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 transcription, replication, DNA repair and recombination machinery. To overcome these barriers, eukaryotic cells possess a number of multi-protein complexes which can alter the chromatin structure and make DNA more accessible. These complexes can be divided into two groups, histone-modifying enzymes and ATP-dependent chromatin remodelling complexes. 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.
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
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 acetyltransferases (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
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 chromatin-remodeling complexes and histone-modifying enzymes. In conjunction with other co-regulators, these chromatin remodelers modify histone-DNA interaction and regulate transcription at specific genomic loci.
2.1. Histone modifications and transcription
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 modifications 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).
There are two major mechanisms underlying the function of histone modifications (Kouzarides, 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 acetylation, a modification associated with transcriptional activation, has been proposed to unfold chromatin structure via neutralization of the basic charges of lysines (Kouzarides, 2007). Indeed,
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 methyltransferases: 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 monomethyl- and 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 mono-methylated 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).
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 condensation 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 receptors 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 phosphorylation 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). Ubiquitination of H2AK119 and H2BK120 are associated with transcriptional repression and activation, respectively (Wang et al., 2006;Zhu
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,
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).
3. The role of chromatin remodelling in the regulation of inflammatory gene expression
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 best-characterized 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
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
3.2. Manipulation of host chromatin remodelling process by bacteria to facilitate infection
Interestingly, intracellular pathogens, such as
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 promoters of IFN-γ -responsive genes thereby mediating the transcription of these genes, including 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 IFN-γ -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. However, 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 expression (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. Co-immunoprecipitation 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 modulator 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,
A recent study has demonstrated that infection with
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
5.1. Inhibition of infection by targeting histone modifying enzymes in the pathogen
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 down-regulation of MyD88-dependent pathway and decreased expression of proinflammatory mediators such as TNF-alpha, IL-1β, and IL-6 (Li et al., 2010;Li
6. Concluding remarks
The activation and suppression of innate immunity are central principles of host-pathogen 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. Phosphorylation 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
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.