Periodontology 2000, Vol. 64, 2014, 95–110 Printed in Singapore. All rights reserved

 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

PERIODONTOLOGY 2000

Modifiable risk factors in periodontal disease Epigenetic regulation of gene expression in the inflammatory response S I L V A N A P. B A R R O S & S T E V E N O F F E N B A C H E R

Epigenetic mechanisms Epigenetics is the study of heritable changes in gene expression that occur without changes in DNA sequence. Epigenetic mechanisms are flexible genomic parameters that can change genome function under an exogenous influence and also provide a mechanism that allows for the stable propagation of gene-activity states from one generation of cells to the next (73). Thus, epigenetics emphasizes heritable changes in gene expression that are distinct from genetic variations in the population (i.e. single nucleotide polymorphisms). Epigenetics is being heralded as probably the most significant interface between the genetic and environmental factors that together give rise to phenotype. Of the three major methods of epigenetic regulation – methylation, histone modification and RNA interference – the best understood genome-wide information currently available pertains to methylation patterns. While classical genetic analyses and the ÔomicÕ technologies (transcriptomic, proteomic and metabolomic) continue to provide important information, new and unexpected data are also emerging from studying various epigenetic processes that do not change the sequence of the DNA itself but modify the way in which genes are expressed during development and ⁄ or under environmental challenges. The original connotations of the term ÔepigeneticsÕ, coined by Conrad H. Waddington (72), encompassed most of what it is now known as

developmental biology, but he stressed that any phenotype was a product of interaction between the inherent genetics of the organism and the influence of the environment – that is, plasticity – in that the central focus was alternative geneexpression states (meta-stable states). Currently, the emphasis is now placed on gene-expression patterns that shift during development but become more stable in end-differentiation states, and that can be further modified by cumulative environmental exposures and, most importantly, can be transmitted through cell divisions (62, 73). Moreover, although still closely related to plasticity, epigenetics is more accurately defined as Ôthe study of stable genetic modifications that result in changes in gene expression and function without a corresponding alteration in DNA sequenceÕ (http:// www.roadmapepigenomics.org/news/item/in-vivoepigenetic-imaging). DNA methylation occurs when a methyl group (-CH3) attaches to cytosine residues within a region of DNA that contains one or more cytosine nucleotide and guanine nucleotide (CG) sequences. In general, methylation of a gene reduces or stops its expression. Patterns of methylation can be inherited from the mother or acquired during life. It has been described that if the genetic code is the hardware for life, the epigenetic code can be seen as the software that determines how the hardware behaves – and as such, it can be rewritten (11). As an epigenetic signaling tool, hypermethylation locks genes in the ÔoffÕ

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position and this silencing is related to the inhibition or down-regulation of mRNA expression. In cancer, DNA hypomethylation often affects more of the genome than does hypermethylation, so that net losses of genomic 5-methylcytosine are seen in many human cancers (24) and are associated with the overexpression of affected genes. Thus, hypomethylation and hypermethylation of DNA are relative terms, denote less or more methylation and can be global or local (17).

Chromatin structure As each DNA molecule within a cell contains all the genetic information necessary to create an entire organism, controlling DNA expression to define a specific cell is all about DNA packaging. This is facilitated by the formation of nucleosomes, localized regions of DNA wrapped around histone octamers that can then form higher-order structures enabling further compaction of DNA (16, 21, 62). In the human genome, each nucleosome consists of an octameric particle of four core histone proteins (H2A, H2B, H3 and H4) that bind and wrap 147 base pairs of DNA around the histone octamer (16). Histones can be covalently modified by reactions such as acetylation, methylation, phosphorylation and ubiquitylation (3, 45). These chromatin modifications are often recognized by effector proteins through which they exert their functions (63, 64). Most of the mammalian genome is packaged as heterochromatin, which is very dense, and only selected regions that are less dense (referred to as euchromatin) are available for transcription. The information on DNA methylation that initiates DNA condensation by histone acetylation and other mechanisms is conserved following DNA replication; thus, the local chromatin structure can be inherited by the next generation of cells. The epigenetic information that can be inherited with chromosomes is embedded in at least two distinct ways: (i) modifications in chromatin proteins, usually caused by modifications in histone (21), which are evenly distributed to the newly synthesized DNA during DNA replication, or (ii) methylation of specific cytosine residues in the DNA whose patterns can be enzymatically copied (9, 44, 74). Hence, both histone modifications and DNA methylation can be easily copied to the newly synthesized DNA strands and transferred as such to subsequent generations. Recent research suggests that histone and DNA methylation are much more dynamic regulators of gene expression than

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previously thought. In chromatin, a complex array of histone modifications, associated proteins, RNAs, higher-order folding conformations and site-specific DNA methylation controls DNA accessibility and recruitment of specific regulatory molecules, thereby regulating most DNA functions, including transcription, replication, recombination and repair (68).

DNA methylation Mammalian cells possess the capacity to epigenetically modify their genomes via DNA methylation. DNA methylation occurs at the 5¢ position of cytosine in CpG dinucleotides (the ÔpÕ indicates that the C nucleotide and the G nucleotide are connected by a phosphodiester bond), replacing a hydrogen atom with a methyl group without interfering with CG base pairing. The postsynthetic addition of methyl groups to the 5¢-position of cytosines alters the appearance of the major groove of DNA to which the DNAbinding proteins bind. These epigenetic ÔmarkersÕ on DNA can be copied after DNA synthesis and result in heritable changes in chromatin structure. Methylation of CpG-rich promoters in mammals prevents transcriptional initiation and ensures the silencing of genes on the inactive X chromosome, imprinted genes and parasitic foreign DNAs (5, 20, 62). The inheritance of DNA CpG methylation is the most well-understood epigenetic mechanism, and several studies have indicated the semiconservative segregation of symmetric CpG methylation and maintenance of DNA methylation. In mammals, CpG methylation is performed by three different DNA methyltransferases. The isoforms DNA methyltransferase 3a and DNA methyltransferase 3b perform the Ôde novoÕ methylation of CpG residues, while DNA methyltransferase 1 is believed to act as a maintenance methyltransferase that is responsible for copying the methylation pattern to the newly synthesized DNA strand during DNA replication (12, 31, 43, 44).

CpG islands CpG dinucleotides, the sites of almost all methylation in mammals, are statistically under-represented in DNA. Clusters of CpGs, called CpG islands, (regions containing >200 CpG sequences) are often found close to genes, with CpG sequences appearing often in the promoters and first exons but also in down-

Epigenetics in periodontal disease

stream regions. Interest in CpG islands grew when it was demonstrated that, in vertebrates, they are enriched in regions of the genome involved in gene transcription, referred to as ÔpromotersÕ (8, 18). DNA methylation usually exerts transcriptional control in two ways. First, methylation can prevent the binding of transcription factors to promoter regions, which is a simple and direct mechanism of steric hindrance. Second, DNA methylation can mediate the binding of methyl-CpG-binding proteins, which recognize methylation sites on DNA (9). Of particular interest are the methyl-CpG-binding proteins MBP1, MBP2 and MBP3 that have been shown to act as transcriptional repressors. Such proteins bind to methylated DNA and regulate genes by blocking the binding of RNA polymerase to the promoter (29, 55). Although several DNA-binding proteins were identified to be sensitive to methylation, the mechanism of methylation recognition is still under investigation. It has been suggested that nucleosomes assembled with nonmethylated DNA appear less stable than those assembled with methylated DNA (38, 39). Molecular dynamics simulations suggest that steric hindrance and hydrophobicity of the methyl groups can lead to reduced flexibility of DNA (15, 48). Apparently, the dynamics of DNA has an influence on protein–DNA binding specificities and it is possible that methylation-induced alteration of local DNA flexibility contributes to the recognition of methylation. Irizarry and collaborators (34) examined DNA methylation on a genome-wide scale in colorectal cancer samples. These investigators reported that ÔCpG shoresÕ, defined as regions within 2000 base pairs distant from CpG islands, are useful predictors for genomic locations that are differentially methylated across different tissues and between cancer samples and normal samples.

Environmental exposures as determinants of the epigenome A combination of physiological stressors and environmental exposures through the lifetime of an organism appears to affect the epigenomic ÔprogramÕ acquired by a cell during differentiation and throughout the cellular lineage lifespan. The epigenomic program might be particularly sensitive to environmental influences during the early stages of development, but significant environmentally induced changes can accumulate over the lifetime of an organism, creating

the Ôaging phenotypeÕ and increasing the risk of aging-associated diseases. As a strategic approach to identify environmental influences on the epigenome and aging-associated epigenomic drift in humans, a comparison between the epigenomes of monozygotic twins was performed as a pioneering study using methylation-sensitive restriction enzymes, an early method for CpG methylation mapping, to obtain epigenome maps in a large cohort of monozygotic twins (22). Although the twins were epigenomically identical during their early years of life in terms of CpG methylation and histone acetylation, the epigenomes significantly diverged with age across various tissue types. These changes in DNA methylation were associated with tissue-specific changes in geneexpression patterns that differentiated between monozygotic twins. The role of the environment was highlighted by the findings indicating that the epigenomes of twins who were older, had different lifestyles and spent less time together, diverged more. Although aging and environmental exposures can affect global genome methylation, new evidence indicates that some promoter methylation sites are targeted by specific toxins and infectious stimuli to modify methylation levels (70).

Virus infection and epigenetic modifications The possibilities of viral DNA integration and of alterations in cellular methylation profiles have been investigated but systematic studies on the consequences of the integration of foreign DNA into established genomes are still in their infancy. Bacteria evolved DNA methylation as a protective mechanism to silence foreign DNA from viruses. Mammals have retained this ability to silence viral sequences, many of which have been incorporated into the host genome. Thus, DNA regions with high recombination rates can be modulated by host DNA methyltransferase activity. Patterns of DNA methylation in the recipient genome can become altered both at the site of foreign DNA integration and also remote from it (28, 35, 53). Although we understand that approximately 8% of the human genome contains viral or viral-like sequences (40), it has become increasingly clear that the methylation state of those viral-related sequences is an important determinant of viral-associated carcinogenesis. As intracellular parasites, viruses have evolved the capability to hijack many host

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processes in order to maximize their own survival, whether it is to increase viral production or to ensure the long-term survival of latently infected host cells. Epigenetic modification of host genes induced by viral infection affecting tumor suppressor genes and host immunity-related genes has been reported involving hepatitis B virus (30, 78). As an example of host-cell-determined methylation by a specific virus, protein X, a multifactorial regulator essential for hepatitis B virus replication, upregulates several DNA methyltransferases, increases promoter CpG methylation and alters the expression of genes known to regulate carcinogenesis, such as E-cadherin and p16 (19). Additionally, it has also been shown that microRNA-152, which represses DNA methyltransferase 1, is down-regulated in patients with hepatitis B virus, causing DNA hypermethylation, a finding that opens the possibility for the therapeutic use of this microRNA to reduce aberrant methylation (32). Fernandez et al., (20) surveying the methylation status of every CpG dinucleotide from the hepatitis B virus and human papillomavirus serotype 16 and 18 genomes and from all the Epstein–Barr virus genome transcription start sites, reported progressive methylation as an acute infection becomes chronic and then progresses to premalignant lesions and invasive cancer. Epstein–Barr virus has been linked to nasopharyngeal carcinoma, Burkitt lymphoma and HodgkinÕs disease. Epstein–Barr virus can establish several types of epigenetic modifications, which sometimes engage in a complex epigenetic crosstalk.

Bacterial infection and the ÔEpigenetic Field EffectÕ Helicobacter pylori, a gram-negative bacterium infecting approximately half of the worldÕs population, has been established as the major risk factor for gastric cancer and is known to be associated with the accumulation of aberrant DNA methylation in gastric epithelial mucosa (71). Several studies have demonstrated that H. pylori infection is associated with the hypermethylation of gene promoters with gene-type-specific methylation profiles. In patients infected with certain H. pylori serotypes, methylation of promoters in several genes in the gastric mucosa is 5–300-times higher than in uninfected individuals. Considering the staging of cancer in the multistep process of carcinogenesis in infected gastric mucosa, the aberrant methylation and subsequent silencing of tumor-suppressor genes,

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including p16, E-cadherin and hMLH1, was found to correlate strongly with subsequent cancer risk (67). In addition, several studies have reported that in the development of gastric cancer the hypomethylation of specific genes can also occur, including many genes that are associated with apoptosis, cell division, proliferation and adhesion (50, 54). To examine the causal link between infection and DNA methylation, Niwa et al. (56). infected Mongolian gerbils with H. pylori and found detectable CpG island methylation 5–10 weeks later. The methylation levels increased until approximately 50 weeks and in some CpG sites reached levels approximately 10–200fold higher than in uninfected animals. Methylation decreased when the infection was eradicated but remained significantly higher than in uninfected controls. Treatment with the immunosuppressant agent cyclosporine, which blocks inflammation without affecting bacterial colonization, prevented this aberrant methylation, indicating that the epigenetic changes occurred as a result of the inflammation accompanying the bacterial infection. This concept was further supported when it was observed that the maintenance of methylation marks on epithelial cancer lines required exogenous interleukin-6 in the culture medium. Thus, it appears that under some circumstances bacterial challenge can serve as a stressor to induce alterations in epigenetic patterns (and subsequent gene expression) and that inflammation represents an important modulator of these patterns. Those findings reveal the establishment of an Ôepigenetic field defect,Õ in which altered DNA methylation may occur as a result of an infection mark in a region that potentially becomes at higher risk for subsequent malignant transformation (56). A molecular basis for the epigenetic field effect has been postulated as an accumulation of genetic and epigenetic alterations in tissues with a normal appearance. The association between infection and epigenetic changes has already been shown in an animal model of infection with Campylobacter rectus, a periodontal pathogen that is phylogenetically closely related (sharing 96% sequence homology and many similar toxins) to H. pylori. C. rectus infection can lead to hypermethylation of the Igf2 gene and to the related down-regulation of insulin-like growth factor 2 expression in mice (10). As an interesting corollary, periodontal inflammation in moderate and severe forms of disease is associated with ulceration of the sulcular epithelium and with higher counts of C. rectus, a pathogen already characterized as a

Epigenetics in periodontal disease

strong predictor of the most severe stages of periodontal disease. Traditionally, cells with a genetic alteration were considered to form a physically continuous patch, producing a genetically altered field (71). This refers to normal-appearing tissues that are regionally epigenetically modified, yielding the epigenetically altered field more susceptible to malignancy. This molecular concept may further support the surgical principle that requires wide margins on excisional biopsies to prevent malignant recurrence.

The potential role of epigenetics in periodontal disease Gene activation in periodontal disease Chronic inflammatory periodontal disease results from an inflammatory response to dental biofilm, and the subgingival biofilm triggers an orchestrated cascade of innate immune mechanisms that create a cytokine network which mediates the pathogenesis of periodontal diseases at the biofilm–gingiva interface (14, 58, 60, 66). Pathogenesis can be compartmentalized by considering the bacterial interaction with host cells (via innate immune activation), the host cellular activation as protective against microbial assault and the acquired immune activation with chronic inflammation and cytokine networks modulating the connective tissue response. All of these mechanisms involve gene-activation pathways, and several are associated with transient modifications of DNA-methylation status and histone acetylation ⁄ phosphorylation states. However, recent findings suggest that (i) the bacteria have the potential to cause alterations in cellular DNA methylation status (10, 23, 33, 54, 56) and (ii) the effects of environment, aging and stress on modifying the expression of periodontal disease appear to involve epigenetic modifications that modify gene expression and disease expression. The periodontal host response is highly complex and has been described as having both protective and destructive elements that may be proactively modified by pathogens (25, 42). The response to various oral bacteria can be initially attributed to the innate immune system, which is the natural host defense against microbes. This innate response includes the recognition of microbial pathogens, usually at the epithelial boundary but possibly also within tissue after tissue invasion, and is mediated by subepithelial dendritic cells, neutrophils, tissue

macrophages, natural killer cells and monocytes, which act as sentinels of the innate immune system. This process requires the coordinated action of several families of proteins (such as toll-like receptors and nucleotide-binding oligomerization domain-like receptors) that are involved in the recognition of bacteria (1). Innate responses do not require previous exposure to the offending organism and are important for initiating the immune response to new exogenous microbial challenges. However, periodontal pathogens are commensal in nature and are acquired shortly after the eruption of the dentition. So, chronic inflammation, mediated by the acquired immunoresponse, strongly influences the nature of the inflammatoryinfiltrate in periodontitis and chronic gingivitis. This does not obviate the importance of the innate response, which largely appears to mediate acute exacerbations of a chronic inflammatory state. While activation of the host inflammatory response acts as an antimicrobial defense mechanism, the bacteria have evolutionarily adapted to this hostile environment and can protect themselves from various host-selective pressures. Recently, new clues have emerged regarding how the bacteria within the biofilm improve self-survival. We now understand that certain biofilm organisms have the capacity to modify the host metabolism, and perhaps also the inflammatory response, by altering the structure of the human host chromosomes (51, 61). Traditional host cellular-activation processes involve the interaction of membrane-bound receptors with bacterial components, principally through tolllike receptors and nucleotide-binding oligomerization domain-containing proteins. Shifts in molecular profiles within the gingival crevicular fluid or periodontal tissues have been attributed to the interactions of biofilm bacteria and their products with epithelial, immunocyte and fibroblastic recognition receptors, including toll-like receptors (6, 69). Biofilm–host interactions also regulate tissue metabolic homeostasis. The inflammatory and metabolic states of the tissues are not only driven by the bacterial stimulus, but the magnitude of the cellular and molecular signature response is further dictated by the host genetic traits and by various systemic exposures, with smoking, obesity and diabetes ⁄ hyperglycemia playing a major role (7, 57). Episodic connective-tissue destruction of the periodontal tissues is believed to be a result of transient fluctuations in both the microbial burden and permutations in the cytokine network (see ref. 60 for review). The activation of toll-like receptors with bacterial components

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such as lipopolysaccharides or fimbriae triggers a cascade of intracellular signaling mechanisms that usually include activation of transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B-cells, which are key transcriptional activators of inflammation. In an inactive state the nucleosomal chromatin at promoters of acute proinflammatory genes is silent but can be rapidly opened following activation by toll-like receptors (6, 69). As an example, tumor necrosis factor alpha transcription is normally restricted by a nucleosome poised over the primary nuclear factor kappa-light-chain-enhancer of activated B-cells site located at its proximal promoter. The activation pathway initiated by recognition of lipopolysaccharide by toll-like receptor-4 initiates the remodeling of this nucleosome, resulting in the elimination of the resident repressor complex from the chromatin, leading to the binding of colony stimulating factor 2 to the promoter element and to the transcription of colony stimulating factor 2 mRNA. Transcription of other cytokine molecules, such as interleukin-1a, tumor necrosis factor alpha and interleukin-6, is also enhanced. The release of these molecules into the extracellular space sends a signal to cells with specific receptors for these inflammatory mediators to carry out the effector function, such as activating fibroblasts to secrete matrix metalloproteinases to degrade the extracellular matrix. In turn, these three mediators (interleukin-1a, tumor necrosis factor alpha and interleukin-6) induce widespread gene expression, including molecules that have been implicated in periodontal disease pathogenesis: adhesion molecules, chemokines, cytokines, cyclooxygenase-2, inducible nitric oxide synthase, matrix metalloproteinases and other proteolytic enzymes (57, 59, 60). Although these epigenetic remodeling events triggered by toll-like receptor activation, for example, are critical for inducing a short-term reversible inflammatory response, there are other epigenetic changes that are more permanent. Recently, we have examined whether periodontal tissues that have been in contact with the biofilm and have signs of gingival inflammation have any evidence of epigenetic field effects. One might anticipate substantial inter-individual epigenomic differences. Therefore, we investigated whether there were differences within a single individual that might be site-specific and associated with inflammation and infection. We used molecular screening methods to detect genome-wide differences in the epigenomic pattern at periodontal sites with inflammation compared with noninflamed sites within the same indi-

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Fig. 1. Representative results of differential methylation hybridization. Comparison was performed between samples collected from periodontally diseased gingival tissue sites and samples collected from healthy gingival tissue sites from the same patient (the reference). Genomic DNA samples of 100 ng were fluorescently labeled with Alexa 647 (red) and Alexa 555 (green) and co-hybridized to a CpG island microarray containing 15 000 CpG island tags. The hybridization output is the measured intensities of the two fluorescence reporters red (indicating periodontal disease) and green (indicating health), overlaid with each other. Yellow spots indicate equal amounts of bound DNA from each amplicon, signifying no methylation difference between diseased and healthy genomes. Spots hybridized predominantly with diseased amplicons which appear as red are indicative of hypermethylated CpG island loci present in the diseased genome. Green spots denote hypomethylation in the diseased genome.

vidual. Figure 1 shows a genome-wide CpG methylation array in which specific regions are CpG rich and the colors reflect hypermethylation or hypomethylation over the entire genome. These alterations in methylation patterns observed upon comparing a healthy site with a diseased site within a single individual suggest that the epigenome may vary from site to site within individuals, as shown in Fig. 1. The CpG islands microarray indicates that many CpG islands are differentially methylated, consistent with an epigenetic field effect occurring in gingival tissue that is chronically inflamed relative to healthy tissue from the same individual. Just visualizing a region of the array, as shown in Fig. 1, it appears that there are many differences in the methylation pattern between healthy and diseased sites. Whether these epigenomic differences are transient or stable cannot be determined from this observation. However, we analyzed multiple patients using diseased or healthy tissues to determine whether periodontal disease has a consistent epigenetic signature. We also conducted a study, using CpG islands microarray analysis, in which we compared periodontally inflamed gingival tissues from subjects with chronic periodontitis with healthy tissues from healthy subjects. The methylation-array data investigated the relative methylation status of approximately 15,000 CpG island clones using methylation-sensitive

Epigenetics in periodontal disease

restriction enzymes. A reference DNA sample was made by pooling DNA samples from 10 healthy subjects. The reference DNA was then used to hybridize against DNA samples from ten individualsÕ diseased gingival biopsies. These biopsies were intrapapillary and comprised epithelium (sulcular, oral and junctional), connective tissues and any inflammatory cell infiltrate present. The data from these 10 arrays were matched for hypermethylation and hypomethylation, and CpG sites were identified by genes adjacent (10 kB upstream and 10 kB downstream) to the transcriptional start site. A list of genes with altered methylation patterns that passed the < 0.05 false discovery rate are listed in Tables 1 and 2, together with the fold changes; and by using significance analysis of microarrays (a qualitative analysis) that indicated which genes were significantly hypomethylated or hypermethylated, other genes also appeared to be hypermethylated in those samples. These genes included SOCS3, VDR, MMP25, BMP4, RUNX3, interleukin-17, TNFRSF18, ZNF277, ZNF501, CADM3 and BDNF. Other genes showed hypomethylation in relation to healthy gingival control samples. Clearly, these alterations in methylation status will need to be confirmed in additional studies. However, the methylation status of some of these CpG regions suggests linkages with inflammatory pathways associated with acute and chronic inflammatory states. The inflammatory genes identified in Table 1 can be identified as linked to an inflammatory network, as shown in Fig. 2. For example, CpG islands adjacent to both chemokine (CX-C motif) ligand 5 and chemokine (C-X-C motif) ligand 3 appear to be hypermethylated. It has already been shown in gingival inflammation that the expression of chemokine (C-X-C motif) ligand 5 mRNA and its levels within the gingival crevice fluid are downregulated (4). Nonetheless, it is premature to conclude from these findings that DNA methylation underlies the observed suppression of chemokine (C-X-C motif) ligand 5, but it raises an important new hypothesis for further investigation. To gain greater perspective of how these methylation patterns might relate to functional changes in gene expression we conducted pathway analyses using those genes-in-play that met a false discovery rate of < 0.05 using Ingenuity Pathway Analyses (IPA). The goal was to define which biologic functions, as classified using gene ontology classifications, appear to be altered in gingival inflammation. These gene ontology findings are summarized in Table 3 for hypermethylated genes and in Table 4 for hypomethylated genes. The number of genes that were significantly differentially methylated under each significant biological

functional domain is listed with its gene ontology ID, gene ontology function and P-value (determined using FisherÕs exact test). Differentially methylated DNA patterns in these CpG island regions were significantly associated with gene pathways that controlled cell differentiation, apoptosis, lipopolysaccharidemediated signaling, oncogenesis and cell adhesion. As hypermethylation is associated with gene downregulation, these data suggest that inflammation promotes silencing of genes related to cell death ⁄ apoptosis. This observation is supported by an extensive literature regarding the changes in cellular integrity during the stress response associated with inflammation (37, 49, 53, 68). It is noteworthy that many genes associated with these CpG regions were hypermethylated relative to those that were hypomethylated. This is in contrast to conditions such as cancer, aging or smoking, which are all associated with a more global (genome-wide) hypomethylation status. It should be emphasized that these conditions can induce a genome-wide hypomethylation, while at the same time induce hypermethylation at very specific sites. Although these proof-of-concept findings provide compelling data supporting the concept of epigenetic field effects, there are many limitations to the interpretation of these findings. First, these identified pathways are based upon changes in the methylation status of CpG island genes that are within, upstream or downstream of the target genes. Thus, this is a rather Ôgranular level of resolutionÕ. Second, as mentioned earlier, the methylation status of CpG islands is not necessarily the best indicator of chromatin availability for the transcription of specific genes. Indeed, we (76, 77) and others (47, 72) have published several studies which demonstrate that the importance of methylation can be limited to one or two CG sequences that are strategically located within an important promoter region. Third, these data represent methylomes from tissues composed of various cell types that are known to differ from each other in health and in chronic inflammatory state. For example, the changes in GATA3 methylation seen in Table 1 may simply reflect an influx of T-helper 2 cells that have hypermethylated GATA3. Thus, we cannot distinguish whether these epigenetic differences represent stable or metastable differences in the methylome of the tissues or whether these alterations in methylation are associated with changes in gene-expression patterns. To investigate the changes in the tissue methylome in chronic gingival inflammation we used another methodology, a PCR-based methylation array (Qiagen, Valencia, CA, USA) that uses bisulfite-treated

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v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog

Vitamin D receptor

Matrix metallopeptidase 25

Bone morphoge- Bone morphogenetic protein 4 increases the levels of osteopontin, bone morphogenetic 2.08 netic protein 4 protein 2, alka phosphatase and core binding factor alpha 1 mRNAs in human periodontal ligament cells

LYN

VDR

MMP25

BMP4

Cell division control protein 42 homolog

Interleukin-17

Runt-related transcription factor 3

Tumor necrosis factor receptor superfamily, member 18

CDC42

IL17

RUNX3

TNFRSF18

This receptor has been shown to have increased expression upon T-cell activation, and it is thought the by CD25+CD4+ regulatory T cells

This gene encodes a member of the runt domain-containing family of transcription factors. It a suppressor, and the gene is frequently deleted or transcriptionally silenced in cancer

This cytokine regulates the activities of nuclear factor kappa-light-chain-enhancer of activated B-ce the expression of interleukin-6, prostaglandin-endoperoxide synthase 2 ⁄ cyclooxygenase-2 and enhance the production of nitric oxide

The protein encoded by this gene is a small GTPase of the Rho-subfamily, which regulates signalin migration, endocytosis and cell cycle progression

Significance analysis of microarrays: qualitative analysis hypermethylated

2.19 In response to bacterial infection or inflammation, the encoded protein is thought to inactivate alpha-1 proteinase inhibitor, a major tissue protectant against proteolytic enzymes released by activated neutro facilitating the transendothelial migration of neutrophils to inflammatory sites.

The receptor belongs to the family of trans-acting transcriptional regulatory factors which 2.07 bind to the vitamin D receptor and regulate calcium metabolism

Member of the Src-family of protein tyrosine kinase, involved in lipopolysaccharide signal 2.17 transducti. Acts as a positive regulator of cell movement while negatively regulating adhesion to stromal cells by inhibiting the intercellular adhesion molecule-1-binding activity of beta-2 integrins

This gene encodes a member of the signal transducer and activator of transcription-induced 1.98 signal transducer and activator of transcription inhibitor. Signal transducer and activator of transcription in family members are cytokine-inducible negative regulators of cytokine signaling. The expression of the gene is induced by various cytokines, including interleukin6, interleukin-10 and interferon-gamma

Suppressor of cytokine signaling

SOCS 3

Hypermethylation fold change

Gene name

Gene symbol

Function ⁄ pathway

Table 1. Genes identified by 15K CpG microarray analysis as presenting altered methylation patterns (using false discovery rate < 0.05) and gene function are indicated. Qualitative assessment of CpG microarray data by Significance Analysis of Microarrays identified genes and direction of methylation alteration: hypermethylation

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GATA binding protein 3 GATA3

This gene encodes a protein which contains two GATA-type zinc fingers, is an important regulator biology

Zinc finger protein 501 ZNF501

Involved in the regulation of transcription: DNA binding and zinc ion binding

Cyclin D1 BCL1 ⁄ CCND1

Regulatory component of the cyclin D1–cyclin-dependent kinase 4 complex that phosphorylates retinoblastoma 1, and regulates the cell cycle during G(1) ⁄ S transition

Cell adhesion molecule 3 CADM3

Involved in cell-to-cell adhesion

Brain-derived neurotrophic factor BDNF

Involved in cell-to-cell adhesion. It is also expressed in leukocyte cell lines, lymphocytes and macrophages

Gene name Gene symbol

Table 1. Continued

Function ⁄ pathway

Hypermethylation fold change

Epigenetics in periodontal disease

Fig. 2. Network involving some of the genes that had a significantly altered methylation status in the Methyl – Profiler DNA Methylation Array analysis. Genes that are depicted in red indicate hypermethylation and the gene in green is hypomethylated. CD276, CD276 molecule; CCR4, chemokine (C-C motif) receptor 4; CXCL3, chemokine (CX-C motif) ligand 3; CXCL5, chemokine (C-X-C motif) ligand 5; CYP2C40, cytochrome P450, family 2, subfamily C, polypeptide 19; ELANE, elastase, neutrophil expressed; GATA3, GATA binding protein 3; GPR44, G proteincoupled receptor 44; IL4, interleukin-4; IL6ST, interleukin-6 signal transducer; NFkB, nuclear factor kappa-lightchain-enhancer of activated B-cells; P38 MAPK, p38 mitogen-activated protein kinase; SOCS6, suppressor of cytokine signaling 6; RUNX3, runt-related transcription factor 3; S1PR3, sphingosine-1-phosphate receptor 3; STAT5A, signal transducer and activator of transcription 5A; WSX1-gp130 Interleukin 27 receptor, alpha; ZBTB32, zinc finger and BTB domain containing 32. 2000-2011 Ingenuity Systems, Inc. All rights reserved.

DNA [see refs (76, 77) for methods] for selected panels of inflammatory cytokines examining for promoterregion methylation. In contrast to the global CpG methylation array analyses shown above, this method is pathway specific and investigates the methylation status of specific CG sites in inflammatory genes of interest. We processed, in our analyses, six healthy and 12 diseased (chronic periodontitis) samples for 24 inflammatory genes on three different arrays, with overlapping genes serving as internal controls. There were 12 genes that were statistically differentially methylated among the 35 surveyed. These genes and the fold methylation are given in Table 5. The network of these inflammatory genes is shown in Fig. 2. Many of these genes match up with predicted methylation status based upon the global CpG island methylation arrays. For example, GATA binding protein 3 and Wnt pathways are confirmed as being hypermethylated.

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104 Interleukin-6

IL6

Interleukin-4 receptor

Interleukin 8

Bone morphogenetic protein 2 Cyclic AMP responsive element-binding protein 3

Matrix metallopeptidase 14 Actin-related protein 1 Tyrosine-protein kinase receptor ETK1 Cyclin-dependent kinase 6 Caspase 7

IL4R

IL8

BMP2

CREB3

MMP14

ARP1

Tyrosine-protein kinase receptor ETK1

CDK6

CASP7

Significance analysis of microarrays: qualitative analysis hypomethylated

Gene name

Gene symbol

1.9

Hypomethylation (fold change)

The precursor of caspase 7 is cleaved by caspase 3 and caspase 10. Caspase 7 is activated by stimuli present at cell death and induces apoptosis

Cyclin-dependent kinase 6 is a catalytic subunit of the protein kinase complex that is important for cell cycle G1 phase progression and G1 ⁄ S transition

Tyrosine-protein kinase receptor ETK1 is a transmembrane receptor involved in the response to lipopolysaccharide, the response to cytokine stimulus with a role in cell adhesion, contact, migration and attachment in B-lymphocyte-derived cell lines and epithelial cells

Actin-related protein 1 is a 42.6 kDa subunit of dynactin. It is involved in nucleotide binding, G2 ⁄ M transition of the mitotic cell cycle, mitotic cell cycle and vesicle-mediated transport

A zinc-dependent endopeptidase involved in extracellular matrix degradation

CREB3 encodes cyclic AMP responsive element-binding protein 3, a transcription factor that is a member of the leucine zipper family of DNA-binding proteins. This protein binds to the cAMPresponse element and regulates cell proliferation. The protein interacts with host cell factor C1, which also associates with the herpes simplex virus protein, VP16, that induces transcription of herpes simplex virus immediate-early genes

Bone morphogenetic protein 2 belongs to the transforming growth factor beta superfamily

Interleukin 8 protein is a member of the CXC chemokine family and one of the major mediators of the inflammatory response. It is secreted by several cell types, functions as a chemoattractant and is also a potent angiogenic factor

A type I transmembrane protein that can bind interleukin 4 and interleukin 13 to regulate IgE production. The encoded protein also can bind interleukin 4 to promote differentiation of Thelper 2 cells

Plays an essential role in the final differentiation of B-cells into immunoglobulin-secreting cells

Function

Table 2. Genes identified by 15K CpG microarray analysis as presenting altered methylation patterns (using false discovery rate < 0.05) and gene function are indicated . Qualitative assessment of CpG microarray data by Significance Analysis of Microarrays identified genes and direction of methylation alteration: hypomethylation

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Epigenetics in periodontal disease

Table 3. Gene ontology classes: associated biological processes and sets of genes differentially methylated in periodontally affected gingival tissues Gene ontology ID

Hypermethylated

P-value

Function

12501

27

0.0001

Programmed cell death

42981

18

0.0005

Regulation of apoptosis

48468

36

0.0005

Cell development

30111

5

0.0006

Regulation of the Wnt receptor signaling pathway

43067

18

0.0006

Regulation of programmed cell death

30693

4

0.0008

Caspase activity

9966

19

0.001

Regulation of signal transduction

5386

13

0.0018

Carrier activity

6916

9

0.002

Anti-apoptosis

30154

45

0.002

Cell differentiation

48869

45

0.002

Cellular developmental process

43066

10

0.0026

Negative regulation of apoptosis

Table 4. Gene ontology classes: associated biological processes and sets of genes differentially methylated in periodontally affected gingival tissues Gene ontology ID

Hypomethylated

P-value

Function

9070

2

0.0078

Protein transporter activity

30155

4

0.0124

Regulation of cell adhesion

To address these points and connections further we interrogated the methylation status of two key genes – prostaglandin-endoperoxide synthase 2 and interferon gamma – using pyrosequencing. Considering the inflammatory states of periodontal diseases in association with epigenetic changes of specific inflammatory genes in gingival tissues, it was demonstrated that the hypermethylation pattern of the prostaglandin-endoperoxide synthase 2 promoter was associated with a lower level of prostaglandinendoperoxide synthase 2 transcription, consistent with a dampening of cyclooxygenase-2 expression that has been reported in gingival samples collected from sites with chronic periodontitis (47, 76). It was also shown that the hypomethylation profile within the interferon gamma promoter region is associated with an increase of interferon gamma transcription in gingival biopsies from patients with chronic periodontitis; however, when an acute inflammatory state was checked using gingival biopsy samples from experimentally induced gingivitis, such an increase of interferon gamma was shown to be independent of the interferon gamma gene promoter methylation modification (77), suggesting that the severity and

chronicity of the infectious inflammatory condition may play a relevant role in the promotion and stability of the methylation changes in the affected tissues. We now understand that the periodontal tissues are epigenetically modified in chronic inflammation and that this occurs locally at the biofilm-gingiva interface around the teeth. However, in order to determine whether the changes in the tissue methylome reflect individual changes in the epigenome of specific cell types, we have focused our study to examine only the sulcular epithelium because those are the cells in close contact with dental biofilm and their products. By using laser capture microdissection, epithelial cells were isolated from the sulcular epithelium (Fig. 3) from periodontally healthy and periodontally diseased patients, and the DNA-methylation differences in periodontally diseased samples compared with periodontally healthy samples were analyzed after DNA isolation (QIAamp DNA Micro Kit; QIAgen) using Methyl-Profiler DNA Methylation qPCR Assays (SABiosciences Corp., Frederick, MD, USA) for panels of inflammatory genes (common cytokines). The findings are shown in Table 6.

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Table 5. Genomic DNA isolated from gingival samples from 10 adult patients with chronic periodontal disease and 10 periodontally healthy subjects analyzed for DNA methylation using Methyl-Profiler DNA Methylation qPCR Assays (SABiosciences Corp., Frederick, MD, USA) for a panel of inflammatory genes Gene symbol

Methylation change (fold)

P-value

Function

CXCL14

2.23

0.008

Potent chemoattractant for neutrophils

CXCL3

1.90

0.020

Chemotactic activity for neutrophilic granulocytes

CXCL5

2.01

0.034

Chemotaxin involved in neutrophil activation

CXCL6

1.76

0.077

Chemotactic for neutrophil granulocytes

S1PR3

3.63

0.026

S1P receptors couple to different G proteins to mediate a range of biological effects, including modulation of cellular survival, proliferation and motility

GATA3

2.46

0.004

Transcriptional activator which binds to the enhancer of the T-cell receptor. GATA3 is present in both human T-helper 2 and T-helper 1 cells

CD276

2.58

0.021

Regulation of T-cell-mediated immune response

ELANE

1.76

0.021

Modifies the functions of natural killer cells, monocytes and granulocytes

CDH13

2.16

0.019

Calcium-dependent cell-adhesion protein

KLF4

1.85

0.052

Involved in the differentiation of epithelial cells

RUNX3

1.39

0.0321

Plays a role during the development of sensory neurons and T cells and regulates transforming growth factor beta signaling in dendritic cells

SFN

1.39

0.0226

ATP-dependent chromatin-remodeling complex that plays role in cell proliferation and differentiation

SFRP2

2.30

0.0460

Modulator of Wnt signaling and of regulation of cell growth and differentiation in specific cell types

IL6ST (GP130)

2.73

0.008

Signal transducer for the interleukin-6 family and regulates osteoclasts and calcium homeostasis

STAT5A

0.58

0.008

Anti-apoptotic activity

TLR2

0.01

0.366

Transmembrane cell-surface receptor, which has a key role in the innate immune system

Bold values indicate P = 0.008

A

B

C

D

Fig. 3. Laser-capture microdissection of epithelial cells performed on gingival biopsy samples from diseased patients and healthy controls. The Zeiss PALM system was used to image, cut (the green line indicates the border of the tissue

to be removed by microdissection and the yellow symbol indicates that the targetted region has been completely encircled for laser excision)and catapult target cells (targetted region has been completely dissected from the tissue).

Although not statistically significant, the trend for hypermethylation of GATA binding protein 3 was observed in epithelial cells, confirming previous findings. Expression of GATA binding protein 3 modulates T-helper 2 cell differentiation and enhances the T-helper 2 cell-specific chromatin acces-

sibility, indicating that GATA binding protein 3 is a key protein that regulates differentiation through chromatin remodeling (52). The hypermethylation of GATA binding protein 3 and interleukin12B genes has also been reported in an in vitro study exposing keratinocytes to Porphyromonas gingivalis (75), and

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Epigenetics in periodontal disease

Fig. 4 Crosstalk in genes that showed an altered methylation pattern in periodontal disease. CXCL3, chemokine (C-X-C motif) ligand 3; CXCL5 chemokine (C-X-C motif) ligand 5; CXCL6, chemokine (C-X-C motif) ligand 6; Elane, Elastase, neutrophil expressed; GATA3, GATA binding protein 3; IL6ST, IL-6 signal transducer, IL6R, interleukin-6 receptor; IL15, interleukin-15; IL13, interleukin-13; IL7, interleukin-7; RUNX3, runt-related transcription factor 3; TYK2, tyrosine kinase 2.

Table 6. Methylation fold changes in the promoter methylation status of various cytokine-producing genes in epithelial cells obtained from Laser Capture Microdissection method (see Fig. 4. caption for definition of these gene names) Gene symbol

Methylation fold-change

P-value

Function

TYK2

1.39

0.0001

Signal transduction through the initiation of type I interferon signaling

IL17C

1.36

0.0033

T-cell-derived cytokine involved in neutrophils, chemotaxis and apoptosis

IL12B

1.37

0.0040

Cytokine produced by antigen-presenting cells that promotes the development of T-helper lymphocyte 1

CCL25

1.32

0.0094

Chemotactic activity for dendritic cells, thymocytes and activated macrophages

CXCL14

1.17

0.0308

Chemoattractant for neutrophils

IL4R

1.50

0.0363

Receptor for both interleukin-4 and interleukin-13

IL13

1.49

0.0626

Cytokine involved in several stages of B-cell maturation and differentiation

GATA3

1.20

0.0640

Transcriptional activator which binds to the enhancer of the T-cell receptor

IL13RA1

1.28

0.0640

Cytokine involved in the activation of the signal transducers and activators of transcription-6 signaling pathways

IL6R

1.13

0.0835

Cytokine that regulates cell growth and differentiation

CXCL5

1.18

0.0895

Chemotaxin involved in neutrophil activation

The methylation levels were calculated by the DCt values and pathways were evaluated using IPA. The interactions of those gene products are indicated in Fig. 4.

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because under Fusobacerium nucleatum challenge epithelial cells in culture presented significant hypomethylation of GATA binding protein 3, the same authors suggested that oral epithelia may respond to the presence of different bacteria with differential epigenetic changes on various genes. Several lines of evidence suggest an interplay between aberrant epigenetic regulation and chronic inflammation. Immune responses and cellular differentiation are tightly controlled by epigenetic modifications, which constitute an additional level of control of gene expression. The differentiation of T cells from progenitor cells to the T-helper 1 or Thelper 2 lineages requires silencing of the genes associated with other lineages, which is accomplished via epigenetic mechanisms (2, 65). Similarly, development of two other important T-cell lineages – T-helper 17 and regulatory T cells – is controlled through epigenetic modifications (36). It is known that the inflammatory reaction has substantial order and constancy as the physiologic changes derived from inflammation have predictable spatial and temporal features. (27). Acute inflammation has been described as being divided into phases that generate distinct clinical phenotypes – an initiation (proinflammatory) phase, an adaptive (anti-inflammatory and reparative) phase and a resolution (restoration of homeostasis) phase (41) – and it has been suggested that epigenetics may partly be responsible for the nuances ranging from hyperinflammation to hypoinflammation and resolution (13). Alterations in the epigenome may also be the underlying molecular basis of certain risk exposures that are known to modify the inflammatory response. This could include obesity, diabetes, smoking and aging – all risk factors or modifiers of periodontal disease. The orderly and stereotypic features of acute systemic inflammation, aligned with multiple receptors and signaling pathways on a similar transcriptome, also predict reprogramming by epigenetics, which would represent the ÔsoftwareÕ that interferes with inflammationÕs ancient DNA code (26, 46). These epigenetic modifications may affect the transcriptional activity of the underlying genes and once epigenetic changes are established, they become relatively stable through rounds of cell division (9, 62). The epigenetic field effects may also explain why periodontal disease preferentially occurs at one specific periodontal site relative to another and why the local tissue will not fully repair or regenerate, thereby persisting as a long-term clinical management problem.

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The importance of epigenetics in periodontal disease has been studied with increasing interest in the field over the last few years. Recent advances in epigenomic approaches allow mapping of the methylation state in the genome with high accuracy, which may help in the identification of biomarkers. Understanding how chronic infection with periodontopathogens in the biofilm-gingiva interface and related inflammation can influence gene-specific epigenetic reprogramming in the periodontal tissues will provide important insights into the mechanisms of bacterial infection-induced cellular changes. The identification of these factors contributing to the initial development of periodontal disease may represent exciting opportunities for the development of novel strategies for preventing and treating periodontal disease.

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