Epigenetics refers to a variety of processes which have long-term effects on gene expression programs without changes in DNA sequence. Key players in epigenetic control are DNA methylation and histone modifications which, in concert with chromatin remodeling complexes, nuclear architecture and microRNAs, define the chromatin structure of a gene and its transcriptional activity. Cellular differentiation is initiated and maintained by epigenetic mechanisms. Although epigenetic marks are established early during development and differentiation, adaptations occur throughout life in response to intrinsic and environmental stimuli and may lead to late life disease and cancer.
Epigenetics refers to mitotically and/or meiotically heritable variations in gene expression that are not caused by changes in DNA sequence. Epigenetic mechanisms regulate all biological processes from conception to death, including genome reprogramming during early embryogenesis and gametogenesis, cell differentiation and maintenance of a committed lineage. Key epigenetic players are DNA methylation and histone post-translational modifications, which interplay with each other, with regulatory proteins and with non-coding RNAs, to remodel chromatin into domains such as euchromatin, constitutive or facultative heterochromatin and to achieve nuclear compartmentalization. Besides epigenetic mechanisms such as imprinting, chromosome X inactivation or mitotic bookmarking which establish heritable states, other rapid and transient mechanisms, such as histone H3 phosphorylation, allow cells to respond and adapt to environmental stimuli. However, these epigenetic marks can also have long-term effects, for example in learning and memory formation or in cancer. Erroneous epigenetic marks are responsible for a whole gamut of diseases including diseases evident at birth or infancy or diseases becoming symptomatic later in life. Moreover, although epigenetic marks are deposited early in development, adaptations occurring through life can lead to diseases and cancer. With epigenetic marks being reversible, research has started to focus on epigenetic therapy which has had encouraging success. As we witness an explosion of knowledge in the field of epigenetics, we are forced to revisit our dogma. For example, recent studies challenge the idea that DNA methylation is irreversible. Further, research on Rett syndrome has revealed an unforeseen role for methyl-CpG-binding protein 2 (MeCP2) in neurons. J. Cell. Physiol. 219: 243–250, 2009. © 2009 Wiley-Liss, Inc.
DNA methylation is associated with gene silencing and occurs on cytosine at CpG dinucleotides across the human genome. Only 1–2% of the genome comprising CpG islands, short CpG-rich regions surrounding promoters, may escape methylation (Suzuki and Bird, 2008). DNA methylation is involved in X chromosome inactivation in females and DNA imprinting, events which result in monoallelic gene expression. It also contributes to genome stability by preventing translocations of repetitive and transposable sequences, and likely plays a dynamic role in development (Miranda and Jones, 2007; Illingworth et al., 2008). The best known imprinting mechanism is the differential DNA methylation of transcription start site-associated CpG islands of paternal and maternal alleles. However, an alternative mode of imprinting was reported where the differential methylation occurs at an intragenic promoter-associated CpG island, resulting in the use of different polyadenylation sites on parental alleles (Wood et al., 2008). About half of the CpG islands are not associated with annotated promoters, but are intra- or intergenic. It has been suggested that these CpG islands mark the transcription start site of non-coding RNAs (Illingworth et al., 2008). This hypothesis is supported by the detection in various tumors of abnormal expression of numerous miRNAs linked to abnormal DNA methylation (Guil and Esteller, 2009). Overall methylation might also affect expression potential as it was reported that, compared to inactive X chromosome, active X chromosome is hypomethylated at promoter regions but hypermethylated at gene bodies (Hellman and Chess, 2007). Mechanisms ruling the targeting of de novo DNA methyltransferases (DNMT3a and DNMT3b) to specific CpG sequences in early development are mostly unknown. In somatic cells, DNA methylation patterns are copied by the maintenance DNA methyltransferase I (DNMTI) positioned at the replication forks, with some cooperation of DNMT3a and DNMT3b (Miranda and Jones, 2007). Thus DNA methylation, maintained through mitosis, is considered a stable epigenetic mark. However, this conventional standpoint is challenged by the existence of active DNA demethylation (Szyf, 2007; Ooi and Bestor, 2008; Szyf et al., 2008), as well as the role of rapid and dynamic DNA methylation/demethylation in long-term memory formation in adult neurons (Miller and Sweatt, 2007). It has been proposed that DNA methylation patterns in many or all cell types result from the balance of methylating and demethylating activities, with both methyltransferases and demethylases being targeted to specific genes by transcription factors acting downstream of signaling pathways yet to be unraveled (Szyf, 2007; Szyf et al., 2008).
Recently it was reported that promoters may be actively methylated and demethylated (Metivier et al., 2008). Analyses of the trefoil factor 1 (TFF1) promoter in breast cancer cells revealed that at the end of each productive transcription cycle, MeCP2, SWI/SNF, DNMT1 and DNMT3a/b were recruited to the TFF1 promoter, resulting in methylation of the TFF1 promoter (Kangaspeska et al., 2008; Metivier et al., 2008; Reid et al., 2009). Demethylation of the CpG sites correlated with the recruitment of DNMT3a/b, which is thought to catalyze the deamination of the methylated C, p68 RNA helicase, thymine DNA glycosylase and base excision repair proteins. Whether other proteins are involved in the demethylation of the TFF1 promoter CpGs remains to be tested. It is of interest to note that thymine DNA glycosylase is associated with estrogen receptor α, other members of the nuclear receptor family, and coactivators SRC-1, p300 and CBP; the latter two being lysine acetyltransferases (Cortazar et al., 2007).
Associated with DNA methylation are methyl-CpG-binding-domain proteins (MBDs), which belong to repressor complexes with histone deacetylase (HDAC) activity (Jones et al., 1998; Nan et al., 1998). HDACs are enzymes that remove the acetyl modification from histones. Histone hyperacetylation is generally associated with chromatin decondensation, accessibility of DNA to binding proteins and increased transcriptional activity, whereas histone hypoacetylation contributes to chromatin condensation and transcriptional repression (Tse et al., 1998; Wang et al., 2001). An in vitro study using chemical ligation to generate a homogeneous recombinant histone preparation demonstrated that K16 acetylation of H4 prevented the formation of the 30-nm chromatin fiber (Shogren-Knaak et al., 2006). Of the histone tails, the H4 tail has a prominent role in compacting the 30 nm fiber. Acetylation of H4 K16 and loss or reduction of linker histones result in a decondensed chromatin fiber (Ridsdale et al., 1990; Robinson et al., 2008). Besides acetylation, histones undergo a variety of reversible post-translational modifications (PTMs), including methylation (me), ubiquitination, ADP-ribosylation and phosphorylation (ph) (Kouzarides, 2007). Enzymes catalyzing reversible histone PTMs are referred to as “writers” (Allis et al., 2007). Amongst them, are K-demethylases, disproving the long-lasting belief that histone methylation was a stable modification. Most core histone PTMs are found within the N- and C-terminal tails. However, sensitive mass spectrometry methods revealed that several PTMs reside in the histone fold domains (Cosgrove et al., 2004; Cosgrove and Wolberger, 2005; Cosgrove, 2007). Some PTMs (active marks) are associated with transcriptionally active chromatin regions, while others (repressive marks) correlate with silent regions. As mentioned above, histone acetylation usually marks active genes as does di- or trimethylation of K4 of H3 (H3K4me2, K4me3) whereas H3K9me2/3 and H3K27me3 constitute repressive marks (Peterson and Laniel, 2004; Sims and Reinberg, 2006).
In delineating the active and repressive marks, the chromatin immunoprecipitation (ChIP) assay has been essential in obtaining this knowledge. Regarding the ChIP technique, a note of caution is warranted. The commonly used ChIP assay includes a formaldehyde cross-linking step to capture the DNA sequence to the protein or modified protein of interest. However, formaldehyde does not necessary cross-link all proteins that are associated with DNA. Indeed, Solomon and Varshavsky (1985) reported that the cross-linking efficiency of formaldehyde was unpredictable. Our results demonstrated that a standard ChIP assay using formaldehyde cross-linking (referred to as X-ChIP) and anti-HDAC2 antibodies would only detect DNA fragments associated with complexes like Sin3 or NuRD containing phosphorylated HDAC2. However, coding regions of genes associated with the more abundant form of unmodified HDAC2 were not detected in the X-ChIP assay (Sun et al., 2007). To monitor the distribution of unmodified HDAC2 in chromatin, the native ChIP assay (also called N-ChIP) or dual cross-linking assay (e.g., DSP-X-ChIP) had to be applied (Zeng et al., 2006; Sun et al., 2007). Thus, it must be kept in mind that the lack of detection of an association between a protein and a DNA sequence does not necessarily mean that this association does not occur.
There is still much to learn about the role of histone PTMs in chromatin structure and function. Histone PTMs function to disrupt chromatin structure and/or provide a “code” for recruitment or occlusion of non-histone chromosomal proteins to chromatin. These recruited proteins are referred to as “readers.” In this reader group are “effectors,” which have activities for further histone PTMs or ATP-dependent chromatin remodeling (Ruthenburg et al., 2007). Added to the complexity is the dynamics of histone PTMs and histone variants (Ausio, 2006; Clayton et al., 2006). H2A, H2B, and H3 have variants that are expressed at the time of DNA synthesis (e.g., H3.1, H2A.1; replication dependent) and those that are expressed throughout the cell cycle (H3.3, H2A.Z; replication independent). H3.1 and H3.3 are components of different histone assembly complexes (Ahmad and Henikoff, 2002; Tagami et al., 2004; Loyola and Almouzni, 2007). H3.3 is located in regulatory regions of genes (Jin and Felsenfeld, 2006; Mito et al., 2007), and H3.3 is enriched in active marks (K4me3 and acetylated K9, 14, 18, and 23) (McKittrick et al., 2004; Loyola et al., 2006; Loyola and Almouzni, 2007). H2A.Z is located at promoters of poised and active genes as well as in facultative heterochromatin (Guillemette and Gaudreau, 2006; Gevry et al., 2007; Mavrich et al., 2008). In facultative heterochromatin H2A.Z is ubiquitinated, and the ubiquitination is catalyzed by RING1b E3 ligase of the polycomb complex, which contains the lysine methyltransferase EZH2 that produced the repressive H3K27me3 mark (Sarcinella et al., 2007).
The activation or repression of mammalian genes involves chromatin remodeling by histone modifying enzymes and ATP-dependent chromatin remodeling complexes (e.g., SWI/SNF). Lysine acetyltransferases (KATs) and HDACs, which catalyze reversible histone acetylation, are among the best understood histone modifying enzymes in terms of multiprotein components, mechanisms of recruitment to regulatory elements of genes and role in transcription. Transcription factors recruit coactivators with KAT activity (e.g., p300/CBP) to regulatory DNA sites, while transcriptional repressors recruit corepressors with HDAC activity (Davie and Moniwa, 2000). In transcriptionally poised and active chromatin regions histone acetylation is a dynamic process, with the steady state of acetylated histones being decided by the relative activities of the recruited KAT and HDAC complexes (Katan-Khaykovich and Struhl, 2002; Davie, 2003). Similarly the steady state of histone phosphorylation is decided by histone kinases and protein phosphatases. The ATP-dependent chromatin remodeling complexes remodel nucleosomes allowing transcription factors and the transcription initiation factors access to regulatory DNA sequences (Langst and Becker, 2004). The temporal order by which histone modifying enzymes and ATP-dependent chromatin remodeling complexes are recruited to DNA is promoter context dependent (Lewis and Reinberg, 2003; Martens et al., 2003; Vermeulen et al., 2003).
Stem Cell Epigenetics
Embryonic stem (ES) cells were first isolated from the inner cell mass (ICM) of developing mouse blastocytes. ICM derived ES cells of mice and human are pluripotent cells with the property of indefinite self-renewal and differentiation into all three germ layers. Transcription factors Oct4, Nanog and Sox2, are among the multiple factors that maintain the pluripotency of ES cells. Similar to active promoters in differentiated somatic cells, Oct4 and Nanog promoters are associated with activating marks such acetylation of H3 and H4 and H3K4me3. However, a subset of key lineage-control genes, such as Pax3 and Msx1, which are not expressed in the ES cells, possess “bivalent” chromatin marks, consisting of an activating mark (H3K4me3) and a repressive mark (H3K27me3). Thus these key lineage-control genes are poised to become fully active with removal of the repressive marks or silenced with removal of the activating marks (Gan et al., 2007).
Genome-wide studies of DNA methylation and chromatin structure in pluripotent ES cells and their differentiated progeny suggest that cellular identity might be a close reflection of cellular epigenetic status. In ES cells, distribution of methylated CpG correlated with distinct histone methylation pattern rather than the underlying sequence context (Meissner et al., 2008). In the human genome, the majority of promoters have a high CpG content (high-CpG-density promoters or HCP), while the remainder have a low CpG content (low-CpG-density promoters or LCP). HCP are associated with two category of genes; housekeeping genes and tightly regulated key developmental genes. In ES cells most HCPs were associated with H3K4me3, with a subset of these being bivalent (H3K4me3 in combination with H3K27me3) (Mikkelsen et al., 2007). The first HCP category was highly expressed in ES cells, while the later was generally silent. The LCPs were associated mainly with tissue specific genes. In contrast to the HCPs, only a small fraction of the LCPs were enriched for H3K4me3 or H3K4me2. Most CpGs of LCPs were methylated, but not the small fraction enriched with H3K4me marks. In ES cells, the presence of H3K4me and absence of H3K9me was a more reliable predictor of DNA methylation than the DNA sequence (Mikkelsen et al., 2007).
During neural precursor cell differentiation, most but not all CpGs of HCP maintained their unmethylated status; however, loss of H3K4me3 and memory of H3K4me correlated with increase in DNA methylation and total loss of H3K4me and H3K27me resulted in DNA hypermethylation (Mikkelsen et al., 2007) (Fig. 1). Most LCPs harboring H3K4me lost this mark during differentiation, while LCP of genes expressed after differentiation gained the H3K4me mark. These data suggest that the enrichment of CpG within a gene regulatory sequence, would determine what category of epigenetic regulation the gene will follow.
Polycomb- (PcG) and trithorax-group (TrxG/MLL) proteins catalyze methylation of H3K27 and H3K4, respectively. PcG proteins are transcriptional silencers and chromatin modifiers implicated in ES cell pluripotency. PcG repression is mediated through two protein complexes Polycomb Repressive Complex 1 (PRC1) and PRC2 that in ES cells silence a wide range of developmentally important genes, including genes that code for transcription factors that are involved in development and differentiation. The multi-protein complex PRC2 trimethylates H3K27, followed by the recruitment of PRC1 which ubiquitinates H2A. In ES cells there are two distinct classes of bivalent domains; one class has association of PRC1 and PRC2, while the other has only PRC2 (Ku et al., 2008). The former class includes genes coding for transcription factors, morphogens or cytokines. The latter class is under-represented in genes coding for transcription factors. During ES cell differentiation, most promoters with bivalent chromatin marks change to a univalent state. Active genes are enriched in H3K4me3 through the action of trxG proteins and loss of H3K27me3 through the action of UTX and JMJD3, which demethylate H3K27me3 (Soshnikova and Duboule, 2008). Genes that remain repressed retain H3K27me3 and lose H3K4me3. PRC2 complex recruits Rbp2 (Jarid1a), an H3K4me3 demethylase. This interaction is required for PRC2-mediated repression of gene expression during ES cell differentiation (Cloos et al., 2008; Pasini et al., 2008).
The preservation of the lineage-specific gene expression profile through cell division includes the preservation of histone PTMs. However, few studies have addressed the question of how histone PTMs are transmitted through mitosis. Unlike most transcription factors which are displaced from mitotic chromosomes (Delcuve et al., 2008), Runx2 maintains occupancy of its target gene promoters during mitosis, and has been suggested to bookmark these genes as transcriptionally poised by preserving active marks such as H4 acetylation and H3K4me2, thus transmitting epigenetic memory to progeny cells (Young et al., 2007). Although levels of highly acetylated H3 and H4 drop significantly at the onset of mitosis (Kruhlak et al., 2001), the bromodomain protein Brd4, which binds to acetylated H3 and H4, also remains bound to mitotic chromosomes and is thought to be involved in preserving acetylated chromatin (Dey et al., 2003; Nishiyama et al., 2006). During mouse oocyte meiosis, histones H3 and H4 become deacetylated while Brd4 is displaced from condensed chromosomes (Nagashima et al., 2007). Conversely, HDAC1 colocalizes with chromosomes during mouse oocyte meiosis (Kim et al., 2003), but is excluded from mitotic chromosomes (Kruhlak et al., 2001; Kim et al., 2003). Thus, both DNA methylation and histone acetylation are wiped out in germ cells, but are maintained through mitosis, with the difference that histones, having several acetylation sites, may be highly acetylated, and a marked decrease in highly acetylated histones occurs when cells enter mitosis (Kruhlak et al., 2001).
Histone H3 Phosphorylation
H3 phosphorylation is a particularly interesting epigenetic mark involved in chromatin remodeling and gene expression. Associated with the induction of specific genes in response to a vast array of stimuli in a variety of cell types, H3 phosphorylation constitutes a crucial intermediate step in the chain of events leading to gene induction upon activation of the ERK and p38 MAPK pathways. Growth factors (EGF) and phorbol esters (e.g., tumor promoter TPA) activate the Ras-MAPK pathway (Ras-Raf-MEK-ERK), while stressors such as UV irradiation, arsenite and acetaldehyde stimulate the p38 MAPK pathway (Hazzalin and Mahadevan, 2002; Dong and Bode, 2006; Lee and Shukla, 2007) (Fig. 2). Mitogen and stress activated protein kinases 1 and 2 (MSK1 and MSK2) are activated by both Ras-MAPK-ERK1/2 and p38 stress kinase pathways (Soloaga et al., 2003), and their substrates include histone H3 (at S10 or S28), nucleosome binding protein HMGN1, p65 subunit of NF-κB and CREB (Arthur, 2008). TPA or EGF stimulation of mouse fibroblasts and human MCF-7 breast cancer cell line results in the phosphorylation of H3 and its variants at S10 and S28 (but not S28 in MCF-7 cells) and HMGN1 at S6, events that have been named the “nucleosomal response” (Strelkov and Davie, 2002; Soloaga et al., 2003; Lim et al., 2004; Dunn and Davie, 2005; Espino et al., 2006). Studies with MSK1/2 knockout cells show a severe reduction in the levels of H3S10ph and H3S28ph in response to TPA. The remaining H3 phosphorylation is due to mitotic phosphorylation by Aurora B of H3 in late G2 phase of the cell cycle (Goto et al., 2002; Soloaga et al., 2003). In Ciras-3 and 10T1/2 cells, H3 phosphorylation events at S10 and S28 do not occur on the same histone tail or even adjacent nucleosomes, and thus are independent and act separately to promote gene expression (Dunn and Davie, 2005; Dyson et al., 2005) (Fig. 2).
TPA/EGF-induced H3S10ph is associated with promoter regions of immediate early (IE) genes (c-jun, c-fos, c-myc) in mouse fibroblasts and the promoter region of TFF1 gene coding for a secreted proinvasive and angiogenic agent in MCF-7 cells (Chadee et al., 1999; Cheung et al., 2000; Clayton et al., 2000; Thomson et al., 2001; Espino et al., 2006). There is also an increase in the steady state of acetylated H3 bound to transcribed chromatin of IE genes c-jun and c-fos following stimulation of the MAPK pathway (Cheung et al., 2000; Thomson et al., 2001). TPA-induction of c-fos is significantly decreased in the double knockout (Soloaga et al., 2003). Although MSK1/2 knockout mice are viable, upon closer examination these animals were found to be defective in stress-related learning and memory processes, with the NMDA-R-mediated H3S10ph and associated c-fos gene induction being abolished in the dentate granule neurons (Reul and Chandramohan, 2007; Chandramohan et al., 2008). Interestingly, the molecular defect in a mouse model for Huntington's disease, a neurodegenerative disease, is a deficiency in MSK1 expression resulting in the lack of induced phosphorylation of H3 S10 and down-regulation of c-fos transcription in the striatum (Roze et al., 2008). MSK1 is also involved in the regulation of clock gene expression in the suprachiasmatic nucleus (Butcher et al., 2005) and in the regulation of inflammatory genes, including interleukin-1, interleukin-8 and COX2 (Beck et al., 2008; Joo and Jetten, 2008).
Several reports suggest that H3 phosphorylation is a key event linking the MAPK signaling cascade with chromatin remodeling. For example, Elk-1 recruits MSK1 to c-fos and egr1 promoters (Zhang et al., 2008). Moreover, the recruitment of a complex with MSK1 activity to the MMTV promoter results in the phosphorylation of H3 S10, causing the displacement of HP1γ and the recruitment of Brg1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, as well as RNA polymerase II (Vicent et al., 2006, 2008).
The steady state of a phosphorylated protein is governed by the net activity of kinases and protein phosphatases. For H3 and HMGN1, the relative activities of MSK1/2 and the protein phosphatase PP1 will determine the steady state level of these phosphorylated proteins. Typically stimulation of the Ras-MAPK by TPA or EGF is transient with phosphorylated ERKs phosphorylating MSK1/2, resulting in the elevated levels of H3S10ph, H3S28ph, and HMGN1S6ph (Dunn et al., 2009). Once the signaling subsides the PP1 activity takes over to reduce phosphorylated H3 and HMGN1 levels. Recently it was demonstrated that the physiological PP1 inhibitor, DARPP-32, controlled the nucleosomal response in midbrain dopamine neurons (Stipanovich et al., 2008). Typically DARPP-32 resides in the cytoplasm, but when phosphorylated, the protein accumulates in the nucleus, inhibiting PP1. Psychostimulant drugs, such as cocaine, which mimic the physiological action of dopamine neurons, promote the nuclear accumulation of DARPP-32. The ensuing nucleosome response and IE gene induction are thought to be important in long-term effects of drugs of abuse and in physiological reward-controlled learning (Stipanovich et al., 2008).
Ciras-3 cells, Ha-ras-transformed 10T1/2 mouse fibroblast cells, are tumorigenic and metastatic (Egan et al., 1987), and have been shown to have elevated levels of phosphorylated ERKs, increased MSK1 activity, but not MSK1 protein, and elevated steady state levels of H3S10ph, H3S28ph, and HMGN1S6ph compared to the non-tumorigenic parental cell line 10T1/2 (Drobic et al., 2004; Dunn et al., 2009). The H3 phosphatase PP1 activity is similar in both cell lines. It was proposed that this elevated activity of MSK and phosphorylated H3 would lead to aberrant gene expression. Recently it has been reported that MSK1 and H3 S10 phosphorylation are required for tumor promoter-induced transformation of JB6 Cl41 cells (Dong and Bode, 2006; Kim et al., 2008). As discussed below, the MSK1/2 mediated induction of IE genes may result in DNA hypermethylation leading to cellular transformation and cancer.
Numerous diseases have been attributed to epigenetic deregulation. Some manifest themselves in early life, for example Fragile X syndrome due to hypermethylation of trinucleotide repeat expansion or Prader–Willi syndrome and Angelman syndrome due to abnormal imprinting (Robertson, 2005). Some diseases are due to mutations affecting “writers” or “readers” of epigenetic marks, for example the immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome due to mutations in the Dnmt3B gene or the Rett syndrome due to mutations in the MECP2 gene (Robertson, 2005). Other diseases, for example type 2 diabetes mellitus or cardiovascular diseases, only become apparent later in life although their epigenetic hallmarks are most likely deposited during development in response to uterine or early life environment. Contributing factors include malnutrition, tobacco smoke or high-sodium diet (Tremblay and Hamet, 2008). In mice, a maternal high dietary intake of methyl donors during gestation has been associated with a higher incidence of asthma in offspring. Decreased transcriptional activity of Runx3, a gene associated with suppression of allergic airway disease, was caused by increased DNA methylation as it could be reversed by treatment with a demethylating agent (Hollingsworth et al., 2008). Epigenetic events are also involved in psychiatric disorders such as addiction, depression, or schizophrenia (Tsankova et al., 2007).
Cancer cells display changes in their DNA methylation program, including global hypomethylation which leads to genetic instability and hypermethylation of CpG islands linked to repression of associated tumor suppressor genes (Gronbaek et al., 2007; Esteller, 2008). Factors promoting these changes in methylation patterns may be environmental elements such as cadium, nickel, arsenic, chronic UV exposure or infectious agents (e.g., Helicobacter pylori), dietary factors such as excessive alcohol intake, folate deficiency or ageing and epigenetic drift (loss of imprinting) (reviewed in Herceg, 2007). Chronic inflammation could also result in altered DNA methylation profiles, as overexpression of interleukin-6 has been associated with overexpression of DNMT1 (Hodge et al., 2005; Meng et al., 2008). Moreover, silencing of tumor suppressor genes by DNA hypermethylation of their regulatory regions is linked with upregulation of the Dmnt1 or Dmnt3b gene expression following the activation of the Ras-MAPK signaling pathway and the induction of IE genes (Yang et al., 1997; Bakin and Curran, 1999; Ordway et al., 2004; Pruitt et al., 2005; Chang et al., 2006; Patra, 2008). The presence of multiple AP-1 response elements with the Dmnt1 5′ region validates the responsiveness of this gene to signals mediated by c-Jun or c-Fos (Bigey et al., 2000).
MeCP2 and Rett Syndrome
The Rett syndrome (RTT) is a severe X-linked postnatal and progressive neurodevelopmental disorder striking mostly girls with a prevalence of ∼1/10,000 female live births. RTT clinical symptoms, starting at 6–18 months of age, include deceleration of head growth, loss of speech and purposeful hand movements, mental retardation, autistic features, anxiety, seizures as well as motor and respiratory abnormalities. RTT is caused by mutations in the gene coding for the methyl-CpG-binding protein 2 (MeCP2) (Amir et al., 1999). MeCP2 is a transcriptional repressor involved in chromatin remodeling as well as a modulator of RNA splicing. As a transcriptional repressor, MeCP2 binds preferentially to methyl-CpG dinucleotides adjacent to A/T-rich sequences and recruits the co-repressor Sin3 complex containing HDAC1 and HDAC2 or other co-repressor complexes (Bienvenu and Chelly, 2006; Chahrour and Zoghbi, 2007). Alternately, MeCP2 achieves chromatin compaction by binding to linker DNA and nucleosomes (Nikitina et al., 2007a,b). A vast array of mutations, deletions or rearrangements has been identified in the MECP2 gene. This genetic variability and the pattern of X chromosome inactivation leading to mosaic expression of the mutant gene give rise to RTT phenotypes with variable severity (Bienvenu and Chelly, 2006; Chahrour and Zoghbi, 2007). It was found that not only loss of MeCP2 function but also gain in MeCP2 dosage lead to RTT-like clinical symptoms (Moretti and Zoghbi, 2006). Although the MECP2 gene is ubiquitously expressed, high MeCP2 levels are specific to postnatal neuronal maturation, explaining why a deficiency in MeCP2 selectively causes neuronal symptoms and results in the defective dendritic branching and altered number of synapses observed in RTT brains (Lasalle, 2004). Studies with several mouse models for RTT confirmed that MeCP2 dysfunction selectively affects postnatal neuronal maturation, and MeCP2 is not a global transcriptional repressor of methylated genes as it was initially assumed. Thus, the identification of MeCP2 target genes has been a main goal among scientists studying RTT, particularly that reversibility of the disease has been demonstrated in RTT mouse models. Indeed, it was shown that reactivating the Mecp2 gene after the onset of disease in RTT mice models can rescue RTT phenotype at least partially (Giacometti et al., 2007; Guy et al., 2007; Jugloff et al., 2008). It is speculated that even in the absence of MeCP2, the epigenetic marks interpreted by MeCP2, are properly formed and following Mecp2 restoration, MeCP2 occupies its designated sites to recover the proper expression profiles in the affected neurons (Bird, 2008). The search for MeCP2 target genes has been complicated by conflicting results, and it seems that the answer to whether a gene is a target for regulation by MeCP2 varies with the cellular and developmental context (Chahrour and Zoghbi, 2007; Lasalle, 2007). One of the identified genes, BDNF coding for the brain-derived neurotrophic factor, has attracted a lot of attention because of its role in neuronal survival and synaptic changes that are basic to memory and learning. It was found that Bdnf expression, repressed by MeCP2, is induced upon neuronal activity-dependent phosphorylation of MeCP2 which results in its dissociation from the Bdnf promoter. It was shown that this phosphorylation event mediates the ability of MeCP2 to regulate dendritic patterning and spine morphogenesis as well as the activity-dependent induction of Bdnf transcription (Zhou et al., 2006). It is believed that MeCP2 role as regulator of the expression of genes such as BDNF is crucial in modulating synaptic function and plasticity. However, another unexpected twist in the understanding of MeCP2 role comes with recent studies suggesting that MeCP2 can serve both as a repressor and as an activator (Yasui et al., 2007; Chahrour et al., 2008). Indeed, in gene expression profiles of the hypothalamus in mice lacking/or overexpressing Mecp2, researchers have found that in 85% of cases MeCP2 acts as an activator (Chahrour et al., 2008). It was shown that MeCP2 is associated with the activating factor CREB1 (cAMP responsive element binding protein 1) at the promoter of an activated gene, but not a repressed gene. Moreover, promoter regions of genes activated by MeCP2 are enriched in undermethylated CpG islands (Chahrour et al., 2008). According to these studies, RTT would be mostly due to loss of transcriptional activation rather than loss of repression.
So far, scientific and clinical research has been focused on the development of demethylating epigenetic drugs in an effort to reactivate tumor suppressor genes. To date, nucleoside analogues inhibitors of DNA methyltransferase, Vidaza® and Dacogen® have been approved by the U.S. Food and Drug Administration to treat myelodysplastic syndromes (MDS). Other small molecule methylation inhibitors are currently being tested for their pharmacological properties (Gronbaek et al., 2007; Vucic et al., 2008).
Histone deacetylation is another epigenetic process that has been targeted for treatment of malignant and other diseases. With the realization that aberrant recruitment of HDACs occurs in cancer cells (Cress and Seto, 2000; Timmermann et al., 2001; Wolffe, 2001), a considerable interest has been aimed at HDAC inhibitors, one of which has been approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (SAHA also known as vorinostat and Zolinza®). Several other HDAC inhibitors are currently in clinical trials as therapeutic agents in the treatment of cancer (Drummond et al., 2005; Riester et al., 2007). Although the HDAC inhibitors so far identified have been sorted into six different structural classes, they are mostly characterized by a common pharmacophore, and affect class I and class II, but not class III HDACs (Marks et al., 2004; Drummond et al., 2005). Class I and class II HDACs share homology in their catalytic sites. Class I HDACs, related to yeast RPD3 deacetylase, are nuclear enzymes including HDAC1 and HDAC2 both found in Sin3, NuRD or Co-Rest complexes. Class II HDACs are related to yeast HDA1 deacetylase and shuttle between the cytoplasm and the nucleus (De Ruijter et al., 2003; Di Gennaro et al., 2004). The mode of action of HDAC inhibitors is poorly understood. They exert their anti-tumor activity by promoting cell cycle arrest, differentiation or apoptosis, via the up or down-regulation of the expression of only a small percentage of genes (<10%) (Drummond et al., 2005). It is noteworthy that the HDAC inhibitor LAQ824 has been reported to affect microRNA levels in a breast cancer cell line (Scott et al., 2006).
Recent studies have identified the chemopreventive agent sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables such as broccoli and broccoli sprouts, as an HDAC inhibitor. It was found that human consumption of 68 g of BroccoSprouts resulted in significant decrease of HDAC activity and increase of acetylated H3 and H4 for at least 3–6 h in peripheral blood mononuclear cells. In mouse models, SFN was also found to have an inhibitory effect on tumor growth that was linked to inhibition of HDAC activity (Dashwood and Ho, 2007; Myzak et al., 2007). The discovery and possibility of using dietary HDAC inhibitors for cancer prevention and therapy is certainly appealing. A note of caution is however warranted as histone acetylation following a short-term exposure to the HDAC inhibitor butyrate has been shown to spread and sequentially expose the whole chromatin (Perry and Chalkley, 1982), with a bona fide possibility for increased susceptibility to DNA modification by carcinogens as well as integration and/or high expression of viral sequences (Perry and Chalkley, 1982; Klehr et al., 1992; Van Lint et al., 1996; Lorincz et al., 2001).
The life of an individual is not only defined by his/her genome, but also by his/her numerous epigenomes, with different epigenomes being generated through development, not only during fetal development but also during the plastic phase of early childhood, and existing in different cell types. Moreover, epigenomes react to environmental influence including maternal care, diet, exposure to toxins and xenobiotics, and epigenetic responses to environmental stimuli may have long-term consequences, even affecting future generations (Szyf et al., 2008). The task ahead of us to decipher all normal epigenomes and dysfunctional epigenomes leading to the vast array of diseases and cancers is colossal. However, thanks to molecular biology techniques such as the ChIP assay (including large-scale variants ChIP-on-chip and ChIP-seq), fluorescent in situ hybridization, DamID (van Steensel and Henikoff, 2003) or bisulfite sequencing, associations of epigenetic marks with specific DNA sequences can be directly elucidated.
This work was supported by the Canadian Institute of Health Research (MOP-9186), CancerCare Manitoba Foundation, Inc., National Cancer Institute of Canada (funds from the Canadian Cancer Society), Canada Research Chair to JRD.