC inhibitors, the expression of a number of IFN-β /STAT1-dependent genes is strongly inhibited by TSA and VPA, suggesting that increased IFN-β production cannot overcome the potent inhibitory effects of HDAC inhibitors. Surprisingly, VPA was shown to increase the mortality of mice infected with *C. albicans* or *K. pneumonia*, but protect mice from toxic shock and severe sepsis in mouse models (Roger *et al.*, 2011). When murine macrophages were treated with TSA and VPA, their ability to kill *Escherichia coli* and *Staphyloccocus aureus* was attenuated, with impaired phagocytosis and production of reactive oxygen and nitrogen species (Mombelli *et al.*, 2011). Together, these data reveal the complex effector mechanisms of HDAC inhibitors and suggest that more studies are required to fully understand this complex process.

#### **6. Concluding remarks**

is a major clinical problem. H3K56 acetylation is mediated by HAT Rtt109 and seems to be much more abundant in yeasts than in mammals (Garcia *et al.*, 2007a;Xie *et al.*, 2009), and close homologues of Rtt109 have not yet been detected in mammals (Bazan, 2008). Therefore, it is expected that Rtt109 might be a unique target for antifungal therapeutics. Indeed, Wurtele and colleagues demonstrated that modulation of the acetylation of H3K56 exhibits potential as an anti-fungal therapy (Wurtele *et al.*, 2010). Interestingly, similar results have been found in a study by Lopes da Rosa *et al* (Lopes da *et al.*, 2010). Wurtele and colleagues showed that deleting Rtt109, an acetyltransferase of H3K56, leads to increased sensitivity to some anti-fungal drugs. Both teams also demonstrated that Rtt109 mutants are considerably less virulent in a mouse model infected with *C.albicans.* Wurtele and colleagues further investigated how the growth of *C. albicans* is affected by chemical modification of H3 *in vitro* and *in vivo*. They have observed that the growth of *C. albicans* is greatly inhibited when HST3, the H3 deacetylase acting on lysine 56, is inhibited by nicotinamide (a form of Vitamin B3 and product of the NAD+ dependent deacetylation reaction). Furthermore, modulation of H3K56 acetylation reduces the virulence of wild-type *C. albicans* in mice when nicotinamide was given in the drinking water of mice to repress HST3 (Wurtele *et al.*, 2010). These results, together with the study by Lopes da Rosa and colleagues, provide basis for targeting H3 modifying enzymes to fight fungal infections. Although important catalytic residues in Rtt109 are much different from those in mammalian homologues, it is still a challenge to find suitable fungal-specific inhibitors of H3

**5.2. Effects of HDAC inhibitors on host defense against bacterial infection**

In a mouse model of septic shock induced by LPS, administration of of suberoylanilide hydroxamic acid (SAHA) (50mg/kg intraperitoneally), improves long-term survival rates of mice whether given before or post a lethal dose of LPS, which may be due to the downregulation of MyD88-dependent pathway and decreased expression of proinflammatory mediators such as TNF-alpha, IL-1β, and IL-6 (Li *et al.*, 2010;Li *et al.*, 2009). Further studies demonstrated that treatment with SAHA increases anti-inflammatory IL-10 levels while decreasing proinflammatory IL-6 and MAP kinase production in the liver of septic shock mice (Finkelstein *et al.*, 2010). In contrast, it has also been shown that treatment with HDAC inhibitors lead to impaired host defense against bacterial infections. Studies have shown that HDAC inhibitors, TSA, SAHA, and VPA, can impair innate immune responses to TLR agonists by down-regulating the expression of genes involved in microbial sensing, such as C-type lectins and adhesion molecules, as well as genes involved in host defense, such as cytokines and chemokines, thereby increasing susceptibility to infection (Roger *et al.*, 2011). Interestingly, while LPS-induced IFN-β production is enhanced by HDAC inhibitors, the expression of a number of IFN-β /STAT1-dependent genes is strongly inhibited by TSA and VPA, suggesting that increased IFN-β production cannot overcome the potent inhibitory effects of HDAC inhibitors. Surprisingly, VPA was shown to increase the mortality of mice infected with *C. albicans* or *K. pneumonia*, but protect mice from toxic shock and severe sepsis in mouse models (Roger *et al.*, 2011). When murine macrophages were treated with TSA and VPA, their ability to kill *Escherichia coli* and *Staphyloccocus aureus* was attenuated, with impaired phagocytosis and production of reactive oxygen and nitrogen species (Mombelli *et al.*, 2011). Together, these

modifying enzymes in the future.

186 Chromatin Remodelling

The activation and suppression of innate immunity are central principles of host-patho‐ gen interaction and need to be very well controlled. To establish persistent infection, intracellular pathogens must acquire efficient mechanisms to evade the host immune response. Interference with host posttranscriptional modifications by bacterial pathogens is a strategy widely used by the pathogens to promote survival and replication during the course of infection. MAPK, IFN-γ and transcription factor NF-κB signaling pathways are common targets for bacteria-induced posttranscriptional modifications (Ribet and Cossart, 2010). Interestingly, in the past few years, evidence has accumulated that targeting of histone modifications and chromatin remodeling, and subsequently subverting the host immune response, is a new and exciting field in the study of host-pathogen interaction. Phosphory‐ lation of H3 and acetylation of H3 and/or H4 at lysine residues are frequently associated with transactivation. Conversely, dephosphorylation and methylation of histones are more often associated with gene suppression (Berger, 2002;Kouzarides, 2007;Verdone *et al.*, 2006). Several strains of bacteria, including *L. monocytogenes*, *C. perfringens*, *S. pneumonia* and *H. pylori*, induce the same dephosphorylation of H3S10, while *S. flexneri* blocks phosphoryla‐ tion of H3S10; all of which lead to decreased phosphorylation of H3S10 and are associat‐ ed with altered host immune response.

The molecular mechanisms by which bacterial infection induces histone modification and chromatin remodeling remain to be understood. For many pathogens, it is very difficult to hypothesize about the extent or the mechanics of epigenetic change they might induce. Currently available data largely provide snapshots of what is happening to the usual host genes studied in an infection model. More comprehensive global studies, such as ChIP-on– chip (chromatin immunoprecipitation coupled with expression microarray technology) for mapping global chromatin modifications, are now necessary and possible. This might provide fundamental clues to better understand the role and mechanism of chromatin regulation in the control of immune gene expression in inflammatory and infectious diseases.

#### **Author details**

Yong Zhong Xu, Cynthia Kanagaratham and Danuta Radzioch

\*Address all correspondence to: danuta.radzioch@mcgill.ca

Department of Medicine, McGill University, Montreal, Canada

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**Chapter 9**

**Rett Syndrome**

Daniela Zahorakova

**1. Introduction**

http://dx.doi.org/10.5772/55020

syndrome will be discussed.

**1.1. History**

Additional information is available at the end of the chapter

Defects in epigenetic mechanisms can give rise to several neurological and behavioral phenotypes. Rett syndrome (MIM 312750) is a pervasive neurodevelopmental disorder that is primarily caused by mutations in a gene encoding methyl-CpG-binding protein 2. The functions of the protein are related to DNA methylation, a key epigenetic mechanism that plays a critical role in gene silencing through chromatin remodeling. Rett syndrome was the first human disorder in which a link between epigenetic modification and neuronal dysfunction was discovered. In this chapter, the clinical features and the molecular pathology of Rett

Rett syndrome was first recognized by the Viennese pediatrician Andreas Rett. In 1965, he observed two girls sitting on their mothers' laps in his waiting room. Both girls were pro‐ foundly intellectually disabled and were continually wringing their hands in the same unusual manner. Dr. Rett recollected seeing such behavior in previous patients and searched for their files with his secretary. They found several girls with a similar developmental history and clinical features. He realized that these symptoms constituted something other than cerebral palsy, which was the usual designation at the time. In 1966, Dr. Rett published the first description of the disorder that now bears his name [1]. His paper, however, remained unnoticed by the medical community until the 1980s, when Swedish child neurologist Bengt Hagberg with colleagues published the same clinical findings and named the disorder Rett syndrome [2]. Later, diagnostic criteria were proposed [3], and Rett syndrome became recognized worldwide by pediatricians, neurologists, geneticists, and neuroscientists. Despite great effort, the genetic cause of the disorder was not determined until more than 30 years after the first clinical account. In 1999, mutations within the methyl-CpG-binding protein 2 gene (*MECP2*) were identified in patients with Rett syndrome [4], which became a turning point in

> © 2013 Zahorakova; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.


**Chapter 9**

## **Rett Syndrome**

[141] Zhu, B, Zheng, Y, Pham, A. D, Mandal, S. S, Erdjument-bromage, H, Tempst, P, & Reinberg, D. (2005). Monoubiquitination of human histone H2B: the factors involved

[142] Zika, E, Greer, S. F, Zhu, X. S, & Ting, J. P. (2003). Histone deacetylase 1/mSin3A dis‐ rupts gamma interferon-induced CIITA function and major histocompatibility com‐

[143] Zupkovitz, G. (2006). Negative and positive regulation of gene expression by mouse

[144] Zurawski, D. V, Mumy, K. L, Faherty, C. S, Mccormick, B. A, & Maurelli, A. T. (2009). Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblasto‐

and their roles in HOX gene regulation. Mol. Cell. *601611*, 20

histone deacetylase 1. Mol. Cell Biol. *79137928*, 26

ma protein. Mol. Microbiol. *350368*, 71

198 Chromatin Remodelling

plex class II enhanceosome formation. Mol. Cell Biol. *30913102*, 23

Daniela Zahorakova

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55020

### **1. Introduction**

Defects in epigenetic mechanisms can give rise to several neurological and behavioral phenotypes. Rett syndrome (MIM 312750) is a pervasive neurodevelopmental disorder that is primarily caused by mutations in a gene encoding methyl-CpG-binding protein 2. The functions of the protein are related to DNA methylation, a key epigenetic mechanism that plays a critical role in gene silencing through chromatin remodeling. Rett syndrome was the first human disorder in which a link between epigenetic modification and neuronal dysfunction was discovered. In this chapter, the clinical features and the molecular pathology of Rett syndrome will be discussed.

#### **1.1. History**

Rett syndrome was first recognized by the Viennese pediatrician Andreas Rett. In 1965, he observed two girls sitting on their mothers' laps in his waiting room. Both girls were pro‐ foundly intellectually disabled and were continually wringing their hands in the same unusual manner. Dr. Rett recollected seeing such behavior in previous patients and searched for their files with his secretary. They found several girls with a similar developmental history and clinical features. He realized that these symptoms constituted something other than cerebral palsy, which was the usual designation at the time. In 1966, Dr. Rett published the first description of the disorder that now bears his name [1]. His paper, however, remained unnoticed by the medical community until the 1980s, when Swedish child neurologist Bengt Hagberg with colleagues published the same clinical findings and named the disorder Rett syndrome [2]. Later, diagnostic criteria were proposed [3], and Rett syndrome became recognized worldwide by pediatricians, neurologists, geneticists, and neuroscientists. Despite great effort, the genetic cause of the disorder was not determined until more than 30 years after the first clinical account. In 1999, mutations within the methyl-CpG-binding protein 2 gene (*MECP2*) were identified in patients with Rett syndrome [4], which became a turning point in

© 2013 Zahorakova; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Rett syndrome research. This discovery allowed the molecular confirmation of clinical cases and contributed to amendments of the diagnostic criteria [5]. Most importantly, this finding started an extensive investigation into the molecular mechanisms that underlie the pathology of Rett syndrome.

#### **1.2. Occurence**

The estimated prevalence of Rett syndrome is 1:10,000 females by the age of 12 years old [6] with no specific ethnic or geographical preference. Rett syndrome is one of the leading genetic causes of profound mental retardation in females, second only to Down syndrome [7]. Male cases are very rare, and their phenotypic manifestations are different from those observed in girls with Rett syndrome.

#### **2. Clinical aspects of Rett syndrome**

#### **2.1. Symptoms and stages**

Rett syndrome, in its classic form, begins to manifest in early childhood and is character‐ ized by neurodevelopmental regression that severely affects motor, cognitive, and commu‐ nications skills.

Prenatal and perinatal periods are usually normal. Affected girls appear to develop normally during the first 6 to 18 months of life and seem to achieve appropriate develop‐ mental milestones. Nevertheless, retrospective analyses of home videos often show that, even during this period, affected female infants display some suboptimal development. This underdevelopment may include subtle motor and behavioral abnormalities, as well as hypotonia and feeding problems. General mobility and eye-hand coordination may be inadequate, and an excess of repetitive hand patting can be observed even during the first year of life. However, the overall developmental pattern is not obviously disturbed. The child is usually quiet and placid, and the parents often describe the child as "very good" [1, 8-11]. The characteristic clinical features appear successively over several stages, forming a distinctive disease progression pattern (Figure 1).

decline of motor and communicative performances is more gradual. Interest in people and objects is diminished, but eye contact may be preserved [11]. Voluntary hand use, such as grasping and reaching out for toys, is replaced with repetitive stereotypic hand movements, the hallmark of Rett syndrome. Patterns consisting of wringing, hand washing, mouthing, clapping, rubbing, squeezing, and other hand automatisms occur during waking hours [1, 16, 17]. Febrile seizures are often present, and epileptic paroxysms occur in most patients [18, 19]. The severity of seizures can vary, ranging from relatively mild or easily controlled by medi‐ cation to severe drug-resistant episodes [20]. Irregular breathing patterns, such as episodes of hyperventilation, breath holding, and aerophagia, usually develop toward the end of the regression period. Panting, spitting, and hypersalivation are also frequent symptoms [8, 21].

stereotypic hand movements, motor abnormalities, mental retardation

**3 4 5 10 20 >20**

decrease/loss of mobility

Parkinsonism

Rett Syndrome

201

http://dx.doi.org/10.5772/55020

loss of acquired hand skills, speech, and social interaction

scoliosis

anxiety

breathing irregularities

seizures

autonomic disfunction

**0.5 1 1.5 <sup>2</sup> age**

apparently normal development

microcephaly, growth arrest, hypotonia

autistic features

**Figure 1.** Onset and progression of Rett syndrome [12]

**(years)**

**Stage I**

**Stage II**

**Stage III**

**Stage IV**

**Stage III: Pseudostationary period** (age of onset: 4-7 years, after stage II). This stage can last for years or decades and is characterized by a relative stabilization of the disorder course. Patients may recover some skills, which were lost during the regression stage. Patients can become more joyful and sociable, and they may use eye pointing as a typical way to commu‐ nicate and to express their needs. Some patients may even learn new words and use simple phrases in a meaningful way. Nevertheless, they continue to suffer from gross cognitive impairments [14]. Despite improved eye contact and non-verbal communication ability, the loss of motor functions further progresses in this stage. Stereotypic hand movements become prominent, as do breathing irregularities. Many patients develop scoliosis, which is often

**Stage I: Early onset stagnation** (age of onset: 6-18 months). Psychomotor development begins to slow, but the general developmental pattern is not significantly abnormal. The deceleration of head growth (which eventually leads to microcephaly), growth retarda‐ tion, and weight loss occur in most patients. The child is delayed or ceases in the acquisi‐ tion of skills. Although babbling and new words may appear, language skills usually remain poor. A girl with Rett syndrome may become irritable and restless, and she may begin to display some autistic features, such as emotional withdrawal and indifference to the surrounding environment [11, 13, 14]

**Stage II: Developmental regression** (age of onset: 1-4 years). This stage may occur over a period of days to weeks and is characterized by a rapid reduction or loss of acquired skills, especially purposeful hand use, speech, and interpersonal contact [15]. In some patients, the

**Figure 1.** Onset and progression of Rett syndrome [12]

Rett syndrome research. This discovery allowed the molecular confirmation of clinical cases and contributed to amendments of the diagnostic criteria [5]. Most importantly, this finding started an extensive investigation into the molecular mechanisms that underlie the pathology

The estimated prevalence of Rett syndrome is 1:10,000 females by the age of 12 years old [6] with no specific ethnic or geographical preference. Rett syndrome is one of the leading genetic causes of profound mental retardation in females, second only to Down syndrome [7]. Male cases are very rare, and their phenotypic manifestations are different from those observed in

Rett syndrome, in its classic form, begins to manifest in early childhood and is character‐ ized by neurodevelopmental regression that severely affects motor, cognitive, and commu‐

Prenatal and perinatal periods are usually normal. Affected girls appear to develop normally during the first 6 to 18 months of life and seem to achieve appropriate develop‐ mental milestones. Nevertheless, retrospective analyses of home videos often show that, even during this period, affected female infants display some suboptimal development. This underdevelopment may include subtle motor and behavioral abnormalities, as well as hypotonia and feeding problems. General mobility and eye-hand coordination may be inadequate, and an excess of repetitive hand patting can be observed even during the first year of life. However, the overall developmental pattern is not obviously disturbed. The child is usually quiet and placid, and the parents often describe the child as "very good" [1, 8-11]. The characteristic clinical features appear successively over several stages, forming

**Stage I: Early onset stagnation** (age of onset: 6-18 months). Psychomotor development begins to slow, but the general developmental pattern is not significantly abnormal. The deceleration of head growth (which eventually leads to microcephaly), growth retarda‐ tion, and weight loss occur in most patients. The child is delayed or ceases in the acquisi‐ tion of skills. Although babbling and new words may appear, language skills usually remain poor. A girl with Rett syndrome may become irritable and restless, and she may begin to display some autistic features, such as emotional withdrawal and indifference to

**Stage II: Developmental regression** (age of onset: 1-4 years). This stage may occur over a period of days to weeks and is characterized by a rapid reduction or loss of acquired skills, especially purposeful hand use, speech, and interpersonal contact [15]. In some patients, the

of Rett syndrome.

girls with Rett syndrome.

**2.1. Symptoms and stages**

nications skills.

**2. Clinical aspects of Rett syndrome**

a distinctive disease progression pattern (Figure 1).

the surrounding environment [11, 13, 14]

**1.2. Occurence**

200 Chromatin Remodelling

decline of motor and communicative performances is more gradual. Interest in people and objects is diminished, but eye contact may be preserved [11]. Voluntary hand use, such as grasping and reaching out for toys, is replaced with repetitive stereotypic hand movements, the hallmark of Rett syndrome. Patterns consisting of wringing, hand washing, mouthing, clapping, rubbing, squeezing, and other hand automatisms occur during waking hours [1, 16, 17]. Febrile seizures are often present, and epileptic paroxysms occur in most patients [18, 19]. The severity of seizures can vary, ranging from relatively mild or easily controlled by medi‐ cation to severe drug-resistant episodes [20]. Irregular breathing patterns, such as episodes of hyperventilation, breath holding, and aerophagia, usually develop toward the end of the regression period. Panting, spitting, and hypersalivation are also frequent symptoms [8, 21].

**Stage III: Pseudostationary period** (age of onset: 4-7 years, after stage II). This stage can last for years or decades and is characterized by a relative stabilization of the disorder course. Patients may recover some skills, which were lost during the regression stage. Patients can become more joyful and sociable, and they may use eye pointing as a typical way to commu‐ nicate and to express their needs. Some patients may even learn new words and use simple phrases in a meaningful way. Nevertheless, they continue to suffer from gross cognitive impairments [14]. Despite improved eye contact and non-verbal communication ability, the loss of motor functions further progresses in this stage. Stereotypic hand movements become prominent, as do breathing irregularities. Many patients develop scoliosis, which is often rapidly progressive and eventually requires surgical treatment. Cold feet and lower limbs, with or without color and atrophic changes, are also common. These conditions occur due to poor perfusion, which is a consequence of altered autonomic control. Sleeping patterns are often disturbed and are characterized by frequent nighttime waking and daytime sleeping. Unexplained night laughing, sudden agitation and crying spells may also be present [11].

**Stage IV: Late motor deterioration** (age of onset: 5-15 years, after stage III). Non-verbal communication and social skills continue to improve gradually. Despite persistent serious cognitive impairment, older patients with Rett syndrome are usually, in contrast to patients with childhood autism, sociable and pleasant with others [22]. Seizures become less frequent and less severe, and stereotypic hand movements become less intense. However, motor deterioration continues with age. Most patients, who previously could walk, become nonam‐ bulatory and wheelchair-dependent. Decreased mobility leads to pronounced muscle wasting and rigidity, and, at older ages, the patients often develop Parkinsonian features [11, 23, 24].

Females with Rett syndrome often survive into adulthood and older age, but their life expectancy is less than that of the healthy population. The estimated annual death rate from Rett syndrome is 1.2%. Approximately 25% of these deaths are sudden and they may occur due to autonomic nervous system disturbances or cardiac abnormalities [25-27].

Many other features are associated with Rett syndrome, but they are not considered diagnostic. The patients are generally small for their age [28], which may be due to poor self-feeding abilities and problems with chewing and swallowing. They often suffer from gastroesophageal reflux and bloating. Decreased intestinal motility often results in severe constipation. Electroencephalogram results tend to be abnormal but without any clear diagnostic pattern. A prolonged QTc interval is observed in many patients and presents a risk for cardiac arrhythmia [26].

#### **2.2. Rett syndrome variants**

At least five atypical variants have been delineated in addition to classic Rett syndrome. These variants do not have all of the diagnostic features, and they are either milder or more severe than the classic form.

The most common atypical variant of Rett syndrome is "forme fruste". This mild variant is characterized by a protracted clinical course with partially preserved communication skills and gross motor functions. Other neurological abnormalities that are typical for Rett syndrome are more subtle and can be easily overlooked in this variant [30]. The mild forms of Rett syndrome also include the late regression variant, which manifests in patients of preschool or early school age [30], and the preserved speech variant (also called the Zappella variant) in which patients have preserved language skills and normal head sizes [31].

whether these variants are separate clinical entities, different from *MECP2*-related Rett

Consider Rett syndrome diagnosis when postnatal deceleration of head growth is observed

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203

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3. Supportive criteria are not required, although often present in classic Rett syndrome

3. Gait abnormalities: impaired ability (dyspraxia) or absence of ability (apraxia)

2. Grossly abnormal psychomotor development in the first 6 months of life

4. Stereotypic hand movements such as hand wringing/squeezing, clapping/tapping, mouthing and washing/

1. Breathing disturbances when awake (hyperventilation, breath-holding, forced expulsion of air or saliva, air

1. Brain injury secondary to trauma (perinatally or postnatally), neurometabolic disease or severe infection that cause

**Required for classic Rett syndrome**

2. All main and all exclusive criteria

2. At least 2 of 4 main criteria 3. 5 out 11 supportive criteria

**Main criteria**

rubbing automatisms

neurological problems

3. Impaired sleep pattern 4. Abnormal muscle tone

6. Scoliosis/kyphosis 7. Growth retardation 8. Small cold hands and feet

5. Peripheral vasomotor disturbances

9. Inappropriate laughing/screaming spells

10. Diminished sensitivity to pain

swallowing)

1. A period of regression followed by recovery or stabilization

1. A period of regression followed by recovery or stabilization

1. Partial or complete loss of acquired purposeful hand skills 2. Partial of complete loss of acquired spoken language

**Supportive criteria for atypical or variant Rett syndrome**

2. Bruxism when awake (grinding or clenching of the teeth)

11. Intense eye communication and eye-pointing behavior

**Table 1.** Diagnostic criteria for Rett syndrome [29]

**Required for atypical or variant Rett syndrome**

**Exclusion criteria for classic Rett syndrome**

Despite a known genetic cause, Rett syndrome remains a clinical diagnosis. Its diagnosis is based on several well-defined criteria (Table 1), which were revised several times over the past

syndrome [11].

**2.3. Diagnostic criteria**

few decades, most recently in 2010 [29].

Severe variants include the early-onset seizure variant (the Hanefeld variant) with the onset of seizures before the age of 6 months [32] and the congenital variant, which is rare and lacks the early period of normal psychomotor development [33]. The Hanefeld variant is often caused by mutations in the *CDKL5* gene [34], and most cases of the congenital variant are related to mutations in the *FOXG1* gene [35]. These genetic abnormalities raise the question of


**Table 1.** Diagnostic criteria for Rett syndrome [29]

whether these variants are separate clinical entities, different from *MECP2*-related Rett syndrome [11].

#### **2.3. Diagnostic criteria**

rapidly progressive and eventually requires surgical treatment. Cold feet and lower limbs, with or without color and atrophic changes, are also common. These conditions occur due to poor perfusion, which is a consequence of altered autonomic control. Sleeping patterns are often disturbed and are characterized by frequent nighttime waking and daytime sleeping. Unexplained night laughing, sudden agitation and crying spells may also be present [11]. **Stage IV: Late motor deterioration** (age of onset: 5-15 years, after stage III). Non-verbal communication and social skills continue to improve gradually. Despite persistent serious cognitive impairment, older patients with Rett syndrome are usually, in contrast to patients with childhood autism, sociable and pleasant with others [22]. Seizures become less frequent and less severe, and stereotypic hand movements become less intense. However, motor deterioration continues with age. Most patients, who previously could walk, become nonam‐ bulatory and wheelchair-dependent. Decreased mobility leads to pronounced muscle wasting and rigidity, and, at older ages, the patients often develop Parkinsonian features [11, 23, 24]. Females with Rett syndrome often survive into adulthood and older age, but their life expectancy is less than that of the healthy population. The estimated annual death rate from Rett syndrome is 1.2%. Approximately 25% of these deaths are sudden and they may occur due to autonomic nervous system disturbances or cardiac abnormalities [25-27].

Many other features are associated with Rett syndrome, but they are not considered diagnostic. The patients are generally small for their age [28], which may be due to poor self-feeding abilities and problems with chewing and swallowing. They often suffer from gastroesophageal reflux and bloating. Decreased intestinal motility often results in severe constipation. Electroencephalogram results tend to be abnormal but without any clear diagnostic pattern. A prolonged QTc interval is observed in many patients and presents a

At least five atypical variants have been delineated in addition to classic Rett syndrome. These variants do not have all of the diagnostic features, and they are either milder or more severe

The most common atypical variant of Rett syndrome is "forme fruste". This mild variant is characterized by a protracted clinical course with partially preserved communication skills and gross motor functions. Other neurological abnormalities that are typical for Rett syndrome are more subtle and can be easily overlooked in this variant [30]. The mild forms of Rett syndrome also include the late regression variant, which manifests in patients of preschool or early school age [30], and the preserved speech variant (also called the Zappella variant) in

Severe variants include the early-onset seizure variant (the Hanefeld variant) with the onset of seizures before the age of 6 months [32] and the congenital variant, which is rare and lacks the early period of normal psychomotor development [33]. The Hanefeld variant is often caused by mutations in the *CDKL5* gene [34], and most cases of the congenital variant are related to mutations in the *FOXG1* gene [35]. These genetic abnormalities raise the question of

which patients have preserved language skills and normal head sizes [31].

risk for cardiac arrhythmia [26].

**2.2. Rett syndrome variants**

than the classic form.

202 Chromatin Remodelling

Despite a known genetic cause, Rett syndrome remains a clinical diagnosis. Its diagnosis is based on several well-defined criteria (Table 1), which were revised several times over the past few decades, most recently in 2010 [29].

### **3. The genetics of Rett syndrome**

#### **3.1. Mapping of the causative gene**

The mode of inheritance of Rett syndrome was difficult to identify because more than 99% of the cases are sporadic, and the patients rarely reproduce. Therefore, the traditional genome-wide linkage analysis was not an applicable method for mapping the disease locus. The lack of males manifesting the classic Rett syndrome phenotype together with the occurrence of families with affected half-sisters suggested an X-linked dominant inheri‐ tance with lethality in hemizygous males [2, 36]. Focused exclusion mapping of the X chromosome in available familial cases was used to narrow down the candidate region, and the subsequent analysis of candidate genes in the patients finally revealed diseasecausing mutations in the *MECP2* gene [4].

heterogeneous, including missense and nonsense mutations, deletions, insertions, duplica‐ tions, splice-site mutations, and large deletions of several exons or the entire *MECP2* gene. More than 99% of the mutations occur *de novo* and mostly originate on the paternal X chro‐ mosome, which explains the high occurrence of Rett syndrome in the female gender [4, 48, 49]. Familial cases of Rett syndrome (mostly affected sisters or maternal half-sisters) are very rare. *MECP2* mutations in these patients are inherited from an asymptomatic or very mildly affected mother, who carries a somatic mutation, but does not manifest the full pathogenic phenotype due to favorable XCI pattern [50, 51]. Another explanation for transmission of a *MECP2* mutation to the next generation is a germline mosaicism for a mutation. It is suggested when the *MECP2* mutation identified in several affected children is not present in somatic cells

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The majority of point mutations in the *MECP2* gene are C>T transitions, presumably resulting from the spontaneous deamination of methylated cytosines [53]. The mutations are scattered throughout the coding sequence and splice sites, with the exception of exon 2. The eight most common mutations, which are also C>T transitions, account for approximately 70% of the Rett syndrome cases. Approximately 10% of cases are due to deletions, which are mostly clustered

Mutations in the *MECP2* gene have long been considered lethal in hemizygous males, and Rett syndrome has been assumed to be exclusively a female disorder. More recently, *MECP2* mutations were not only identified in males but also in females with phenotypes different from Rett syndrome. *MECP2*-related disorders thus represent a broad spectrum of phenotypes in

The estimated frequency of *MECP2* mutations in boys with mental retardation is 1.3-1.7% [54]. Typical Rett syndrome features have been observed almost exclusively in boys with Klinefelter syndrome (47,XXY) [55] or somatic mosaicism for a *MECP2* mutation [56, 57]. Other pheno‐ types include severe congenital encephalopathy with death in the first years of life [58-60] and mild to severe intellectual disability with or without various neurological and psychiatric symptoms [54, 61]. The most common *MECP2* mutations detected in males are duplications of the whole *MECP2* gene (and usually genes in its vicinity). This finding indicates that, besides the lack, an overabundance of fully functional MeCP2 protein is also harmful to the CNS. *MECP2* duplication syndrome is usually very mild or does not manifest in females. In boys, this syndrome is characterized by infantile hypotonia, severe mental retardation, loss of

In females, *MECP2* mutations have been detected in patients with mild mental retardation, learning disabilities or autism [65, 66]. More severe cases include severe mental retardation

speech, recurrent respiratory infections, seizures, and spasticity [62-64].

with seizures and Angelman-like syndrome [67, 68].

of their parents [51, 52].

both genders.

in the terminal segment of the coding region [12].

**3.4.** *MECP2* **mutations in males and other disorders**

#### **3.2.** *MECP2* **gene**

The *MECP2* gene (MIM 300005) is located on Xq28 and undergoes X chromosome inactivation (XCI) in females [37, 38]. The gene spans approximately 76 kb and consists of four exons, which encode methyl-CpG-binding protein 2 (MeCP2). Alternative splicing of exon 2 and several polyadenylation signals in a conserved and unusually long 3' untranslated region (3'UTR) give rise to eight different transcripts regulated in a tissue-specific and developmental stage-specific manner [39-42]. For example, the shortest transcript (1.8 kb) is predominant in adult muscles, heart, blood, and liver. The longest transcript (10.2 kb) occurs at the highest levels in the brain [41, 42]. The unique expression patterns of each transcript suggest a specific biological significance, such as a role in mRNA stability, nuclear export, folding, and sub-cellular localization, thus affecting the levels of the resulting protein [39]. The longest transcript also has one of the longest 3' UTR tails in the human genome (8.5 kb), with several blocks of highly conserved residues between the human and mouse genomes. These findings argue in favor of a potential regulatory role of the 3' UTR of the *MECP2* gene [43].

#### **3.3.** *MECP2* **mutations**

Mutations in the *MECP2* gene are identified in 90-95% of classic Rett syndrome patients [4, 44]. Because only the coding region and the adjacent non-coding parts of the gene are routinely analyzed, it is highly probable that mutations in more remote regulatory elements are responsible for the rest of the cases. The frequency of *MECP2* mutations in patients with atypical Rett syndrome variants varies considerably between studies. However, the frequency is generally lower (only 20-70% of patients have *MECP2* mutations) than in the classic form [44-46], suggesting that mutations of regulatory elements or other genes are involved more often in atypical Rett syndrome than in the classic Rett syndrome. The identification of *CDKL5* mutations in the Hanefeld variant and *FOXG1* mutations in the congenital variant strongly support the latter idea.

According to the Human Gene Mutation Database [47], more than 550 mutations have been identified in the *MECP2* gene in patients with Rett syndrome. The spectrum of mutations is heterogeneous, including missense and nonsense mutations, deletions, insertions, duplica‐ tions, splice-site mutations, and large deletions of several exons or the entire *MECP2* gene. More than 99% of the mutations occur *de novo* and mostly originate on the paternal X chro‐ mosome, which explains the high occurrence of Rett syndrome in the female gender [4, 48, 49]. Familial cases of Rett syndrome (mostly affected sisters or maternal half-sisters) are very rare. *MECP2* mutations in these patients are inherited from an asymptomatic or very mildly affected mother, who carries a somatic mutation, but does not manifest the full pathogenic phenotype due to favorable XCI pattern [50, 51]. Another explanation for transmission of a *MECP2* mutation to the next generation is a germline mosaicism for a mutation. It is suggested when the *MECP2* mutation identified in several affected children is not present in somatic cells of their parents [51, 52].

The majority of point mutations in the *MECP2* gene are C>T transitions, presumably resulting from the spontaneous deamination of methylated cytosines [53]. The mutations are scattered throughout the coding sequence and splice sites, with the exception of exon 2. The eight most common mutations, which are also C>T transitions, account for approximately 70% of the Rett syndrome cases. Approximately 10% of cases are due to deletions, which are mostly clustered in the terminal segment of the coding region [12].

#### **3.4.** *MECP2* **mutations in males and other disorders**

**3. The genetics of Rett syndrome**

causing mutations in the *MECP2* gene [4].

a potential regulatory role of the 3' UTR of the *MECP2* gene [43].

**3.2.** *MECP2* **gene**

204 Chromatin Remodelling

**3.3.** *MECP2* **mutations**

support the latter idea.

The mode of inheritance of Rett syndrome was difficult to identify because more than 99% of the cases are sporadic, and the patients rarely reproduce. Therefore, the traditional genome-wide linkage analysis was not an applicable method for mapping the disease locus. The lack of males manifesting the classic Rett syndrome phenotype together with the occurrence of families with affected half-sisters suggested an X-linked dominant inheri‐ tance with lethality in hemizygous males [2, 36]. Focused exclusion mapping of the X chromosome in available familial cases was used to narrow down the candidate region, and the subsequent analysis of candidate genes in the patients finally revealed disease-

The *MECP2* gene (MIM 300005) is located on Xq28 and undergoes X chromosome inactivation (XCI) in females [37, 38]. The gene spans approximately 76 kb and consists of four exons, which encode methyl-CpG-binding protein 2 (MeCP2). Alternative splicing of exon 2 and several polyadenylation signals in a conserved and unusually long 3' untranslated region (3'UTR) give rise to eight different transcripts regulated in a tissue-specific and developmental stage-specific manner [39-42]. For example, the shortest transcript (1.8 kb) is predominant in adult muscles, heart, blood, and liver. The longest transcript (10.2 kb) occurs at the highest levels in the brain [41, 42]. The unique expression patterns of each transcript suggest a specific biological significance, such as a role in mRNA stability, nuclear export, folding, and sub-cellular localization, thus affecting the levels of the resulting protein [39]. The longest transcript also has one of the longest 3' UTR tails in the human genome (8.5 kb), with several blocks of highly conserved residues between the human and mouse genomes. These findings argue in favor of

Mutations in the *MECP2* gene are identified in 90-95% of classic Rett syndrome patients [4, 44]. Because only the coding region and the adjacent non-coding parts of the gene are routinely analyzed, it is highly probable that mutations in more remote regulatory elements are responsible for the rest of the cases. The frequency of *MECP2* mutations in patients with atypical Rett syndrome variants varies considerably between studies. However, the frequency is generally lower (only 20-70% of patients have *MECP2* mutations) than in the classic form [44-46], suggesting that mutations of regulatory elements or other genes are involved more often in atypical Rett syndrome than in the classic Rett syndrome. The identification of *CDKL5* mutations in the Hanefeld variant and *FOXG1* mutations in the congenital variant strongly

According to the Human Gene Mutation Database [47], more than 550 mutations have been identified in the *MECP2* gene in patients with Rett syndrome. The spectrum of mutations is

**3.1. Mapping of the causative gene**

Mutations in the *MECP2* gene have long been considered lethal in hemizygous males, and Rett syndrome has been assumed to be exclusively a female disorder. More recently, *MECP2* mutations were not only identified in males but also in females with phenotypes different from Rett syndrome. *MECP2*-related disorders thus represent a broad spectrum of phenotypes in both genders.

The estimated frequency of *MECP2* mutations in boys with mental retardation is 1.3-1.7% [54]. Typical Rett syndrome features have been observed almost exclusively in boys with Klinefelter syndrome (47,XXY) [55] or somatic mosaicism for a *MECP2* mutation [56, 57]. Other pheno‐ types include severe congenital encephalopathy with death in the first years of life [58-60] and mild to severe intellectual disability with or without various neurological and psychiatric symptoms [54, 61]. The most common *MECP2* mutations detected in males are duplications of the whole *MECP2* gene (and usually genes in its vicinity). This finding indicates that, besides the lack, an overabundance of fully functional MeCP2 protein is also harmful to the CNS. *MECP2* duplication syndrome is usually very mild or does not manifest in females. In boys, this syndrome is characterized by infantile hypotonia, severe mental retardation, loss of speech, recurrent respiratory infections, seizures, and spasticity [62-64].

In females, *MECP2* mutations have been detected in patients with mild mental retardation, learning disabilities or autism [65, 66]. More severe cases include severe mental retardation with seizures and Angelman-like syndrome [67, 68].

#### **3.5. MeCP2 protein**

The MeCP2 protein is ubiquitously expressed, but it is particularly abundant in the brain [41, 69]. The protein levels are low during embryogenesis, but they progressively increase during postnatal neuronal maturation and synaptogenesis [70-75]. High expression of MeCP2 in mature neurons implies its involvement in postmitotic neuronal functions, such as the modulation of neuronal activity and plasticity [12].

(amino acids 173-193 and 255-271) mediate transportation of the protein into the nucleus [85]. The C-terminal domain (amino acids 325-486) facilitates binding to DNA [86] and it most likely increases protein stability [87]. This domain also contains conserved poly-proline motifs that

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The original model suggested that MeCP2 is a global transcriptional repressor [89], and it was based on *in vitro* experiments in which MeCP2 inhibited transcription from methylated promoters. Briefly, the protein binds to the promoters of target genes via its MBD, and the TRD then recruits the co-repressor Sin3A and HDACs [90, 91]. These interactions lead to the deacetylation of histones, resulting in chromatin condensation and the repression of down‐ stream genes. In addition to Sin3A, other co-repressors, such as c-Ski and N-CoR, may interact with MeCP2 [92]. The compaction of chromatin can also be promoted through direct interac‐ tion with the C-terminal domain [93], which is an example of HDAC-independent MeCP2 mediated transcriptional repression. The TRD may also directly interact with transcription factor IIB; therefore, MeCP2 may silence transcription by interfering with the assembly of the transcriptional preinitiation complex [94]. Additional factors interacting with the TRD include Brahma, which is a catalytic component of the SWI/SNF-related chromatin-remodeling complex (at least in NIH 3T3 cells) [95], DNA methyltransferase 1 [96], and ATRX, a SWI2/

Surprisingly, transcriptional profiling studies did not reveal major gene expression changes caused by the lack of functional MeCP2 protein [98, 99]. These observations, together with additional evidence, implied that MeCP2 regulates the transcription of tissue-specific genes in specific brain regions during certain developmental stages instead of acting as a global repressor [98, 100-102]. However, recent studies suggest that MeCP2 reduces genome-wide transcriptional noise, potentially by repressing spurious transcription of repetitive elements [103, 104]. Surprisingly, MeCP2 also interacts with the transcriptional activator CREB, and its genomic distribution is often associated with actively transcribed genes [105, 106]. MeCP2 apparently has dual roles in transcriptional regulation as a repressor and as an activator, and

MeCP2 additionally acts as an architectural chromatin protein that is involved in chromatin remodeling and nucleosome clustering, which is consistent with the fact that the majority of MeCP2-binding sites are located outside of genes [105, 107]. MeCP2 can bind *in vitro* to

For further complexity, MeCP2 may also be involved in RNA splicing. Its interaction with the RNA-binding protein Y box-binding protein 1 (YB-1) has been observed, and MeCP2-deficient

MeCP2 functions are undeniably much more complex than initially anticipated (Figure 3),

it performs different downstream responses depending on the context.

although the precise mechanisms of their regulation remain unknown.

mice showed aberrant alternative splicing patterns [110].

chromatin fibers and compact them into higher order structures [93, 108, 109].

can bind to group II WW domain splicing factors [88].

**3.6. MeCP2 function**

SNF2 DNA helicase/ATPase [97].

MeCP2 occurs in two isoforms that arise from alternative splicing of exon 2, and they differ only by their N-termini (Figure 2). MeCP2 e1 (498 amino acids), generated by exons 1, 3, and 4, is the dominant MeCP2 isoform in the brain [76-78]. The MeCP2 e2 isoform (486 amino acids) is encoded by exons 2, 3, and 4. Both isoforms were initially assumed to be functionally equivalent, but recent observations imply that additional isoform-specific functions may exist. This idea is strongly supported by the fact that no mutations in exon 2 have been found in Rett syndrome patients, which contrasts with the finding of identified mutations in exon 1. Thus, defects in MeCP2 e2 may lead to non-Rett phenotypes or fatally affect embryo viability [79, 80].

MBD: methyl-CpG-binding domain, TRD: transcriptional repression domain, C-ter: C-terminal domain, yellow boxes: nuclear localization signals.

**Figure 2.** *MECP2* gene structure and the isoforms MeCP2 e1 and MeCP2 e2 with different N-termini due to alternative splicing of exon 2 and different translation start sites.

Apart from different N-terminal regions, both isoforms share the same amino acid sequence, including at least three functional domains and two nuclear localization signals (Figure 2). The methyl-CpG-binding domain (MBD) (amino acids 78-162) mediates binding to symmetrically methylated CpG dinucleotides [81, 82], with a preference for CpGs with adjacent A/T-rich motifs [83]. The MBD also binds to unmethylated four-way DNA junctions, which suggests the role of MeCP2 in higher-order chromatin interactions [84]. The transcriptional repression domain (TRD) (amino acids 207-310) interacts with numerous proteins, such as co-repressor factors and histone deacetylases, HDAC1 and HDAC2. The nuclear localization signals (NLS) (amino acids 173-193 and 255-271) mediate transportation of the protein into the nucleus [85]. The C-terminal domain (amino acids 325-486) facilitates binding to DNA [86] and it most likely increases protein stability [87]. This domain also contains conserved poly-proline motifs that can bind to group II WW domain splicing factors [88].

#### **3.6. MeCP2 function**

**3.5. MeCP2 protein**

206 Chromatin Remodelling

**MeCP2 e1 ATG**

**MeCP2 e2 ATG**

MVAGMLGLR

splicing of exon 2 and different translation start sites.

MAAAAAAAPSGGGGGGEEERL

*MECP2*

**MeCP2 e2**

nuclear localization signals.

**MeCP2 e1**

modulation of neuronal activity and plasticity [12].

**1 2 3 4**

The MeCP2 protein is ubiquitously expressed, but it is particularly abundant in the brain [41, 69]. The protein levels are low during embryogenesis, but they progressively increase during postnatal neuronal maturation and synaptogenesis [70-75]. High expression of MeCP2 in mature neurons implies its involvement in postmitotic neuronal functions, such as the

MeCP2 occurs in two isoforms that arise from alternative splicing of exon 2, and they differ only by their N-termini (Figure 2). MeCP2 e1 (498 amino acids), generated by exons 1, 3, and 4, is the dominant MeCP2 isoform in the brain [76-78]. The MeCP2 e2 isoform (486 amino acids) is encoded by exons 2, 3, and 4. Both isoforms were initially assumed to be functionally equivalent, but recent observations imply that additional isoform-specific functions may exist. This idea is strongly supported by the fact that no mutations in exon 2 have been found in Rett syndrome patients, which contrasts with the finding of identified mutations in exon 1. Thus, defects in MeCP2 e2 may lead to non-Rett phenotypes or fatally affect embryo viability [79, 80].

MBD: methyl-CpG-binding domain, TRD: transcriptional repression domain, C-ter: C-terminal domain, yellow boxes:

**Figure 2.** *MECP2* gene structure and the isoforms MeCP2 e1 and MeCP2 e2 with different N-termini due to alternative

Apart from different N-terminal regions, both isoforms share the same amino acid sequence, including at least three functional domains and two nuclear localization signals (Figure 2). The methyl-CpG-binding domain (MBD) (amino acids 78-162) mediates binding to symmetrically methylated CpG dinucleotides [81, 82], with a preference for CpGs with adjacent A/T-rich motifs [83]. The MBD also binds to unmethylated four-way DNA junctions, which suggests the role of MeCP2 in higher-order chromatin interactions [84]. The transcriptional repression domain (TRD) (amino acids 207-310) interacts with numerous proteins, such as co-repressor factors and histone deacetylases, HDAC1 and HDAC2. The nuclear localization signals (NLS)

**polyA (1.8 kB)**

**polyA (5.4 kB)**

**polyA (7.5 kB)**

**polyA (10.2 kB)** The original model suggested that MeCP2 is a global transcriptional repressor [89], and it was based on *in vitro* experiments in which MeCP2 inhibited transcription from methylated promoters. Briefly, the protein binds to the promoters of target genes via its MBD, and the TRD then recruits the co-repressor Sin3A and HDACs [90, 91]. These interactions lead to the deacetylation of histones, resulting in chromatin condensation and the repression of down‐ stream genes. In addition to Sin3A, other co-repressors, such as c-Ski and N-CoR, may interact with MeCP2 [92]. The compaction of chromatin can also be promoted through direct interac‐ tion with the C-terminal domain [93], which is an example of HDAC-independent MeCP2 mediated transcriptional repression. The TRD may also directly interact with transcription factor IIB; therefore, MeCP2 may silence transcription by interfering with the assembly of the transcriptional preinitiation complex [94]. Additional factors interacting with the TRD include Brahma, which is a catalytic component of the SWI/SNF-related chromatin-remodeling complex (at least in NIH 3T3 cells) [95], DNA methyltransferase 1 [96], and ATRX, a SWI2/ SNF2 DNA helicase/ATPase [97].

Surprisingly, transcriptional profiling studies did not reveal major gene expression changes caused by the lack of functional MeCP2 protein [98, 99]. These observations, together with additional evidence, implied that MeCP2 regulates the transcription of tissue-specific genes in specific brain regions during certain developmental stages instead of acting as a global repressor [98, 100-102]. However, recent studies suggest that MeCP2 reduces genome-wide transcriptional noise, potentially by repressing spurious transcription of repetitive elements [103, 104]. Surprisingly, MeCP2 also interacts with the transcriptional activator CREB, and its genomic distribution is often associated with actively transcribed genes [105, 106]. MeCP2 apparently has dual roles in transcriptional regulation as a repressor and as an activator, and it performs different downstream responses depending on the context.

MeCP2 additionally acts as an architectural chromatin protein that is involved in chromatin remodeling and nucleosome clustering, which is consistent with the fact that the majority of MeCP2-binding sites are located outside of genes [105, 107]. MeCP2 can bind *in vitro* to chromatin fibers and compact them into higher order structures [93, 108, 109].

For further complexity, MeCP2 may also be involved in RNA splicing. Its interaction with the RNA-binding protein Y box-binding protein 1 (YB-1) has been observed, and MeCP2-deficient mice showed aberrant alternative splicing patterns [110].

MeCP2 functions are undeniably much more complex than initially anticipated (Figure 3), although the precise mechanisms of their regulation remain unknown.

**Gene Function Reference** *UBE3A* member of ubiquitin proteasome pathway, transcriptional co-activator [118] *GABRB3* neurotransmission (GABA-A receptor) [118] *PCDHB1* cell adhesion [119] *PCDH7* cell adhesion [119]

*Sgk1* hormone signaling (regulation of renal functions and blood pressure) [120] *Fkbp5* hormone signaling (regulation of glucocorticoid receptor sensitivity) [120] *Uqcrc1* member of mitochondrial respiratory chain [121]

*ID1, ID2, ID3, ID4*transcription factors (involved in cell differentiation and neural development) [122]

*FXYD1* ion channel regulator [123] *IGFBP3* hormone signaling (regulation of cell proliferation and apoptosis) [124] *GDI1* regulation of GDP/GTP exchange [112] *APLP1* enhancer of neuronal apoptosis [112] *CLU* Extracellular molecular chaperone [112] *Crh* neuropeptide (regulation of neuroendocrine stress response) [125]

Mutations in the *MECP2* gene are not likely to act in a dominant-negative mechanism because only one allele is active in each female cell due to XCI. The functional consequences of missense mutations on the function of the MeCP2 protein are sometimes especially difficult to predict. This difficulty is because testing the protein's various functions can be problematic because there are still many MeCP2 roles that are as yet unknown or not fully understood. Generally, mutations in the NLS prevent the transportation of the protein to the nucleus. Mutations located within the MBD reduce the affinity of the protein for methylated DNA [87, 126]. However, several mutant proteins with mutations in the MBD have been shown to bind to heterochromatin [127]. Proteins with an intact MBD but with a mutated TRD retain their ability to bind to methylated DNA, but they have impaired repressing activity [87]. Other mutations may affect the stability or the structure (secondary or tertiary) of the protein, and they may

[113, 114]

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[116]

*Bdnf* neuronal development and survival, neuronal plasticity, learning and memory

*Dlx5* neuronal transcription factor (probably involved in control of GABAergic

(brain derived neurotropic factor)

differentiation)

**Table 2.** Some of MeCP2 target genes.

**3.8. The effect of** *MECP2* **mutations on MeCP2 function**

interfere with other functions of the protein.

**Figure 3.** Representation of multiple MeCP2 roles [111].

#### **3.7. MeCP2 target genes**

A comprehensive knowledge of the target genes that are controlled by MeCP2 is essential for understanding the pathomechanisms of Rett syndrome and subsequently developing effective therapeutic strategies. Multiple studies have attempted to identify genes with altered expres‐ sion in neuronal and nonneuronal tissues from Rett syndrome patients and mouse models, but these studies have often yielded conflicting results [102, 106, 112, 113]. Nevertheless, several candidate target genes have been proposed (Table 2). One of the most extensively studied target genes is the brain-derived neurotrophic factor gene (*Bdnf*), which has been shown to be up and down regulated in an activity-dependent manner through MeCP2 phosphorylation in mice [113-115]. Other targets, such as the imprinted genes *Dlx5* and *Dlx6*, also revealed a novel mode of gene repression mediated by MeCP2 through the formation of a silent chromatin loop (Figure 3) [116, 117].


**Table 2.** Some of MeCP2 target genes.

**Figure 3.** Representation of multiple MeCP2 roles [111].

a silent chromatin loop (Figure 3) [116, 117].

A comprehensive knowledge of the target genes that are controlled by MeCP2 is essential for understanding the pathomechanisms of Rett syndrome and subsequently developing effective therapeutic strategies. Multiple studies have attempted to identify genes with altered expres‐ sion in neuronal and nonneuronal tissues from Rett syndrome patients and mouse models, but these studies have often yielded conflicting results [102, 106, 112, 113]. Nevertheless, several candidate target genes have been proposed (Table 2). One of the most extensively studied target genes is the brain-derived neurotrophic factor gene (*Bdnf*), which has been shown to be up and down regulated in an activity-dependent manner through MeCP2 phosphorylation in mice [113-115]. Other targets, such as the imprinted genes *Dlx5* and *Dlx6*, also revealed a novel mode of gene repression mediated by MeCP2 through the formation of

**3.7. MeCP2 target genes**

208 Chromatin Remodelling

#### **3.8. The effect of** *MECP2* **mutations on MeCP2 function**

Mutations in the *MECP2* gene are not likely to act in a dominant-negative mechanism because only one allele is active in each female cell due to XCI. The functional consequences of missense mutations on the function of the MeCP2 protein are sometimes especially difficult to predict. This difficulty is because testing the protein's various functions can be problematic because there are still many MeCP2 roles that are as yet unknown or not fully understood. Generally, mutations in the NLS prevent the transportation of the protein to the nucleus. Mutations located within the MBD reduce the affinity of the protein for methylated DNA [87, 126]. However, several mutant proteins with mutations in the MBD have been shown to bind to heterochromatin [127]. Proteins with an intact MBD but with a mutated TRD retain their ability to bind to methylated DNA, but they have impaired repressing activity [87]. Other mutations may affect the stability or the structure (secondary or tertiary) of the protein, and they may interfere with other functions of the protein.

#### **3.9. Genotype-phenotype correlation**

The severity of the clinical manifestations in Rett syndrome patients is widely variable and is relevant beyond the context of classic vs. atypical variants. Therefore, much effort has been devoted to uncovering the relationships between various *MECP2* mutations and the variability of clinical features. Such knowledge may provide information on the likely clinical profile of new cases with specific *MECP2* mutations and may be useful in designing specific preventive therapeutic interventions.

Rehabilitation programs and physical therapy help to control and improve balance and movement, maintain flexibility and strengthen muscles. These programs should be adapted to the patient's individual state and needs. Proper physical therapy is also important for preventing joint contractures and other deformities, such as scoliosis. Occupational therapy is recommended for improving purposeful hand use and to attenuate stereotypic hand movements. Particular care should be taken to preserve and maintain alternative commu‐ nication (eye contact, eye pointing, facial expressions, signs, etc.) and thereby improving social interactions. Receiving necessary nutrients and maintaining an adequate weight may result in improved growth. To ensure appropriate caloric and nutritional intake, a highfat, high-calorie diet or gastrostomy feeding may be required. Sufficient intake of fluid and high-fiber food is necessary to prevent constipation. The patients with cardiac conduction defects (such as prolonged QTc intervals) should avoid certain medications, which may worsen the condition. These medications include several antipsychotics (thioridazine, tricyclic antidepressants), certain antiarrhythmics (quinidine, sotalol, amiodarone), and

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Early diagnosis and intervention, together with life-long management focused on each patient's specific needs, can significantly improve the health, quality of life, and longevity of

Much progress has been made in the identification of the multiple roles of the MeCP2 protein in the brain since its discovery. Nevertheless, many mysteries still remain in understanding the precise mechanisms of how *MECP2* mutations affect protein function and subsequently contribute to the pathogenesis of Rett syndrome. A significant phenotypic overlap between Rett syndrome and several neurodevelopmental disorders implies that a common pathogenic process may induce or at least contribute to these conditions. The identification of pathogenic *MECP2* mutations in a portion of patients without the classic Rett syndrome phenotype strengthens this theory. Understanding the molecular pathology underlying Rett syndrome will therefore shed more light on the role of epigenetic modifications in neuronal development and function, and it may provide insight into the pathogenesis of other neurodevelopmental

I would like to thank prof. Pavel Martasek, MD, Ph.D. for helpful discussions and critical reading of the manuscript. The author was supported by grants NT 13120-4/2012, MZCR RVO-

antibiotics (erythromycin) [147].

patients with Rett syndrome.

**5. Conclusion**

disorders.

**Acknowledgements**

VFN64165/2012, P24/LF1/3, and UNCE 204011/2012.

The general genotype-phenotype correlations were confirmed by numerous studies. As expected, patients with early truncating mutations (specifically p.R168X, p.R270X, p.R255X), large deletions of several exons, or the entire *MECP2* gene usually have the most severe clinical presentations. A milder phenotype is often associated with late truncating mutations that do not affect the MBD or the TRD, such as p.R294X. Interestingly, late truncating mutations together with the missense mutation p.R133C, which is located in the MBD, are frequently detected in patients with a preserved speech variant of Rett syndrome [128-133].

Despite some overall trends, considerable variability in clinical severity is often observed among patients with the same *MECP2* mutation [128, 130, 134]. Such variations may be caused by a different XCI pattern. Favorable skewing of XCI has been observed in some patients with milder phenotypes [135-137] and in asymptomatic carrier mothers [58, 135, 138]. However, XCI cannot be used as the single predictor because, according to several studies, it has limitations in explaining all of the differences of Rett syndrome severity [139-141]. Other modulation factors have been considered, such as *BDNF* [142, 143] and *APOE* [144].

#### **3.10. Genetic counseling**

A negative result from the *MECP2* analysis (usually including analysis of the entire coding region and copy number analysis of large deletions/duplications) does not rule out the diagnosis of Rett syndrome because regulatory and non-coding regions are not routinely analyzed. The recurrence risk in a family with a single Rett syndrome case and an otherwise negative family history is very low (less than 0.5%) because the majority of *MECP2* mutations arise *de novo*. Mothers of the patients, however, should be tested for *MECP2* mutations found in their daughters to rule out the possibility of being asymptomatic carriers. In such case, the recurrence risk is 50%. Prenatal diagnosis may be performed even in pregnancies of non-carrier mothers due to the likelihood of germline mosaicism.

#### **4. Management of Rett syndrome**

Currently, there is no effective cure for Rett syndrome. However, hopes for developing a targeted therapy have risen following the announcement of a study that rescued the patho‐ logical phenotype in a mouse model after postnatal reactivation of *Mecp2* [145, 146]. Treatment strategies are currently symptomatic and preventive. These strategies are aimed at ameliorat‐ ing specific symptoms, such as seizures, mood disturbances, sleeping and feeding problems, as well as maintaining and improving motor and communication functions.

Rehabilitation programs and physical therapy help to control and improve balance and movement, maintain flexibility and strengthen muscles. These programs should be adapted to the patient's individual state and needs. Proper physical therapy is also important for preventing joint contractures and other deformities, such as scoliosis. Occupational therapy is recommended for improving purposeful hand use and to attenuate stereotypic hand movements. Particular care should be taken to preserve and maintain alternative commu‐ nication (eye contact, eye pointing, facial expressions, signs, etc.) and thereby improving social interactions. Receiving necessary nutrients and maintaining an adequate weight may result in improved growth. To ensure appropriate caloric and nutritional intake, a highfat, high-calorie diet or gastrostomy feeding may be required. Sufficient intake of fluid and high-fiber food is necessary to prevent constipation. The patients with cardiac conduction defects (such as prolonged QTc intervals) should avoid certain medications, which may worsen the condition. These medications include several antipsychotics (thioridazine, tricyclic antidepressants), certain antiarrhythmics (quinidine, sotalol, amiodarone), and antibiotics (erythromycin) [147].

Early diagnosis and intervention, together with life-long management focused on each patient's specific needs, can significantly improve the health, quality of life, and longevity of patients with Rett syndrome.

#### **5. Conclusion**

**3.9. Genotype-phenotype correlation**

therapeutic interventions.

210 Chromatin Remodelling

**3.10. Genetic counseling**

mothers due to the likelihood of germline mosaicism.

**4. Management of Rett syndrome**

The severity of the clinical manifestations in Rett syndrome patients is widely variable and is relevant beyond the context of classic vs. atypical variants. Therefore, much effort has been devoted to uncovering the relationships between various *MECP2* mutations and the variability of clinical features. Such knowledge may provide information on the likely clinical profile of new cases with specific *MECP2* mutations and may be useful in designing specific preventive

The general genotype-phenotype correlations were confirmed by numerous studies. As expected, patients with early truncating mutations (specifically p.R168X, p.R270X, p.R255X), large deletions of several exons, or the entire *MECP2* gene usually have the most severe clinical presentations. A milder phenotype is often associated with late truncating mutations that do not affect the MBD or the TRD, such as p.R294X. Interestingly, late truncating mutations together with the missense mutation p.R133C, which is located in the MBD, are frequently

Despite some overall trends, considerable variability in clinical severity is often observed among patients with the same *MECP2* mutation [128, 130, 134]. Such variations may be caused by a different XCI pattern. Favorable skewing of XCI has been observed in some patients with milder phenotypes [135-137] and in asymptomatic carrier mothers [58, 135, 138]. However, XCI cannot be used as the single predictor because, according to several studies, it has limitations in explaining all of the differences of Rett syndrome severity [139-141]. Other

A negative result from the *MECP2* analysis (usually including analysis of the entire coding region and copy number analysis of large deletions/duplications) does not rule out the diagnosis of Rett syndrome because regulatory and non-coding regions are not routinely analyzed. The recurrence risk in a family with a single Rett syndrome case and an otherwise negative family history is very low (less than 0.5%) because the majority of *MECP2* mutations arise *de novo*. Mothers of the patients, however, should be tested for *MECP2* mutations found in their daughters to rule out the possibility of being asymptomatic carriers. In such case, the recurrence risk is 50%. Prenatal diagnosis may be performed even in pregnancies of non-carrier

Currently, there is no effective cure for Rett syndrome. However, hopes for developing a targeted therapy have risen following the announcement of a study that rescued the patho‐ logical phenotype in a mouse model after postnatal reactivation of *Mecp2* [145, 146]. Treatment strategies are currently symptomatic and preventive. These strategies are aimed at ameliorat‐ ing specific symptoms, such as seizures, mood disturbances, sleeping and feeding problems,

as well as maintaining and improving motor and communication functions.

detected in patients with a preserved speech variant of Rett syndrome [128-133].

modulation factors have been considered, such as *BDNF* [142, 143] and *APOE* [144].

Much progress has been made in the identification of the multiple roles of the MeCP2 protein in the brain since its discovery. Nevertheless, many mysteries still remain in understanding the precise mechanisms of how *MECP2* mutations affect protein function and subsequently contribute to the pathogenesis of Rett syndrome. A significant phenotypic overlap between Rett syndrome and several neurodevelopmental disorders implies that a common pathogenic process may induce or at least contribute to these conditions. The identification of pathogenic *MECP2* mutations in a portion of patients without the classic Rett syndrome phenotype strengthens this theory. Understanding the molecular pathology underlying Rett syndrome will therefore shed more light on the role of epigenetic modifications in neuronal development and function, and it may provide insight into the pathogenesis of other neurodevelopmental disorders.

#### **Acknowledgements**

I would like to thank prof. Pavel Martasek, MD, Ph.D. for helpful discussions and critical reading of the manuscript. The author was supported by grants NT 13120-4/2012, MZCR RVO-VFN64165/2012, P24/LF1/3, and UNCE 204011/2012.

#### **Author details**

Daniela Zahorakova\*

Address all correspondence to: Daniela.Zahorakova@lf1.cuni.cz

Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine Charles Uni‐ versity and General Teaching Hospital, Prague, Czech Republic

[10] Einspieler C, Kerr AM, Prechtl HF. Abnormal general movements in girls with Rett disorder: the first four months of life. Brain and Development 2005;27 Suppl 1: S8-

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[11] Smeets EE, Pelc K, Dan B. Rett Syndrome. Molecular Syndromology 2011;2(3-5):

[12] Chahrour M, Zoghbi HY. The story of Rett syndrome: from clinic to neurobiology.

[13] Nomura Y, Segawa M. Natural history of Rett syndrome. Journal of Child Neurology

[14] Shahbazian MD, Zoghbi HY. Rett syndrome and MeCP2: linking epigenetics and neuronal function. American Journal of Human Genetics 2002;71(6): 1259-72.

[15] Nomura Y. Early behavior characteristics and sleep disturbance in Rett syndrome.

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**Author details**

212 Chromatin Remodelling

Daniela Zahorakova\*

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versity and General Teaching Hospital, Prague, Czech Republic

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Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine Charles Uni‐

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## *Edited by Danuta Radzioch*

The term "chromatin remodelling" is widely used to describe changes in chromatin structure which is controlled by histone-modifying enzymes, chromatin remodelling complexes, non-histone DNA-binding proteins and noncoding RNAs. Many human diseases such as cancer, various genetic syndromes, autism and infectious disease have been linked to the disruption of these control processes by genetic, environmental or microbial factors. Therefore, to unravel the mechanisms by which they operate is one of the most exciting and rapid developing fields of modern biology and will contribute to new ways in treatment of these diseases. The chapters in this book will focus on recent advances in our understanding of the mechanisms that govern the dynamic structural of chromatin, thereby providing important insights into gene regulation, DNA repair, and human diseases.

Chromatin Remodelling

Chromatin Remodelling

*Edited by Danuta Radzioch*

Photo by Rost-9D / iStock