Maternal care, the epigenome and phenotypic differences ... - CiteSeerX

Reproductive Toxicology 24 (2007) 9–19

Review

Maternal care, the epigenome and phenotypic differences in behavior Moshe Szyf a,b,∗ , Ian Weaver b , Michael Meaney b,∗∗ a

Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montréal, Québec H3G 1Y6, Canada b Douglas Hospital Research Center, 6875 LaSalle Blvd, McGill Program for the Study of Behaviour, Genes and Environment, GRIP University of Montreal, Montréal, Québec H4H 1R3, Canada Received 11 April 2007; received in revised form 26 April 2007; accepted 2 May 2007 Available online 10 May 2007

Abstract The genome is programmed by the epigenome, which is comprised of chromatin and a covalent modification of DNA by methylation. Epigenetic patterns are sculpted during development to shape the diversity of gene expression programs in the different cell types of the organism. The epigenome of the developing fetus is especially sensitive to maternal nutrition, and exposure to environmental toxins as well as psychological stress. It is postulated here that not only chemicals but also exposure of the young pup to social behavior, such as maternal care, could affect the epigenome. Since epigenetic programming defines the state of expression of genes, epigenetic differences could have the same consequences as genetic polymorphisms. We will propose here a mechanism linking maternal behavior and epigenetic programming and we will discuss the prospect that similar epigenetic variations generated during early life play a role in generating inter-individual differences in human behavior. We speculate that exposures to different environmental toxins, which affect the epigenetic machinery might alter long-established epigenetic programs in the brain. © 2007 Published by Elsevier Inc. Keywords: DNA methylation; DNA demethylase; Epigenetics; Histone acetylation; Maternal care; Stress; Glucocorticoid receptor; CBP; MBD2 NGFIA

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Epigenetics and inter-individual differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Genes, gene expression programs and phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The epigenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The histone code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Histone modifying enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Chromatin remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Targeting of chromatin modifying enzymes to specific genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. DNA methylation and demethylation enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. The DNA methylation pattern is reversible; DNA demethylation enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Targeting DNA methylation and demethylation; chromatin and DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of epigenetic programming by maternal care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The maternal care model and its implication on epigenetics as both a mediator and effector of behavior . . . . . . . . . . . . . . . . . . . . . 3.2. Maternal care epigenetically programs stress responses in the offspring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗

Corresponding author at: Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montréal, Québec H3G 1Y6, Canada. Tel.: +1 514 398 7107; fax: +1 514 398 6690. ∗∗ Corresponding author. E-mail addresses: [email protected] (M. Szyf), [email protected] (M. Meaney). 0890-6238/$ – see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.reprotox.2007.05.001

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3.3. Epigenetic programming by maternal care is reversible in the adult animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Mechanisms leading from maternal care to epigenetic programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic programming early in life and human behavior and health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Specific challenges for studying epigenetic programming of human behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Epigenetics and inter-individual differences

2. The epigenome

1.1. Genes, gene expression programs and phenotype

2.1. Chromatin

Different cell types execute distinctive plans of gene expression, which are highly responsive to developmental, physiological, pathological and environmental cues. The combinations of mechanisms, which confer long-term programming to genes and could bring about a change in gene function without changing gene sequence are termed here epigenetic. Epigenetic programming occurs during development to generate the complex patterns of gene expression characteristic of complex organisms such as humans, however epigenetic programs in difference from the genetic sequence itself are somewhat dynamic and responsive to different environmental exposures during fetal development as well as early in life. Epigenetic programs are potentially dynamic even later in life. Thus, many of the phenotypic variations seen in human populations might be caused by differences in long-term programming of gene function rather than the sequence per se. Any analysis of inter-individual phenotypic diversity should take into account epigenetic variations in addition to genetic sequence polymorphisms [1]. Some critical environmental exposure could alter the progression of epigenetic programming during development both in utero as well as postnatally. Thus, variation in environmental exposures during these critical periods could result in epigenetic and therefore phenotypic differences later in life. It stands to reason that exposure to nutritional deprivation and chemical toxins would affect the epigenetic machinery during development. Recent data suggests however that psychosocial exposures early in life could also impact on the epigenome resulting in differences in epigenetic program and as a consequence in behavioral differences later in life [2]. Thus, certain behavioral pathologies might be a consequence of early in life exposures which altered epigenetic programming. It is important to understand the mechanisms driving variations in epigenetic programming in order to identify the behavioral pathologies that result from such mechanisms. In difference from genetic mechanisms, epigenetic mechanisms are dynamic and potentially reversible and are therefore amenable to therapeutic intervention [3]. Drugs, which target the epigenetic machinery, are currently tested in clinical trials in cancer [4,5] and psychiatry disorders [6]. Moreover, once we understand the rules through which different environmental exposure modify the epigenetic processes, we might be able to design behavioral strategies to prevent and reverse deleterious environmentally driven epigenetic alterations.

The epigenome consists of the chromatin and its modifications as well as a covalent modification by methylation of cytosine rings found at the dinucleotide sequence CG (Fig. 1) [7]. The epigenome determines the accessibility of the transcription machinery. Inaccessible genes are therefore silent whereas accessible genes are transcribed. We therefore distinguish between open and closed configurations of chromatin [8–12]. Recently another new level of epigenetic regulation by small non-coding RNAs termed microRNA has been discovered [13]. microRNAs regulate gene expression at different levels; silencing of chromatin, degradation of mRNA and blocking translation. microRNAs were found to play an important role in cancer [14] and could potentially play an important role in behavioral pathologies as well [15]. 2.2. The histone code The DNA is wrapped around a protein-based structure termed chromatin. The basic building block of chromatin is the nucleosome, which is formed of an octamer of histone proteins. There are five basic forms of histone proteins termed H1, H2A, H2B H3 and H4 [16] as well as other minor variants, which are involved in specific functions such as DNA repair or gene activation [17]. The octamer structure of the nucleosome is composed of a H3–H4 tetramer flanked on either side with a H2A–H2B dimer [16]. The N-terminal tails of these histones are extensively modified by methylation [18], phosphorylation, acetylation [19] and ubiquitination [20]. The state of modification of these tails plays an important role in defining the accessibility of the DNA wrapped around the nucleosome core. Different histone variants, which replace the standard isoforms also play a regulatory

Fig. 1. The reversible DNA methylation reaction. DNA methyltransferases (DNMT) catalyze the transfer of methyl groups from the methyl donor Sadenosylmethionine to DNA releasing S-adenosylhomocysteine. Demethylases release the methyl group from methylated DNA as either methanol or formaldehyde.


role and serve to mark active genes in some instances [21]. The specific pattern of histone modifications was proposed to form a “histone code”, that delineates the parts of the genome to be expressed at a given point in time in a given cell type [22]. A change in histone modifications around a gene will change its level of expression and could convert an active gene to become silent resulting in “loss of function” or switch a silent gene to be active leading to “gain of function”. 2.3. Histone modifying enzymes The most-investigated histone modifying enzymes are histone acetyltransferases (HAT), which acetylate histone H3 at the K9 residue as well as other residues and H4 tails at a number of residues, and histone deacetylases (HDAC), which deacetylate histone tails [23]. Histone acetylation is believed to be a predominant signal for an active chromatin configuration [24,25]. Deacetylated histones signal inactive chromatin, chromatin associated with inactive genes. Many repressors and repressor complexes recruit HDACs to genes, thus causing their inactivation [26]. Histone tail acetylation is believed to enhance the accessibility of a gene to the transcription machinery whereas deacetylated tails are highly charged and believed to be tightly associated with the DNA backbone and thus limiting accessibility of genes to transcription factors [23]. Histone modification by methylation is catalyzed by different histone methyltransfareses. Some specific methylation events are associated with gene silencing and some with gene activation. For example methylation of the K9 residue of H3-histone tails is catalyzed by the histone methyltransferase SUV3-9 and is associated with silencing of the associated gene [27]. Particular factors recognize histone modifications and further stabilize an inactive state. For example, the heterochromatin associated protein HP-1, binds H3-histone tails methylated at the K9 residue and precipitates an inactive chromatin structure [27]. Recently described histone demethylases remove the methylation mark causing either activation or repression of gene expression [28,29]. 2.4. Chromatin remodeling Chromatin remodeling complexes, which are ATP dependent alter the position of nucleosomes around the transcription initiation site and define its accessibility to the transcription machinery [12]. It is becoming clear now that there is an interrelationship between chromatin modification and chromatin remodeling. For example, the presence of BRG1 the catalytic subunit of SWI/SNF-related chromatin remodeling complexes is required for histone acetylation and regulation of ␤-globin expression during development [30]. 2.5. Targeting of chromatin modifying enzymes to specific genes A basic principle in epigenetic regulation is targeting. Histone modifying enzymes are generally not gene specific. Specific transcription factors and transcription repressors recruit his-

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tone modifying enzymes to specific genes and thus define the gene-specific profile of histone modification [22]. Specific transacting factors are responsive to cellular signaling pathways. Signal transduction pathways, which are activated by cell-surface receptors could thus serve as conduits for epigenetic change linking the environmental trigger at cell surface receptors with gene specific chromatin alterations and reprogramming of gene activity. For example, numerous signaling pathways including those triggered by G protein coupled cell surface receptors in the brain alter the concentration of cAMP in the cell. One of the transcription factors, which respond to increased cAMP is CREB (cAMP response element binding protein). CREB binds cAMP response elements in certain genes. CREB also recruits CREB binding protein CBP. CBP is a HAT, which acetylates histones [31]. Thus, elevation of cAMP levels in response to an extracellular signal would result in a change in the state of histone acetylation in specific genes. Obviously environmental or physiological events, which interfere at any point along the signaling pathway, might result in chromatin alterations. An example of such a pathway that leads from maternal behavior to long-term programming of gene expression in the hippocampus will be discussed in detail here [1]. 2.6. DNA methylation In addition to chromatin, which is associated with DNA, the DNA molecule itself is chemically modified by methyl residues at the 5 position of the cytosine rings in the dinucleotide sequence CG in vertebrates [7] (Fig. 1). What distinguishes DNA methylation in vertebrate genomes is the fact that not all CGs are methylated in any given cell type [7]. Distinct CGs are methylated in different cell types, generating cell type specific patterns of methylation. Thus, the DNA methylation pattern confers upon the genome its cell type identity [7]. Since DNA methylation is part of the chemical structure of the DNA itself, it is more stable than other epigenetic marks and thus it has extremely important diagnostic potential [32] which is yet to be taken advantage of in behavioral disorders. The DNA methylation pattern is established during development and is then maintained faithfully through life by the maintenance DNA methyltransferase [33]. The DNA methylation reaction was believed to be irreversible, thus the common consensus was that the only manner by which methyl residues could be lost was through replication in the absence of DNA methyltransferase by passive demethylation [33]. Recent data supports the idea that similar to chromatin modification, DNA methylation is also potentially reversible [34] even in postmitotic tissues [35]. Recent results suggest that the DNA methylation pattern is highly dynamic in neurons and plays a critical role in memory and fear conditioning [36,37]. DNA methylation patterns in vertebrates are distinguished by their correlation with chromatin structure. Active regions of the chromatin, which enable gene expression, are associated with hypomethylated DNA whereas hypermethylated DNA is packaged in inactive chromatin [7,38]. It is generally accepted that DNA methylation plays an important role in regulating gene expression (Fig. 2). DNA methylation

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Fig. 2. Two mechanisms of silencing gene expression by DNA methylation. An expressed gene (transcription indicated by horizontal arrow) is usually associated with acetylated histones and is unmethylated. An event of methylation would lead to methylation by two different mechanisms. The methyl group (CH3) interferes with the binding of a transcription factor which is required for gene expression resulting in blocking of transcription. The second mechanism shown in the bottom right is indirect. Methylated DNA attracts methylated DNA binding proteins such as MeCP2, which in turn recruits co-repressors such as SIN3A, histone methyltransferases such as SUV39 that methylates histones and histone deacetylases (HDAC), which remove the acetyl groups from histone tails. Methylated histones (K9 residue of histone tails) recruit heterochromatin proteins such as HP1, which contributes to a closed chromatin configuration and silencing of the gene.

in distinct regulatory regions is believed to mark silent genes. A recent whole epigenomic screening of three human chromosomes suggests that a third of the genes analyzed show inverse correlation between the state of DNA methylation at the 5 regulatory regions and gene expression [39]. There is now overwhelming evidence indicting that aberrant silencing of tumor suppressor genes by DNA methylation is a common mechanism in cancer [40]. DNA methylation silences gene expression by two principal mechanisms. The first mechanism involves direct interference of a methyl residue in a recognition element for a transcription factor with the binding of the transcription factor resulting in silencing of gene expression [41,42]. A second mechanism is indirect. A certain density of DNA methylation moieties in the region of the gene attracts the binding of methylated-DNA binding proteins such as MeCP2 [43]. MeCP2 recruits other proteins such as SIN3A and histone modifying enzymes, which lead to formation of a “closed” chromatin configuration and silencing of gene expression [43]. Several methylated-DNA binding proteins such as MBD1, MBD2 and MBD3 suppress gene expression by a similar mechanism [44–46]. 2.7. DNA methylation and demethylation enzymes The DNA methylation reaction is catalyzed by DNA methyltransferase(s) (DNMT) (Fig. 1) [38]. Methylation of DNA occurs immediately after replication by a transfer of a methyl moiety from the donor S-adenosyl-l-methionine (AdoMet) in a reaction catalyzed by DNA methyltransferases (DNMT)

(Fig. 1). Three distinct phylogenic DNA methyltransferases were identified in mammals. DNMT1 shows preference for hemimethylated DNA in vitro, which is consistent with its role as a maintenance DNMT, whereas DNMT3a and DNMT3b methylate unmethylated and methylated DNA at an equal rate which is consistent with a de novo DNMT role [47]. Two additional DNMT homologs were found. DNMT2 whose substrate and methylation activity is unclear [48] and DNMT3L which belongs to the DNMT3 family of DNMTs by virtue of its sequence and is essential for the establishment of maternal genomic imprints but lacks key methyltransferase motifs, and is possibly a regulator of methylation rather than an enzyme that methylates DNA [49]. Knock-out mouse data indicates that DNMT1 is responsible for a majority of DNA methylation marks in the mouse genome [50] as well as the human genome [51] whereas DNMT3a and DNMT3b are responsible for some but not all de novo methylation during development [52]. 2.8. The DNA methylation pattern is reversible; DNA demethylation enzymes It was a long held belief that the DNA methylation pattern is solely dependent on DNMTs and that the reverse reaction cannot occur. Thus, it was believed that DNA methylation pattern could be altered only during cell division when new unmethylated DNA is synthesized and serves as a substrate for maintenance DNMT. If DNA methylation only happens when DNMT is copying DNA methylation patterns during cell division as suggested by the classic model there is no requirement for DNMTs in


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Fig. 3. A pathway from an environmental cue to demethylation of DNA. Environmental cues activate signaling pathways in the cell which turn-on transcription factors. These transcription factors recruit histone acetyltransferases to the gene. Histone acetylation (Ac) inhibitor of histone deacetylase facilitates the interaction of demethylases with methylated DNA resulting in demethylation of DNA.

post-mitotic neurons. Nevertheless, DNMTs are present in postmitotic neurons [53] and there is data suggesting that DNMT levels in neurons change in certain pathological conditions such as schizophrenia [54]. The presence of DNMT in neurons would make sense only if the DNA methylation is dynamic in postmitotic tissues and is a balance of methylation and demethylation reactions [3]. Without active demethylation there is no need for DNA methylation activity in neurons. We have proposed a while ago that the DNA methylation pattern is a balance of methylation and demethylation reactions that are responsive to physiological and environmental signals and thus forms a platform for gene–environment interactions [55] (Fig. 3). There is a long list of data from both cell culture and early mouse development supporting the hypothesis that active methylation occurs in both embryonal and somatic cells. Active demethylation was reported for the myosin gene in differentiating myoblast cells [56], Il2 gene upon T cell activation [57], the interferon ␥ gene upon antigen exposure of memory CD8 T cells [58] and in the glucocorticoid receptor gene promoter in adult rat brains upon treatment with the HDAC inhibitor TSA [35]. The main challenge of the field is identifying the enzymes responsible for demethylation. The biochemical properties of the enzymes responsible for active demethylation are controversial. One proposal has been that a G/T mismatch repair glycosylase also functions as a 5-methylcytosine DNA glycosylase, recognizes methyl cytosines and cleaves the bond between the sugar and the base. The abasic site is then repaired and replaced with a non-methylated cytosine resulting in demethylation [59]. An additional protein with a similar activity was recently identified, the methylated DNA binding protein 4 (MBD4) [60]. While such mechanism can explain site-specific demethylation,

global demethylation by a glycosylase would involve extensive damage to DNA that would compromise genomic integrity. Another report has proposed that methylated binding protein 2 MBD2 has demethylase activity. MBD2b (a shorter isoform of MBD2) was shown to directly remove the methyl group from methylated cytosine in methylated CpGs [61]. This enzyme was therefore proposed to reverse the DNA methylation reaction. However, other groups disputed this finding [45]. Very recent data suggests that active demethylation early in embryogenesis as well as in somatic cells is catalyzed by a nucleotide excision repair mechanism, whereby methylated cytosines are replaced by unmethylated cytosines, which involves the growth arrest and damage response protein GADD45A and the DNA repair endonuclease XPG [62]. The main problem with this mechanism is that it involves extensive damage to the DNA. Although a number of biochemical processes were implicated in demethylation, it is unclear how and when these different enzymes participate in shaping and maintaining the overall pattern of methylation and how these activities respond to different environmental exposures in the brain. This remains one of the most important unresolved questions in the field. 2.9. Targeting DNA methylation and demethylation; chromatin and DNA methylation Methylation and demethylation enzymes have to be targeted to specific genes to either preserve or change the pattern of methylation. The gene-specificity of chromatin modification state is defined by sequence-specific trans-acting factors that recruit chromatin modifying enzymes to specific genes. Chromatin configuration then gates the accessibility of genes to either

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DNA methylation or demethylation machineries [63,64]. In support of this hypothesis we have previously shown that the histone deacetylase inhibitor (HDACi) trichostatin A, which causes histone hyperacetylation also causes active DNA demethylation (see Fig. 3) [63]. Histone modification enzymes interact with DNA methylating enzymes and participate in recruiting them to specific targets. A growing list of histone modifying enzymes has been shown to interact with DNMT1, such as HDAC1 and HDAC2 and the histone methyltransferases SUV3-9 and EZH2, a member of the multi-protein Polycomb complex PRC2, which methylates H3 histone at the K27 residue [65–68]. DNMT3a was recently also shown to interact with EZH2 which targets the DNA methylation-histone modification multi-protein complexes to specific sequences in DNA [68].Trans-acting repressors target both histone modifying enzymes and DNMTs to specific cisacting signals such as the promyelocytic leukemia PML-RAR fusion protein that engages histone deacetylases and DNMTs to its target binding sequences and produces de novo DNA methylation of adjacent genes [69]. There are also documented interactions between proteins, which read the DNA methylation and histone methylation marks and either histone or DNA modifying enzymes. The methylated DNA binding protein MeCP2 interacts with the HMT SUV3-9 [67] and in plants it was shown that Chromomethylase3 (CMT3), a plant CNG specific DNMT interacts with an Arabidopsis homologue of HP1, a protein which binds histone H3 methylated at Lysine 9 [70]. Transcription factors recruit HATs to specific genes causing gene specific acetylation and thus we propose could facilitate their demethylation. There are examples in literature indicat-

ing that enhancers are required for active demethylation. For example, the intronic kappa chain enhancer and the transcription factor NF-kappaB are required for B cell specific demethylation of the kappa immunoglobulin gene [71]. The demethylation of the maize Suppressor-mutator (Spm) transposon is mediated by the transposon-encoded transcriptional activator TnpA protein [57]. We will discuss below how maternal care is employing this mechanism to program gene expression through recruitment of the transcription factor NGFI-A to one of the GLUCOCORTICOID RECEPTOR (GR) gene promoters in the hippocampus [72]. This is a mechanism, which could potentially mediated between external signals from the environment and demethylation of specific genes in neurons. In summary, we propose that DNA methylation and chromatin structure are found in a dynamic balance through life. The direction of the balance is maintained and defined by sequence specific factors, which deliver histone modification and DNA modification enzymes to genes. These factors are responsive to signaling pathways in the cell. The state of this equilibrium is defined during development and in the process of cellular differentiation. Physiological or environmental signals, which alter the signaling pathways in the cell, would result in tilting of this balance by activating or suppressing specific trans-acting factors. This proposed mechanism provides a basis for understanding how the environment in early life typified by maternal care defines epigenetic programming of gene expression programs in the brain (see model in Fig. 4) [1]. We will summarize below the evidence for a pathway which links the environment including the social environment early in life and stable programming of gene expression in the brain.

Fig. 4. Behavioral gene programming by maternal care. The sequence of events leading from maternal licking and grooming behavior to epigenetic programming of the GR exon 17 promoter. CBP, a HAT (cAMP recognition element binding protein CREB); M, methylated CG; Ac, acetylated H3-histone.


3. Mechanisms of epigenetic programming by maternal care 3.1. The maternal care model and its implication on epigenetics as both a mediator and effector of behavior The best-documented case to date of epigenetic programming triggered by the social environment is the long-term impact that maternal care has on expression of the glucocorticoid receptor gene in the hippocampus of the offspring in the rat. In the rat, the adult offspring of mothers that exhibit increased levels of pup licking/grooming (i.e., High LG mothers) over the first week of life show increased hippocampal GR expression, enhanced glucocorticoid feedback sensitivity, decreased hypothalamic corticotrophin releasing factor (CRF) expression and more modest HPA stress responses compared to animals reared by Low LG mothers [73,74]. Cross-fostering studies suggest direct effects of maternal care on both gene expression and stress responses [73,74]. These studies supported an epigenetic mechanism rather than a genetic mechanism for the effect of maternal care, since the fostering mother and not the biological mother defined the stress response of its adult offspring [73,74]. The critical question was obviously that of mechanism? How could the behavior of the caregiver cause a stable change in gene expression in the offspring long after the caregiver is gone? We postulated an epigenetic mechanism. We hypothesized that the maternal behavior of the caregiver triggered an epigenetic change in the brain of the offspring [1]. This model has two nodal implications on our understanding of the relationship between behavior and epigenetics. First, social behavior of one subject can affect epigenetic programming in another subject. Thus, our model provides a molecular mechanism mediating the effects of nurture on nature. Second, epigenetic programming can have long-term impact on behavior as well as on inter-individual differences in behavior and other health outcomes. 3.2. Maternal care epigenetically programs stress responses in the offspring We have previously published evidence to support the hypothesis that epigenetic mechanisms mediate the maternal effect on stress-response. Increased maternal LG is associated with demethylation of the nerve growth factor-inducible protein A (NGFI-A) transcription factor response element located within the exon 17 GR promoter [35] (Fig. 4). The difference in the methylation status of this CpG site between the offspring of High and Low LG mothers emerges over the first week of life, is reversed with cross-fostering, persists into adulthood and is associated with altered histone acetylation and NGFI-A binding to the GR promoter [35]. Thus, maternal care affects the chromatin, DNA methylation and transcription factor binding to the GR exon 17 promoter illustrating the basic principles of epigenetic regulation discussed above. We have also shown that maternal care early in life affected the expression of hundreds of genes in the adult hippocampus [75]. Thus, these data illustrate the profound effect of the social environment early in life on gene

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expression programming throughout life. These results have quite tantalizing implications. They imply that differences in maternal care early in life can result in gene expression changes, which remain persistent into adulthood in numerous genes. This illustrates the potential power of epigenetic processes in modulating our genomic inheritance. An important implication of this mechanism is that exposures to different chemicals and toxins which interact with the epigenetic machinery during this critical period might have a profound impact on behavior later in life by interfering with the maternal care driven epigenetic programming. Future experiments need to address this possibility. 3.3. Epigenetic programming by maternal care is reversible in the adult animal Although epigenetic programming by maternal care is limited to a critical window of time after birth, is highly stable and results in long-term changes in gene expression, it is nevertheless potentially reversible because the steady-state methylation pattern is defined by a dynamic equilibrium of methylation-demethylation reactions [3]. We have previously proposed as discussed above that increasing histone acetylation by a HDAC inhibitor such as TSA would tilt the balance of the DNA methylation equilibrium towards demethylation (Fig. 3) [63,76]. Treating adult offspring of low LG-ABN maternal care with TSA reversed the epigenetic marks on the GR exon 17 promoter; histone acetylation increased, the gene was demethylated and there was increased occupancy of the promoter with the transcription factor NGFIA, resulting in increased GR exon 17 promoter expression. The epigenetic reversal was accompanied with a behavioral change so that the stress response of the TSA treated adult offspring of low LG-ABN was indistinguishable from the offspring of high LG-ABN [77]. The combination of reversibility and stability is one of the appealing aspects of epigenetics. This was the first illustration of reversal of early life behavioral programming by pharmacological modulation of the epigenome during adulthood. Histone acetylation could be altered not only by pharmacological modulation but also by neurotransmitter activation of signaling pathways whose downstream targets include histone acetyltransferases. Thus, behavioral interventions which lead to firing of neurons and consistent and repetitive activation of signaling pathways might also lead to a change in DNA methylation of specific genes in the adult brain. In addition, drugs that are used for entertainment or therapeutic process, or other toxic exposures, which reach the CNS might potentially alter DNA methylation patterns of genes in the brain by similar mechanisms. An interesting example of such a drug is the anti-epileptic and mood stabilizing agent valproic acid. This drug was found to be a HDAC inhibitor. We have shown that valproate triggered replication independent DNA demethylation in tissue culture [78,79] and valproate was shown to inhibit DNA methylation in the brain in an animal model [80]. Other HDAC inhibitors are now being tested as potential inducers of genes silenced by methylation in schizophrenia [6]. It is possible also to reverse the DNA methylation equilibrium in the hippocampus in the opposite direction by increasing

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DNA methylation. The methyl donor S-adenosyl methionine (SAM) inhibits the demethylation reaction [81]. Thus, changing SAM levels would alter the DNA methylation equilibrium by either increasing the rate of the DNA methylation reaction or by inhibiting the demethylation reaction or both. Systemic injection of methionine was previously shown to increase SAM concentrations in the brain [80]. Injection of methionine to the brain led to hypermethylation and reduced expression of the GR exon 17 expression in the adult hippocampus of offspring of high LG-ABN and reversal of its stress response to a pattern which was indistinguishable from offspring of low LG-ABN [82]. In summary, maternal epigenetic programming early in life could be reversed later in life in both directions. Methionine is especially interesting since the levels of methionine in cells are influenced by diet. Thus, this might provide an example of a potential link between dietary intake and alterations in epigenetic programming in the brain. 3.4. Mechanisms leading from maternal care to epigenetic programming Our working hypothesis is that maternal care triggers a signaling pathway, which activates certain transcription factors directing the epigenetic machinery (DNA and chromatin modifying enzymes) to specific targets in the genome. Maternal LG in early life elicits a thyroid hormone-dependent increase in serotonin (5-HT) activity at 5-HT7 receptors, and the subsequent activation of cyclic adenosine 3 , 5 monophosphate (cAMP) and cAMP-dependent protein kinase A (PKA) [83–85]. This is accompanied by increased hippocampal expression of NGFI-A transcription factor. The GR exon 17 promoter region contains a binding site for NGFI-A [86]. NGFI-A was previously shown to interact with the transcriptional co-activator and histone acetyl transferase CREB Binding Protein CBP. Signaling pathways that result in increased cAMP also activate CBP. Recruitment of CBP to the GR exon 17 promoter in response to maternal care could explain the increased acetylation and demethylation observed in offspring of high LG-ABN [35]. To test a causal link between NGFI-A binding and epigenetic reprogramming of the GR exon 17 promoter we resorted to cell culture experiments. Our results show that an expression vector expressing high levels of NGFI-A is co-transfected with the methylated GR exon 17 promoter–luciferase, the transcription activity of the promoter is induced, there is an increased recruitment of NGFI-A to the promoter as expected, increased recruitment of CBP, increased histone acetylation and methylation mapping indicated that the GR exon 17 promoter was demethylated. We suggest that the role that NGFI-A plays in regulation of the GR exon 17 promoter is bimodal. Under low concentrations of NGFI-A, binding to the target sequence is inhibited by DNA methylation. However, under conditions of high NGFI-A activity some NGFI-A interacts with the methylated GR exon 17 promoter launching a cascade of events leading to demethylation of the promoter. Therefore, increased activation of NGFI-A triggered by a repetitive and frequent behavior such as maternal LG leads to binding of NGFI-A to the methy-

lated promoter and recruitment of CBP. We proposed that the recruitment of CBP led to increased histone acetylation that resulted in demethylation [72]. This sequence of events is consistent with our working hypothesis on the relationship between histone acetylation and DNA demethylation [63,87]. Thus, we show that similar to acetylation induced in response to pharmacological administration of TSA, targeted acetylation by recruitment of a transcription factor leads to demethylation of DNA [72]. In summary, our studies establish a first working hypothesis of how maternal behavior can result in an epigenetic reprogramming in the offspring. Neurotransmitter release results in activation of a signaling pathway that leads to recruitment of particular transcription factors such as NGFI-A to their recognition elements in front of specific genes. The transcription factors recruit histone acetyltransferases that in turn reprogram the chromatin and facilitate demethylation. The remaining question was to identify the protein(s) involved in demethylation of DNA. We previously proposed that the methylated DNA binding protein 2 (MBD2) was a DNA demethylase and could bring about DNA demethylation in vitro [61]. Other groups [88] hotly contested the in vitro demethylation activity of MBD2 but more recent data from our laboratory supported a demethylation role for MBD2 [81,89]. We therefore tested the hypothesis that MBD2 mediated the demethylation of GR exon 17 promoter. Our results indicate that MBD2 binds the GR exon 17 promoter in the hippocampi of day 6 pups and that this binding is increased with high maternal LG-ABN. Using a transient transfection assay we show that ectopically expressed MBD2 transcriptionally activates in vitro methylated GR exon 17 promoter-luciferase promoter, increases the interaction of CBP and increases histone acetylation. A combination of ChIP and bisulfite mapping of DNA methylation indicated that MBD2 bound GR exon 17 promoter molecules were demethylated at a CG site found in the NGFI-A recognition element. We showed that binding of NGFI-A to its response element was required for MBD2 action since a mutation which abolished NGFI-A binding also prevented MBD2 binding, activation of gene expression and demethylation. Using a double ChIP approach, which involves immunoprecipitation sequentially with both NGFI-A and MBD2 antibodies, we show that both proteins simultaneously bind the same GR exon 17 promoter molecule (Weaver et al., unpublished data). Our data is consistent with the hypothesis that NGFI-A facilitates the accessibility of the sequence to MBD2 leading to target specific demethylation (Fig. 4). This provides a general paradigm for gene specific epigenetic programming by sequence specific factors, which reside downstream to signal transduction pathways (Fig. 4). In summary, our study suggests a possible conduit for social behavior such as maternal care to affect epigenetic programming of specific genes in brain of other subjects. It also illustrates the critical impact that social exposures early in life as well as exposure to other toxins and environmental hazards which target the epigenetic machinery might have on gene expression programming and behavior that could last into adulthood.


4. Epigenetic programming early in life and human behavior and health 4.1. Specific challenges for studying epigenetic programming of human behavior A fundamental question that remains to be answered is whether a mechanism similar to the mechanism described in the rat operates in generating inter-individual differences in human behavior (Fig. 5). The hypothesis is obviously attractive; social adversity in early childhood similar to low LG-ABN might result in aberrant epigenetic programming causing changes in gene expression, which will stably impact on behavior later in life. Similarly, strong environmental exposures later in life might reverse or alter epigenetic programming of genes regulating human behavior. The main impediment in studying epigenetic programming in living human is obviously the inaccessibility of the brain to epigenetic analysis. The critical question is whether epigenetic alterations, which are relevant to human behavior occur in peripheral tissue. The best candidates are probably white blood cells. Cytokines secreted by different types of blood cells were shown to interact with receptors in the CNS and crosstalk between the immune system and the brain have been proposed for some time to play a role in human behaviors such as stress response [90–92]. IL-1␤ was proposed to interact with the HPA axis activities and thus it stands to reason that differences in IL-1␤ might impact on stress responsivity as well as other behaviors. Notably, several cytokines were previously shown to be regulated by DNA methylation [57,93–96] and expression of IL-1␤ was induced by treatment of human monocyte cell lines with the DNA methylation inhibitor 5-azacytidine [97]. However, although these candidate genes are interesting, a nonbiased approach might identify other unanticipated candidates. Thus, whole epigenome analyses should enable the identification of hitherto unknown epigenetic markers of human behavior patterns. The question of whether there are markers of epigenetic alteration in peripheral tissues is obviously of utmost importance for the progress of the study of epigenetics in human behavior.

Fig. 5. A scheme for environmental driven epigenetic states and inter-individual phenotypic variance in behavior and susceptibility to disease in humans.

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5. Summary Recent data from the rat maternal care model charts a pathway leading from the behavior of the mother to long-term programming of gene expression in the offspring. This pathway involves the firing of neurotransmitter receptors in response to the behavior, signaling pathways, which activate sequence specific transcription factors, which recruit histone acetyltransferases to the gene, thus facilitating the accessibility of the methylated gene to demethylases [64]. This results in acetylation of chromatin and recruitment of DNA demethylases such as MBD2 leading to demethylation and stable activation of this gene. These data point to a thought provoking notion that epigenetic processes play a role in shaping human behavior in response to different levels of social adversity early in life and later during adulthood. Since several environment agents are known to interfere with the epigenetic machinery, this data raises the possibility that exposure to these agents during periods of epigenetic reprogramming early in life might alter behavior later in life. In addition, the model discussed here suggests that epigenetic programming in the brain though stable is nevertheless reversible upon different exposures. Thus, later life exposures to environmental chemicals might alter stable epigenetic programming and as a consequence behavior in the adult. Acknowledgements These studies were supported by a grant from the Canadian Institutes for Health Research (CIHR) to M.J.M. and M.S. and from the National Cancer Institute of Canada to MS. M.J.M. is supported by a CIHR Senior Scientist award and the project was supported by a Distinguished Investigator Award to M.J.M. from the National Alliance for Research on Schizophrenia and Affective Disorders (NARSAD). References [1] Meaney MJ, Szyf M. Maternal care as a model for experience-dependent chromatin plasticity? Trends Neurosci 2005;28:456–63. [2] Meaney MJ, Szyf M. Environmental programming of stress responses through DNA methylation: life at the interface between a dynamic environment and a fixed genome. Dialogues Clin Neurosci 2005;7:103–23. [3] Szyf M. Towards a pharmacology of DNA methylation. Trends Pharmacol Sci 2001;22:350–4. [4] Weidle UH, Grossmann A. Inhibition of histone deacetylases: a new strategy to target epigenetic modifications for anticancer treatment. Anticancer Res 2000;20:1471–85. [5] Kramer OH, Gottlicher M, Heinzel T. Histone deacetylase as a therapeutic target. Trends Endocrinol Metab 2001;12:294–300. [6] Simonini MV, Camargo LM, Dong E, Maloku E, Veldic M, Costa E, et al. The benzamide MS-275 is a potent, long-lasting brain regionselective inhibitor of histone deacetylases. Proc Natl Acad Sci USA 2006;103:1587–92. [7] Razin A. CpG methylation, chromatin structure and gene silencing—a three-way connection. EMBO J 1998;17:4905–8. [8] Groudine M, Eisenman R, Gelinas R, Weintraub H. Developmental aspects of chromatin structure and gene expression. Prog Clin Biol Res 1983;134:159–82. [9] Marks PA, Sheffery M, Rifkind RA. Modulation of gene expression during terminal cell differentiation. Prog Clin Biol Res 1985;191:185–203.

18


[10] Ramain P, Bourouis M, Dretzen G, Richards G, Sobkowiak A, Bellard M. Changes in the chromatin structure of Drosophila glue genes accompany developmental cessation of transcription in wild type and transformed strains. Cell 1986;45:545–53. [11] Grunstein M. Histone acetylation in chromatin structure and transcription. Nature 1997;389:349–52. [12] Varga-Weisz PD, Becker PB. Regulation of higher-order chromatin structures by nucleosome-remodelling factors. Curr Opin Genet Dev 2006;16:151–6. [13] Bergmann A, Lane ME. HIDden targets of microRNAs for growth control. Trends Biochem Sci 2003;28:461–3. [14] Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302:1–12. [15] Vo N, Klein ME, Varlamova O, Keller DM, Yamamoto T, Goodman RH, et al. A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc Natl Acad Sci USA 2005;102:16426–31. [16] Finch JT, Lutter LC, Rhodes D, Brown RS, Rushton B, Levitt M, et al. Structure of nucleosome core particles of chromatin. Nature 1977;269:29–36. [17] Sarma K, Reinberg D. Histone variants meet their match. Nat Rev Mol Cell Biol 2005;6:139–49. [18] Jenuwein T. Re-SET-ting heterochromatin by histone methyltransferases. Trends Cell Biol 2001;11:266–73. [19] Wade PA, Pruss D, Wolffe AP. Histone acetylation: chromatin in action. Trends Biochem Sci 1997;22:128–32. [20] Shilatifard A. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. Annu Rev Biochem 2006. [21] Henikoff S, McKittrick E, Ahmad K. Epigenetics, histone H3 variants, and the inheritance of chromatin states. Cold Spring Harb Symp Quant Biol 2004;69:235–43. [22] Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:1074–80. [23] Kuo MH, Allis CD. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 1998;20:615–26. [24] Perry M, Chalkley R. Histone acetylation increases the solubility of chromatin and occurs sequentially over most of the chromatin. A novel model for the biological role of histone acetylation. J Biol Chem 1982;257: 7336–47. [25] Lee DY, Hayes JJ, Pruss D, Wolffe AP. A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 1993;72:73–84. [26] Wolffe AP. Histone deacetylase: a regulator of transcription. Science 1996;272:371–2. [27] Lachner M, O’Carroll D, Rea S, Mechtler K, Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 2001;410:116–20. [28] Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 2004;119:941–53. [29] Tsukada Y, Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, Tempst P, et al. Histone demethylation by a family of JmjC domaincontaining proteins. Nature 2006;439:811–6. [30] Bultman SJ, Gebuhr TC, Magnuson T. A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in beta-globin expression and erythroid development. Genes Dev 2005;19:2849–61. [31] Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 1996;87:953–9. [32] Beck S, Olek A, Walter J. From genomics to epigenomics: a loftier view of life. Nat Biotechnol 1999;17:1144. [33] Razin A, Riggs AD. DNA methylation and gene function. Science 1980;210:604–10. [34] Ramchandani S, Bhattacharya SK, Cervoni N, Szyf M. DNA methylation is a reversible biological signal. Proc Natl Acad Sci USA 1999;96:6107–12. [35] Weaver IC, Cervoni N, Champagne FA, D’Alessio AC, Sharma S, Seckl JR, et al. Epigenetic programming by maternal behavior. Nat Neurosci 2004;7:847–54.

[36] Miller CA, Sweatt JD. Covalent modification of DNA regulates memory formation. Neuron 2007;53:857–69. [37] Levenson JM, Roth TL, Lubin FD, Miller CA, Huang IC, Desai P, et al. Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J Biol Chem 2006;281:15763–73. [38] Razin A, Cedar H. Distribution of 5-methylcytosine in chromatin. Proc Natl Acad Sci USA 1977;74:2725–8. [39] Eckhardt F, Lewin J, Cortese R, Rakyan VK, Attwood J, Burger M, et al. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat Genet 2006;38:1378–85. [40] Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K, Herman JG. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 2001;10:687–92. [41] Comb M, Goodman HM. CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucleic Acids Res 1990;18:3975–82. [42] Inamdar NM, Ehrlich KC, Ehrlich M. CpG methylation inhibits binding of several sequence-specific DNA-binding proteins from pea, wheat, soybean and cauliflower. Plant Mol Biol 1991;17:111–23. [43] Nan X, Campoy FJ, Bird A. MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 1997;88: 471–81. [44] Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol Cell Biol 1998;18:6538–47. [45] Ng HH, Zhang Y, Hendrich B, Johnson CA, Turner BM, ErdjumentBromage H, et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet 1999;23:58–61 [see comments]. [46] Fujita N, Takebayashi S, Okumura K, Kudo S, Chiba T, Saya H, et al. Methylation-mediated transcriptional silencing in euchromatin by methylCpG binding protein MBD1 isoforms. Mol Cell Biol 1999;19:6415–26. [47] Okano M, Xie S, Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 1998;19:219–20 [letter]. [48] Vilain A, Apiou F, Dutrillaux B, Malfoy B. Assignment of candidate DNA methyltransferase gene (DNMT2) to human chromosome band 10p15.1 by in situ hybridization. Cytogenet Cell Genet 1998;82:120. [49] Bourc’his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the establishment of maternal genomic imprints. Science 2001;294:2536–9. [50] Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992;69:915–26. [51] Chen T, Hevi S, Gay F, Tsujimoto N, He T, Zhang B, et al. Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells. Nat Genet 2007. [52] Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99:247–57. [53] Goto K, Numata M, Komura JI, Ono T, Bestor TH, Kondo H. Expression of DNA methyltransferase gene in mature and immature neurons as well as proliferating cells in mice. Differentiation 1994;56:39–44. [54] Veldic M, Guidotti A, Maloku E, Davis JM, Costa E. In psychosis, cortical interneurons overexpress DNA-methyltransferase 1. Proc Natl Acad Sci USA 2005;102:2152–7. [55] Ramchandani S, Bhattacharya SK, Cervoni N, Szyf M. DNA methylation is a reversible biological signal. Proc Natl Acad Sci USA 1999;96:6107–12 [see comments]. [56] Lucarelli M, Fuso A, Strom R, Scarpa S. The dynamics of myogenin sitespecific demethylation is strongly correlated with its expression and with muscle differentiation. J Biol Chem 2001;276:7500–6. [57] Bruniquel D, Schwartz RH. Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat Immunol 2003;4:235–40. [58] Kersh EN, Fitzpatrick DR, Murali-Krishna K, Shires J, Speck SH, Boss JM, et al. Rapid Demethylation of the IFN-{gamma} Gene Occurs in Memory but Not Naive CD8 T Cells. J Immunol 2006;176:4083–93. [59] Jost JP. Nuclear extracts of chicken embryos promote an active demethylation of DNA by excision repair of 5-methyldeoxycytidine. Proc Natl Acad Sci USA 1993;90:4684–8.

M. Szyf et al. / Reproductive Toxicology 24 (2007) 9–19 [60] Zhu B, Zheng Y, Hess D, Angliker H, Schwarz S, Siegmann M, et al. 5-Methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex. Proc Natl Acad Sci USA 2000;97:5135–9. [61] Bhattacharya SK, Ramchandani S, Cervoni N, Szyf M. A mammalian protein with specific demethylase activity for mCpG DNA. Nature 1999;397:579–83 [see comments]. [62] Barreto G, Schafer A, Marhold J, Stach D, Swaminathan SK, Handa V, et al. Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature 2007. [63] Cervoni N, Szyf M. Demethylase activity is directed by histone acetylation. J Biol Chem 2001;276:40778–87. [64] D’Alessio AC, Szyf M. Epigenetic tete-a-tete: the bilateral relationship between chromatin modifications and DNA methylation. Biochem Cell Biol 2006;84:463–76. [65] Fuks F, Burgers WA, Brehm A, Hughes-Davies L, Kouzarides T. DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat Genet 2000;24:88–91. [66] Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat Genet 2000;25:269–77. [67] Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T. The methyl-CpGbinding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem 2003;278:4035–40. [68] Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, et al. The Polycomb group protein EZH2 directly controls DNA methylation. Nature 2005. [69] Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M, et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 2002;295:1079–82. [70] Jackson JP, Lindroth AM, Cao X, Jacobsen SE. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 2002;416:556–60. [71] Lichtenstein M, Keini G, Cedar H, Bergman Y. B cell-specific demethylation: a novel role for the intronic kappa chain enhancer sequence. Cell 1994;76:913–23. [72] Weaver IC, D’Alessio AC, Brown SE, Hellstrom IC, Dymov S, Sharma S, et al. The transcription factor nerve growth factor-inducible protein a mediates epigenetic programming: altering epigenetic marks by immediate-early genes. J Neurosci 2007;27:1756–68. [73] Liu D, Diorio J, Tannenbaum B, Caldji C, Francis D, Freedman A, et al. Maternal care, hippocampal glucocorticoid receptors, and hypothalamicpituitary-adrenal responses to stress. Science 1997;277:1659–62. [74] Francis D, Diorio J, Liu D, Meaney MJ. Nongenomic transmission across generations of maternal behavior and stress responses in the rat. Science 1999;286:1155–8. [75] Weaver IC, Meaney MJ, Szyf M. Maternal care effects on the hippocampal transcriptome and anxiety-mediated behaviors in the offspring that are reversible in adulthood. Proc Natl Acad Sci USA 2006;103:3480–5. [76] Cervoni N, Detich N, Seo SB, Chakravarti D, Szyf M. The oncoprotein Set/TAF-1␤, an inhibitor of histone acetyltransferase, inhibits active demethylation of DNA, integrating DNA methylation and transcriptional silencing. J Biol Chem 2002;277:25026–31. [77] Weaver IC, Diorio J, Seckl JR, Szyf M, Meaney MJ. Early environmental regulation of hippocampal glucocorticoid receptor gene expression: characterization of intracellular mediators and potential genomic target sites. Ann NY Acad Sci 2004;1024:182–212. [78] Detich N, Bovenzi V, Szyf M. Valproate induces replication-independent active DNA demethylation. J Biol Chem 2003;278:27586–92. [79] Milutinovic S, D’Alessio AC, Detich N, Szyf M. Valproate induces widespread epigenetic reprogramming which involves demethylation of specific genes. Carcinogenesis 2006.

19

[80] Tremolizzo L, Carboni G, Ruzicka WB, Mitchell CP, Sugaya I, Tueting P, et al. An epigenetic mouse model for molecular and behavioral neuropathologies related to schizophrenia vulnerability. Proc Natl Acad Sci USA 2002;99:17095–100. [81] Detich N, Hamm S, Just G, Knox JD, Szyf M. The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of SAdenosylmethionine. J Biol Chem 2003;278:20812–20. [82] Weaver IC, Champagne FA, Brown SE, Dymov S, Sharma S, Meaney MJ, et al. Reversal of maternal programming of stress responses in adult offspring through methyl supplementation: altering epigenetic marking later in life. J Neurosci 2005;25:11045–54. [83] Meaney MJ, Aitken DH, Sapolsky RM. Thyroid hormones influence the development of hippocampal glucocorticoid receptors in the rat: a mechanism for the effects of postnatal handling on the development of the adrenocortical stress response. Neuroendocrinology 1987;45: 278–83. [84] Meaney MJ, Diorio J, Francis D, Weaver S, Yau J, Chapman K, et al. Postnatal handling increases the expression of cAMP-inducible transcription factors in the rat hippocampus: the effects of thyroid hormones and serotonin. J Neurosci 2000;20:3926–35. [85] Laplante P, Diorio J, Meaney MJ. Serotonin regulates hippocampal glucocorticoid receptor expression via a 5-HT7 receptor. Brain Res Dev Brain Res 2002;139:199–203. [86] McCormick JA, Lyons V, Jacobson MD, Noble J, Diorio J, Nyirenda M, et al. 5 -Heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events. Mol Endocrinol 2000;14:506–17. [87] Cervoni N, Detich N, Seo SB, Chakravarti D, Szyf M. The oncoprotein Set/TAF-1beta, an inhibitor of histone acetyltransferase, inhibits active demethylation of DNA, integrating DNA methylation and transcriptional silencing. J Biol Chem 2002;277:25026–31. [88] Ng HH, Zhang Y, Hendrich B, Johnson CA, Turner BM, ErdjumentBromage H, et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat Genet 1999;23:58–61. [89] Detich N, Theberge J, Szyf M. Promoter-specific activation and demethylation by MBD2/demethylase. J Biol Chem 2002;277:35791–4. [90] Takao T, Tracey DE, Mitchell WM, De Souza EB. Interleukin-1 receptors in mouse brain: characterization and neuronal localization. Endocrinology 1990;127:3070–8. [91] Takao T, Culp SG, Newton RC, De Souza EB. Type I interleukin1 receptors in the mouse brain-endocrine-immune axis labelled with [125I]recombinant human interleukin-1 receptor antagonist. J Neuroimmunol 1992;41:51–60. [92] Takao T, Culp SG, De Souza EB. Reciprocal modulation of interleukin1 beta (IL-1 beta) and IL-1 receptors by lipopolysaccharide (endotoxin) treatment in the mouse brain-endocrine-immune axis. Endocrinology 1993;132:1497–504. [93] Reid TR, Merigan TC, Basham TY. Resistance to interferon-alpha in a mouse B-cell lymphoma involves DNA methylation. J Interferon Res 1992;12:131–7. [94] Fitzpatrick DR, Shirley KM, Kelso A. Cutting edge: stable epigenetic inheritance of regional IFN-gamma promoter demethylation in CD44highCD8+ T lymphocytes. J Immunol 1999;162:5053–7. [95] Northrop JK, Thomas RM, Wells AD, Shen H. Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells. J Immunol 2006;177:1062–9. [96] Falek PR, Ben-Sasson SZ, Ariel M. Correlation between DNA methylation and murine IFN-gamma and IL-4 expression. Cytokine 2000;12:198–206. [97] Kovacs EJ, Oppenheim JJ, Carter DB, Young HA. Enhanced interleukin1 production by human monocyte cell lines following treatment with 5azacytidine. J Leukoc Biol 1987;41:40–6.