Tài liệu Báo cáo khoa học: DNA methylation-mediated nucleosome dynamics and oncogenic Ras signaling pptx

REVIEW ARTICLE DNA methylation-mediated nucleosome dynamics and oncogenic Ras signaling Insights from FAS, FAS ligand and RASSF1A Samir K Patra1,* and Moshe Szyf2 Cancer Epigenetics Research, Kalyani, India Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada Keywords apoptosis; cancer; DNA methylation; epigenetics; FAS; FAS ligand; H-Ras; K-Ras; nucleosome dynamics; RASSF1A Correspondence S K Patra, Cancer Epigenetics Research, Kalyani (B-7 ⁄ 183), Nadia, West Bengal, India Fax: +91 332 582 8460 Tel: +91 943 206 0602 E-mail: skpatra_99@yahoo.com *Present address Division of Biochemistry, Department of Experimental Medicine, University of Parma, Italy (Received June 2008, revised August 2008, accepted 22 August 2008) doi:10.1111/j.1742-4658.2008.06658.x Cytosine methylation at the 5-carbon position is the only known stable base modification found in the mammalian genome The organization and modification of chromatin is a key factor in programming gene expression patterns Recent findings suggest that DNA methylation at the junction of transcription initiation and elongation plays a critical role in suppression of transcription This effect is mechanistically mediated by the state of chromatin modification DNA methylation attracts binding of methylCpG-binding domain proteins that trigger repression of transcription, whereas DNA demethylation facilitates transcription activation Understanding the rules that guide differential gene expression, as well as transcription dynamics and transcript abundance, has proven to be a taxing problem for molecular biologists and oncologists alike The use of novel molecular modeling methods is providing exciting insights into the challenging problem of how methylation mediates chromatin dynamics New data implicate lipid rafts as the coordinators of signals emanating from the cell membrane and are converging on the mechanisms linking DNA methylation and chromatin dynamics This review focuses on some of these recent advances and uses lipid-raft-facilitated Ras signaling as a paradigm for understanding DNA methylation, chromatin dynamics and apoptosis DNA methylation and chromatin modification and remodeling are currently center stage in studies of the epigenetic regulation of genome function in normal physiology, disease states and development [1–25] Several isoforms of enzymes catalyzing both DNA and histone modifications have been characterized Concomitant with differentiation, cell-type-specific patterns of DNA methylation and histone modification are generated and are believed to program cell-type-specific physiological functions, including memory formation in neurons [2,18,20] These elaborate epigenetic programs may be difficult to reverse and rebuild during animal cloning procedures, because the signals and mechanisms for gene-specific hypermethylation and global demethylation patterns are not completely understood [2] In eukaryotes, the chromatin is organized as euchromatin and heterochromatin Euchromatin encompasses the majority of single-copy genes, it replicates during early S phase and contains acetylated histones Heterochromatin is composed of long Abbreviations aSMAase, acid sphingomyelinase; DISC, death-inducing signaling complex; DNMTs, DNA methyltransferases; FADD, FAS-associated death domain; FASL, FAS ligand; gld, generalized lymphoproliferative disorder; lpr, lymphoproliferative disorder; MAPK, mitogen activated protein kinase; MBD, methyl-CpG-binding domain proteins; MGMT, O6-methylguanine methyltransferase; RESE, Ras epigenetic silencing effectors; TNF, tumor necrosis factor FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5217 DNA methylation-mediated nucleosome dynamics S K Patra and M Szyf stretches of DNA repeats, replicates in late S phase and contains lower levels of acetylated histones and higher levels of DNA methylation [1–3,5–7,9,13–17,21– 24] Cytosine methylation is implicated in controlling transcription, maintaining genome stability, parental imprinting and X chromosome inactivation [1,2,21, 24,25] The DNA methylation-mediated repression of several genes, including those encoding proteins involved in cell-cycle regulation and apoptosis, is a major cause of tumor development and cancer progression [1–9] In addition to the gene-specific hypermethylation of several genes in many cancers, genomes of tumor cells are globally hypomethylated and several genes critical for tumor metastasis and progression are activated by demethylation [2,3,6,11–16] The enzymes and cofactors responsible for demethylation in cancer cells are currently unknown It is known, however, that during early development asymmetric DNA demethylation of the paternal genome is observed just after fertilization [2,17–19] Similar to the global hypomethylation observed in cancer, the enzymes responsible for this demethylation are unknown Although the DNA methylation pattern is programmed during development, it remains highly sensitive to both the chemical and social environment [10] Studies have suggested that bioactive components of food, both essential and nonessential nutrients, can modify DNA methylation patterns in complex ways For example, consumption of a methyl-deficient diet led to hypomethylation of specific CpG sites within several oncogenes (such as c-myc, c-fos and c-H-Ras), resulting in high expression of these genes [26] Recent studies have shown that tea catechins are effective inhibitors of human DNA methyltransferase (DNMT)mediated DNA methylation in vitro, and re-expression of a few genes in cultured cancer cells is observed in response to tea catechins [27,28] Thus, DNA methylation may be viewed as an interface between the environment and the human genome It stands to reason that it might play critical role in several human pathologies, in particular age-related disease DNA methylation enzymes In mammalian cells, DNA methylation is catalyzed by two classes of DNMT DNA-methyltransferase-1 (DNMT1; EC 2.1.1.37) is essential for maintaining DNA methylation patterns in proliferating cells and is also involved in establishing new DNA methylation patterns; de novo methylation Members of the second class of methyltransferases, DNMT3a and DNMT3b are required for de novo methylation during embryonic 5218 development [2,25], whereas DNMT3L cooperates with the DNMT3 family to establish maternal imprints in mice [29] DNMT1 and DNMT3B interact among themselves [30] and DNMT3A interacts with histone methyltransferases SETDB1 in the promoters of silenced gene during cancer development [16] Catalytic mechanisms of DNMTs involve the formation of a covalent bond between a cysteine residue in the active site of the enzyme and carbon (C6) of cytosine in DNA The mechanisms involved have been described recently [2,25,31–39] Very recent data suggest that DNMTs may also be involved in the deamination of methylated cytosines to thymines [31,32] The mismatched thymidine is then removed by base ⁄ nucleotide excision repair resulting in repair to an unmethylated cytosine [2] This has been proposed to serve as a mechanism for dynamic DNA methylation [31,32] A different type of DNA methyltransferase is O6-methylguanine DNA methyltransferase (MGMT; EC 2.1.1.63) This enzyme does not methylate DNA but is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine O6-Methylguanine often mispairs with thymine during replication Following DNA replication this would result in conversion of a guanine–cytosine (GC) pair to an adenine–thymine (AT) pair Thus, repairing O6-methylguanine adducts is essential for the integrity of the genome Interestingly this DNA methyltransferase is regulated by DNA methylation Hypermethylation of the MGMT promoter is associated with loss of MGMT expression ⁄ function in many tumor types [1,4,40] MGMT hypermethylation is an example of the emerging field of pharmacoepigenomics The impact of chemotherapy would be dependent on the epigenetic state of cardinal genes such as MGMT [1,41] Knowing the state of methylation of critical repair genes is critical for the proper planning of a chemotherapeutic protocol Removal of the methyl group (MeC-DNA demethylation) from critical positions in promoters is essential for the transcription of many genes A long line of evidence suggests that active enzymatic MeC-DNA demethylation occurs in nonreplicating cells to induce the transcription of specific genes at distinct time points [2] The mechanisms of demethylation are unknown and the enzymes involved are not firmly established One possible mechanism is through formation of a cytosine–Michael adduct ⁄ complex with MBD2 protein but this would require a cofactor [2,42– 48] which is not known The current notion of Michael adduct chemistry is that in such types of complex the SN2 mechanism would not occur In principle, water added across the 4C–5C double bond with the FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS S K Patra and M Szyf hydroxyl group attacking carbon 4, followed by elimination of ammonia will yield thymidine [2,25,34,46] Epigenetic consequences of DNA modifications – nucleosome dynamics There is a bilateral relationship between DNA methylation and chromatin structure [2,16,49–58] Promoters of genes and important regulatory sequences are associated with hyperacetylated histones, whereas silent genes are associated with hypoacetylated histones Acetylated histones are associated with unmethylated DNA and are rarely present in methylated DNA regions [59] In addition to histone acetylation, which plays a critical role in gene regulation, other histone modifications such as methylation, phosphorylation and ubiquitination play a similar role in regulating genome functions [49–56] A combinatorial arrangement of these modifications is believed to constitute a ‘histone code’ Methylation of DNA and deacetylation of histones H3 and H4, combined with methylation of K27 residue on the H3-histone tail in upstream regulatory regions leads to inactivation ⁄ repression of gene expression, whereas selective acetylation of histones H1, H3, H4, methylation of H3K4 and DNA demethylation are associated with activation of transcription [2,5,6,12,16,22,23] (Fig 1) How does DNA methylation signal for repression of transcription? Repression of transcription may occur through different mechanisms One simple mechanism is that DNA methylation interferes with the binding of transcriptional activators [24,25,53,55,59] A second mechanism involves recruitment of methyl-CpG-binding domain proteins (MBDs), such as MBD1, MBD2, MBD3, MBD4 and MeCP2 MBDs recruit co-repressor complexes to methylated genes, which include histone-modifying enzymes such as histone deacetylases and histone methyltransferases precipitating an inactive chromatin structure [16,21,39,49,51,56] This mechanism provides an explanation for the correlation between DNA methylation and inactive chromatin configuration DNA methylation and histone modification act in concert to program gene expression Figure presents a model of the inhibition of gene expression by DNA methylation Cytosine methylation at the DNA sequence d(GGCGCC)2 triggers an extended eccentric double-helix structure called E-DNA Like B-DNA, E-DNA has a long helical rise and the base is perpendicular to the helix axis The 3¢-endo sugar conformation provides the characteristic deep major groove and shallow minor groove of A-DNA [60] Analysis of the hydration pattern around methylated CpG sites in DNA methylation-mediated nucleosome dynamics crystal structures of A-DNA decamers at three high ˚ resolutions (1.7, 2.15 and 2.2 A) reveals that the methyl groups of cytosine residues are well hydrated with a higher amount of Mg2+ in their vicinity [61], which facilitates the interaction of MBD proteins and chromatin remodeling machines with the MeCpG sites The MBD–MeCpG complex then brings about deacetylation of histones H3 and H4 [2,22] by recruiting class I histone deacetylases, which may be co-recruited with DNA-topoisomerase II [62] (Fig 1) Indeed, it has been shown experimentally that methylation of DNA brings about general deacetylation of histones H3 and H4, prevents methylation at H3K4 and induces methylation of H3 K9 [2,52–56] Histone H3K4 trimethylation is associated with transcriptionally active genes [59,63–73] MeCP2 has also been shown to recruit the histone methyltransfaerase SUV39 which targets H3 K9 [74] Okitsu & Hsieh observed a tight correlation between depletion of H3K4Me2 and regions of DNA methylation, and proposed that DNA methylation dictates a closed chromatin structure devoid of H3K4Me2 [59] The recent discovery of histone demethylases has challenged the originally held belief that histone methylation is static Histone demethylases specific for mono-, di- and trimethylated histone H3K4 are now known and their structures have been described [54,70–73,75–78] It is possible that the recently discovered histone demethylase LSD1 also participates in maintaining methylated regions of DNA devoid of H3K4 methylation [56,78] DNA methylation immediately downstream of the transcription start site has a dramatic impact on transcription, affecting transcription elongation rather than initiation Recent findings suggest that DNA methylation at the junction of transcription initiation and elongation is most critical in transcription suppression and this effect is mechanistically mediated through chromatin structure [53,56,59,78] Although some important ideas have been suggested in other studies [64,68–70], it is still difficult to predict the effect of methylated DNA segments on transcription because differences in the size and position of the methylated DNA regions may differentially affect transcription For example, although a methylated coding region positioned kb downstream of the promoter has little impact on transcription initiation, as observed by Lorincz et al [65], the same methylated sequence might have a much larger impact on transcription initiation if positioned immediately downstream of the promoter [54,64,68–70,78] The context seems to be critical [56] Recent data suggest that not only can DNA methylation direct the formation of inactive chromatin structure, but also that histone signals can direct DNA FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5219 DNA methylation-mediated nucleosome dynamics S K Patra and M Szyf Fig Cytosine base in DNA – the amazing switch for the regulation of gene expression and chromatin remodeling Cytosine, extended from the sugar-phosphate backbone (black circles–pink lines) and expanded manifold beyond the scale, is the only base in mammalian chromosomes which is stably modified by methylation at the carbon-5 position (formation of -MeCpG-) after replication DNA methylation inhibits gene expression affecting chromatin structure [2,6,22,59–71], because the presence of methyl groups on DNA affect the structure of DNA and the interaction of other proteins and enzymes with local nucleosomes [2,60] Methylation of DNA (MeCpG-) brings about a general hydration of DNA [61], which facilitates the methyl-CpG-sequence binding (MBD) proteins to recognize the MeCpG- sites in nucleosomes for remodeling into a repressive complex DNMT-MeCpG- influence deacetylation of histones H3 and H4 by recruiting class I histone deacetylases (HDACs); prevents methylation at H3K4, and induce methylation of H3 K9 in eukaryotes [56–69] HDACs may be co-recruited with DNA-topoisomerase II [62] Histone H3K4 methylation is associated with transcriptionally active nucleosomes of chromatin in which K4 of H3 are trimethylated, whereas H3 K27 methylation is associated with inactive chromatin [56,59,63–68,79] Methylation of histones is reversible and histone demethylases specific for di- and trimethylated histone H3K4 are discovered; for example, LSD1 represses transcription through demethylation of H3K4 Me3 [72,75,77] Okitsu & Hsieh [59] observed a tight correlation between the depletion of H3K4Me2 in the regions of DNA methylation Conversely, the level of H3K4Me2 remains high in the unmethylated DNA regions regardless of the presence of RNA Pol II It can be proposed that MeCpG- dictates a closed chromatin structure that is devoid of H3K4Me2 and inhibits transcription, and that the presence of H3K4me2 marks an open chromatin structure that would permit transcription if all other conditions for active transcription are fulfilled [56,58] In early development, genomic methylation is erased and the somatic methylation pattern is re-established at the time of implantation The initiation of DNA demethylation-dependent nuclear processes is highly dependent on unfolding of chromatin structure In this context, acetylation of lysine ⁄ arginine of histone tails of H3 and ⁄ or H4 at the respective MeCpG-rich nucleosome depends on histone acetyl transferases (HATs) [48–50] In addition to methylation, H3 K9, H3 K14, H3 K23 and H3 K27 are also prone to acetylation, whereas as H3 K18 is only acetylated [22,49–59,63–69] This implies that nucleosome position is biased by the DNA sequence to facilitate access to initiation factors and activators by hundreds of histone modification (deacetylation, demethylation and also phosphorylations at serine ⁄ threonine residues) Also, activation of specialized domains by removal of loosely associated mobile proteins, including HMG, HP and H1, partly regulates the expression of independent genes modulating the access of the above factors [2,22] Note: all the modifications mentioned here are not require for activation of a particular type of gene Histone decoding, DNA modifications and the accessory factors are predominantly dependent on the types of signals a cell receives for activation ⁄ repression of a specific gene or for a particular class of gene 5220 FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS S K Patra and M Szyf methylation For example, methylation of tumor suppressor genes in cancer usually occurs in regions of DNA associated with H3-histone K27 methylation, a suppressive histone mark [2,12,22,54] The histone methyltransferase EZH2 recruits the DNA methyltransferase to targets of EZH2 in the genome [1] Thus, there is a bilateral relationship between DNA methylation and inactive chromatin configuration DNA demethylation is also tightly associated with chromatin structure; histone acetylation of H3 and H4 histone tails is a hallmark of active chromatin configuration and transcriptionally active regions of the genome [22,48,52,53,55,57–59] Hypomethylation of DNA [2,11,14] is found in regions associated with hyperacetylated histones, and pharmacological histone acetylation could induce DNA demethylation [48,79–81] Thus, DNA demethylation, like DNA hypermethylation, has a bilateral relationship with chromatin modification Ras oncogenes and oncoproteins Ras, Rho, Rab, Arf and Ran are the five major classes of monomeric GTPases whose biological functions are regulated by the Ras family GTPases The cellular Ras oncogene encodes a 21-kDa guanine nucleotide-binding protein, which plays a role in the regulation of growth and differentiation in eukaryotic cells [1,82] Despite profound improvements in our understanding of the molecular and cellular mechanisms of action of the Ras proteins, the expanding list of downstream effectors and the complexity of the signaling cascades that they regulate suggest that much remains to be learnt [83] The study of Ras proteins and their functions in cell physiology has led to many insights not only into tumorigenesis but also into many developmental disorders [82–84] Although Ras binds both GDP and GTP with very high affinity, the GTP-bound form is active and the GDP-bound form is inactive The rate of intrinsic nucleotide exchange and GTPase activity is very slow Ras–GDP predominates in resting cells, but when Ras is activated, specific guanine nucleotide exchange factors enhance nucleotide exchange, increasing the Ras–GTP complex Ras–GTP then activates downstream effectors such as Raf-1 GTPase-activating protein, however, causes the precipitation and accumulation of inactive Ras–GDP within a cell and may deregulate cellular physiology when overexpressed [1,82–90] The retroviral oncogene, V-Ras, encodes a protein that differs from the C-Ras product by a point mutation that maintains this Ras protein constitutively active [83] The Ras-related GTPase, Rho is required DNA methylation-mediated nucleosome dynamics for transmission of a proliferative signal by Ras If Rho is inhibited, the constitutively active Ras induces the cyclin-dependent kinase inhibitor p21(Waf1) ⁄ Cip1, which blocks entry into the DNA synthesis phase of the cell cycle Rho activity suppresses induction of p21(Waf1) ⁄ Cip1 by Ras, thus overcoming the block on entry into the S phase of the cell cycle Cells lacking p21(Waf1) ⁄ Cip1 activity not require Rho for the induction of DNA synthesis by activated Ras [1,83] Lipid rafts and Ras signaling Palmitoylation of N-Ras, K-Ras-4A and H-Ras, but not K-Ras-4B, in their C-terminal hypervariable regions is commonly required for their membrane relocation After relocation to the membrane, H-Ras and K-Ras-4A are translocated to lipid rafts; however, K-Ras-4B remains in the non-raft portion of the membrane [83] Hypervariable regions are responsible for targeting the isoforms to different microdomains in membrane Ras proteins localize to different plasma membrane microdomains, lipid rafts, formed by segregation of lipids based on their dissimilar biophysical properties [83,91] A comprehensive model of how Ras proteins are clustered for amplification, internalized and transmit their signals has recently been proposed [1] Eisenberg et al [92], employing fluorescence recovery after photobleaching, demonstrated coupling between membrane domains (rafts) in the external and internal leaflets of the plasma membrane and showed that this coupling modulated transbilayer signal transduction Ras circulates between the Golgi, the endoplasmic reticulum and the plasma membranes H-Ras localized in the membranes of the endoplasmic reticulum and Golgi apparatus is activated by epidermal growth factor [1,91–93] After post-translational modification in the endoplasmic reticulum, K-Ras is transported to the plasma membrane by a desorption–absortion mechanism [92,94] Ras detachment from lipid rafts requires GTP hydrolysis [92,93] H-Ras and N-Ras are transported to different sub-compartments by vesicular traffic, or by a nonvesicular pathway involving a constitutive deacylation–reacylation cycle [1,83,92,95–97] Inter-relationship of genetics and epigenetics in Ras oncogenic signaling There is a bilateral relationship between genetic and epigenetic mechanisms in the activation of oncogenic Ras signaling Activation of K-Ras and H-Ras in human cancers results in DNA hypermethylation FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5221 DNA methylation-mediated nucleosome dynamics S K Patra and M Szyf of target genes [1,6,31,83,98] Epigenetic deregulation of critical repair genes can, however, increase the rate of mutation of Ras genes (Fig 2) For example, silencing of the repair methyltransferase MGMT would result in an increased rate of mutation of Ras and other oncogenes Figure represents a scheme of how the loss of MGMT expression would result in G to A transitions in the K-Ras oncogene and in p53 [1,41,99] Indeed, MGMT promoter hypermethylation is significantly pronounced in tumours that also bear a G to A mutation in p53 suggesting a link between epigenetic and genetic events and apoptosis related diseases [99] Genetic activation of Ras would also cause a change in the state of methylation of several genes Constitutive activation of Ras induces DNMT1 expression at the transcriptional level through activation of cJUN [100–102] The excess of unscheduled DNMT levels would target certain genes for hypermethylation It is believed that promoters marked by H3-K27 methylation are targets of hypermethylation perhaps through recruitment of DNMTs by EZH2 [103,104] Indeed, targeting the Ras signaling pathway by drugs such as methotrexate and inhibitors of ERK ⁄ mitogen activated protein kinase (MAPK) decreases DNA methylation in malignant hematologic diseases and colon cancer cells indicating a causal relationship between Ras signaling and DNA methylation [105–108] Ras activation would affect the DNA-methylation state and chromatin dynamics in the other direction as well Expression of v-H-Ras in mouse embryonal P19 cells resulted in genome-wide demethylation of certain genes, including a skeletal muscle-specific gene, adrenal cortex (c21)-specific gene, ubiquitous genes and exogenously introduced sequences [100,109] Hence, DNA demethylase might be a potential downstream effector of Ras signaling [1,2] Also, stimulation of the Ras– MAPK pathway leads to chromatin modification by histone H3 serine 10 and 28 phosphorylation in an acetylation-dependent and -independent fashion [55] In summary, activation of the Ras-signaling pathway would trigger the methylation aberrations and histone modifications that are a hallmark of cancer: regional DNA hypermethylation and global hypomethylation An attractive hypothesis is that K-Ras or H-Ras signals originating from the membrane at different lipid raft-anchored Ras pools would have distinct effects on DNA methylation and demethylation [1,2,91,92], and histone phosphorylation and acetylation machineries [55] Interestingly, this relationship between lipid rafts, Fig Epigenetic silencing of repair genes affects genetics O6-methylguanine DNAmethyltransferase (MGMT) gene silencing through promoter methylation demonstrates how the loss of MGMT expression results in G to A transitions of the K-Ras oncogene (the most frequently mutated isoform of Ras), and of p53 [1,5,6,41,99] MGMT, a DNA repair protein, removes mutagenic and cytotoxic adducts from the O6 position of guanine [41,183] O6-methylguanine often mispairs with thymine during replication, and it results in conversion from a guanine– cytosine (GC) pair to an adenine–thymine (AT) pair if the adduct is not removed 5222 FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS S K Patra and M Szyf Ras and epigenetic states might be bilateral as well because many lipid raft component encoding genes are known to be regulated by DNA methylation [2,91,110] FAS, FAS ligand, FAS-associated death domain and lipid raft-mediated FAS signaling FAS-triggered apoptosis is another critical process which is an effector of Ras signaling and is tightly associated with epigenetic deregulation The 36 kDa cell surface cytokine receptor, FAS (TNFRSF6 ⁄ FAS ⁄ APT1 ⁄ APO1 ⁄ CD95, OMIM 134637) contains a 16-amino acid signal sequence followed by a mature protein of 319 amino acids that contains a solo transmembrane domain and two specialized functional domains; a FAS death domain and a FAS ligand (FASL) binding domain [111] FAS-associated death domain (FADD, OMIM 602457) protein is the universal adaptor-protein for apoptosis This FADD mediates signaling of all known death domain-containing members of the tumor necrosis factor (TNF) receptor superfamily [112] The FADD gene contains two exons and spans  3.6 kb [113] Northern blot analysis revealed that FADD was expressed as a 1.6-kb mRNA in many fetal and adult tissues [114] The death domain of FADD is 25–30% identical to those of FAS and the TNF receptor, TNFR1 (OMIM 191190) Natural ligands, cognate agonist antibodies and interactions of FADD with FAS at their respective death domains trigger apoptosis through FAS and TNFRI High expression of FADD in mammalian cells induces apoptosis, which can be blocked by Crma, a Pox-virus gene product that also blocks FAS-induced apoptosis [115,116] The FAS protein shows structural homology with a number of cell surface receptors, including TNFR1 and the low-affinity nerve growth factor receptor It has been shown that following activation of T cells, the FAS receptor is rapidly induced The interaction between FAS and FASL induces cell death that occurs in a cell-autonomous manner, similar to the classic apoptotic sequence [117,118] FAS activates caspase by inducing the cleavage of the caspase zymogen to its active subunits and by stimulating the denitrosylation of its active site thiol [119] Myc-induced apoptosis requires interaction between FAS and FASL on the cell surface [120] Hueber et al established the dependence of Myc on FAS signaling for its potent cell killing activity [120,121] The pathway leading to apoptosis by FAS cross-linking with FASL results in the formation of a death-inducing DNA methylation-mediated nucleosome dynamics signaling complex (DISC) composed of FAS, the signal adaptor protein FADD, and procaspase and 10, and the caspase ⁄ 10 regulator C-FLIP [91,122,123] Yeh et al [115] proposed that the interaction of FADD and FAS through their C-terminal death domains unmasks the N-terminal effector domain of FADD, allowing it to recruit caspase (CASP-8; 601763) to the FAS signaling complex This results in activation of a cysteine protease cascade, which leads to cell death Apoptosis triggered by infection, radiation or chemotherapeutic drugs is also mediated by FAS This process involves modification, placement in membrane, aggregation in lipid rafts and internalization of the FAS–DISC complex [91,124–130] Internalization of FAS with either lipid rafts or an endosomal compartment may determine which signaling pathways are involved When internalization of FAS is blocked, the receptor cannot induce apoptosis and instead remains fully engaged, most probably in activating nonapoptotic ⁄ proliferative pathways [112,115,125,127,131] Inter-relation of genetic and epigenetic alterations in cancer It is now evident, as discussed above, that changes occurring in cancer cells, including chromosomal instability, an increased propensity for mutation, activation of oncogenes, silencing of tumor suppressor genes and inactivation of DNA repair systems are a result of both genetic and epigenetic abnormalities The correlation between the status of -CpG-island hypermethylation and ⁄ or mutations in critical genes shows that, for virtually every tumor type, both genespecific hypermethylation and distinct genetic alterations over time are major driving forces in neoplastic development But naturally occurring mutations of specific genes in somatic cells are infrequent, because under normal conditions maintenance of genomic integrity is guarded by a complex array of DNA monitoring and repair enzymes Karyotypic order is also guaranteed by other molecular guards, such as cell-cycle check-points that operate at critical times during mitotic division Together, these systems ensure that mutations are rare events, so rare indeed that the multiple mutations known to be present in tumor cells, which are necessary for cancer progression, are low probability events within a normal human life span However, during oncogenesis epigenetic silencing of genes encoding DNA repair proteins (for example, MGMT) may cause retention of mutants as well as encourage neo-mutants [1–6,41] The FAS apoptotic pathway is one of the most promising targets of this process FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5223 DNA methylation-mediated nucleosome dynamics S K Patra and M Szyf FAS mutations in cancer and other diseases Several lines of evidence have highlighted that perhaps all tumor cells express FAS, but in many cases the gene is mutated encoding a nonfunctional protein Some cancers such as papillary thyroid carcinoma, however, express functional FAS [132] Analysis of the entire FAS coding region in micro-dissected biopsy samples from 21 burn scar-related squamous cell carcinomas revealed somatic point mutations in all of the splice sites from three patients [133] The mutations were located in all three domains of the protein: the death domain, ligand-binding domain and transmembrane domain of the FAS gene Analyses of the other FAS alleles in tumors carrying the N239D and C162R mutations indicated loss of heterozygosity, and expression of FAS was confirmed in all tumors with FAS mutations [91,133] In contrast to the situation in burn scar-related squamous cell carcinoma, no mutations were detected in 50 cases of conventional squamous cell carcinoma [133] This difference in mutation of the FAS gene is interesting because burn scar-related squamous cell carcinoma is usually more aggressive than conventional squamous cell carcinoma It was therefore suggested that somatic mutations in the FAS gene may contribute to the development and ⁄ or progression of burn scar-related squamous cell carcinoma The following mutations in the gene encoding FAS were identified: a 957A-to-G transition resulting in an N239D substitution in the FAS death domain; a 547A-to-G transition resulting in an N102S substitution in the FAS ligand-binding domain; a 726T-to-C transition resulting in a C162R substitution in the FAS transmembrane domain [133] Zhang et al [134] genotyped 1000 Han Chinese lung cancer (211980) patients and 1270 controls for two functional polymorphisms in the promoter regions of the FAS and FASL genes, -1377G-to-A (134637.0021) and -844T-to-C (134638.0002), respectively Compared with noncarriers, there was an increased risk of developing lung cancer for carriers of either the FAS -1377AA or the FASL -844CC genotype; carriers of both homozygous genotypes had a more than fourfold increased risk [134] Their results further support the concept that the inactivation of FAS- and FASL-triggered apoptosis pathway plays an important role in human carcinogenesis [91,135] A heterozygous mutation in the FAS gene in five unrelated children (134637.0001–134637.0005), with a rare autoimmune lymphoproliferative (lpr) syndrome was identified by Fisher et al [136] The disease is 5224 characterized by massive nonmalignant lymphadenopathy, heightened autoimmunity and expanded populations of TCR-CD3(+)CD4())CD8()) lymphocytes, resulting from defective FAS-mediated T-lymphocyte apoptosis While delineating the prognostic markers for the disorder, Sneller et al [137] further analyzed one of the patients studied by Fisher et al., and pointed out its resemblance to autosomal recessive lpr ⁄ gld (generalized lymphoproliferative disorder) mouse The lpr and gld mice bear mutated genes for FAS and FASL, respectively The murine autosomal recessive lpr phenotype is characterized by lymphadenopathy, hypergammaglobulinemia, multiple autoantibodies and the accumulation of large numbers of nonmalignant CD4, CD8 and T cells Affected mice usually develop a systemic lupus erythematosus-like autoimmune disease, and a defect in the negative selection of self-reactive T lymphocytes in the thymus The mouse lpr phenotype is identical to the phenotype displayed by human patients bearing mutated FAS [138,139] Epigenetic downregulation of apoptosis – the role of the Ras signaling pathway In addition to genetic mutations in FAS as discussed above, FAS is silenced by epigenetic mechanisms in several cancers Several lines of evidence suggest that the trigger for methylation of the FAS gene is activation of the Ras fi Raf fi MEK fi ERK fi Elk signaling pathway [1,91,98] Methylation of the FAS gene is associated with loss of FAS expression in antigen-specific cytotoxic T cells [140] There is evidence for involvement of DNA methylation in silencing of FAS–FASL signaling and loss of apoptosis [141] Silencing of FASL and TRAIL-R1, TRAIL-R2 and Caspase-8 expression by DNA methylation has been linked to resistance of small cell lung cancer cells to FASL and TRAIL induced apoptosis [142] FAS promoter methylation in prostatic and bladder carcinomas and respective cell lines correlates with downregulation of FAS expression [143] H-Ras is linked to the silencing of FAS-triggered apoptosis through DNA methylation Peli et al [144] reported that oncogenic H-Ras downregulated FAS by DNA methylation It was suggested that the phosphatidylinositol 3-kinase pathway was involved in mediating this effect of RAS The involvement of phosphatidylinositol 3-kinase points to the possibility that some of the known anti-apoptotic effects of PKB ⁄ Akt kinase may be mediated, at least in part, by the downregulation of FAS expression through DNA FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS S K Patra and M Szyf methylation [144] It remains to be seen whether this effect of the Ras signaling pathway on DNA methylation is brought about by increase of DNMT1 as previously reported [102] or through activation of factor(s) that recruits DNMT to specific targets such as apoptosis The epigenetic downregulation of apoptosis pathways involves additional genes in various types of tumors In neuroblastomas and neurobalstoma cell lines, which are resistant to apoptosis induced by TRAIL, CASP-8 and the FLIP gene, and in tissues adjacent to tumors the CASP-8 gene is hypermethylated [145] The FLIP protein is a negative regulator of CASP-8, and the methylation of CASP-8 and FLIP genes is somewhat correlated [91,125,145] Mechanisms of silencing of FAS in response to Ras activation How does Ras activation cause methylation and epigenetic silencing of FAS and other apoptosis-related genes? Activated Ras epigenetically silences FAS expression in mouse NIH3T3 cells [143], and in human K-Ras transformed cell line, HEC1A [98] Twentyeight ‘Ras epigenetic silencing effector’ (RESE) genes were discovered in a genome-wide functional screen [98] Nine RESEs were found to be bound to different regions of the FAS promoter in K-Ras-transformed NIH3T3 cells [98], whereas in nontransfected NIH3T3 cells only one RESE (NPM2) was associated with the FAS promoter It was therefore proposed that these nine RESEs were recruited to specific regions of FAS promoter in response to expression of oncogenic K-Ras and are involved in the recruitment of DNMT1 and other chromatin modifiers to the promoter, resulting in DNA methylation and epigenetic silencing In support of this hypothesis, knockdown of any of the 28 RESEs in K-Ras-transformed NIH3T3 cells resulted in an absence of DNMT1 on the FAS promoter, demethylation of the FAS promoter and induction of FAS expression What are the biochemical and cellular functions of other RESEs? Among the 28 RESE proteins discovered using a functional genomics approach there are transcriptional activators and repressors (CTCF, EID1, E2F1, RCOR2 and TRIM66 ⁄ TIF1D), sequence-specific DNA-binding proteins (SOX14, ZCCHC4 and ZFP345B), histone methyltransferases (DOT1L, EZH2 and SMYD1), histone deacetylase (HDAC9), histone chaperones (ASF1A and NPM2), DNMT1 and several Polycomb group proteins (BMI1, EED and EZH2) Several recent studies have linked Polycomb proteins to abnormal DNA methylation and DNA methylation-mediated nucleosome dynamics gene silencing [1,146–148] It is surprising that one of the nuclear RESEs is BAZ2A ⁄ TIP5, previously known to be involved only in repression of RNA polymerase I-directed ribosomal gene transcription [149] A number of RESEs were substantially upregulated at the transcriptional or post-transcriptional level in K-Ras-transformed NIH3T3 cells compared with nontransformed NIH3T3 cells, explaining at least in part, how K-Ras activates this silencing pathway (Fig 3A) Treatment of K-Ras NIH3T3 cells with the demethylating drug 5-aza-CdR resulted in FAS re-expression, supporting the hypothesis that FAS is regulated by DNA methylation in Ras-transformed cells [98] Epigenetic inactivation of the RAS effector homolog RASSF1 Genes encoding human RAS effector homolog, RASSF1 (OMIM, 605082) family proteins, along with several putative tumor-suppressor genes are located at chromosome 3p21 [83,150–152] RASSF1 produce eight transcripts, A–H, derived from alternative splicing and promoter usage [152–154] The RASSF1 gene contributes to the spatiotemporal regulation of mitosis through a number of regulatory mechanisms that cooperate to restrict the activity of APC ⁄ C to a specific period in the cell cycle [153–155] Mechanistic roles for RASSF1A in inducing apoptosis in cancer cells and solid tumors are emerging [156–158] RASSF1A function was missing in a variety of solid tumors and cancer cell lines, including small cell lung cancer and prostate [150–156] DNA methylation of the CpG island promoter sequence of RASSF1A was implicated in its silencing [16,155] RASSF1A is the most frequently methylated gene in both primary tumors and cell lines and in a group of nine genes mapped in 175 primary pediatric tumors and 23 tumor cell lines RASSF1A methylation was tumor specific and absent in adjacent nonmalignant tissues [157] RASSF1A gene silencing is also associated with aberrant methylation and histone deacetylation in a variety of other cancers [158–163] RASSF2 methylation and inactivation is a consequence as well of K-Ras-induced oncogenic transformation [164] Apart from Ras-regulated methylation of RASSF1A, Ras and RASSF1A have direct physical interaction in cellular physiology [155,160] Lipid raft facilitated Ras signaling and chromatin modification Clustering of raft-associated receptors, like epidermal growth factor receptor, facilitates the early step of FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5225 DNA methylation-mediated nucleosome dynamics 5226 S K Patra and M Szyf FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS S K Patra and M Szyf DNA methylation-mediated nucleosome dynamics Fig Epigenetic signaling for proliferation or death emanating from the plasma membrane microdomains (A) Lipid raft-dependent (H-Ras and K-Ras-4A) and -independent (K-Ras-4B) signaling A model scheme proposed for the modulation of transbilayer signaling by clustering of raft protein H-Ras, in which external clustering (antibody or ligand mediated) enhances the association of internal leaflet proteins with the stabilized clusters, promoting either enhancement or inhibition of signaling Clustering raft-associated epidermal growth factor receptor proteins may facilitate the early step, whereby H-Ras is converted to an activated, GTP-loaded state but inhibit the ensuing step of downstream signaling via the MEK ⁄ ERK pathway GTP hydrolysis releases the raft-resident H-Ras [83,90–93] which ensures one of the downstream proliferative signals for DNA methylation-associated repression of cell-cycle control and pro-apoptotic genes (e.g FAS) [1–4] (B) Radiation- or drug-induced activation of genes Chemotherapeutic drugs like aplidine, edelfosin and green tea catechins (for example, EGCG) affect membrane raft composition, on the one hand [91,172–178] and the DNA-methylation status [26–28] of many genes (e.g FAS and CLU), on the other hand EGCG can bind to GpC-rich regions of DNA Radiation can damage the respective genes, thereby inviting repair enzymes [2] This facilitates repair-based DNA demethylation [2,47] and the induction of genes for transcription For exampe, FAS gene is known to be repressed by DNA methylation, for which a signal is transduced by H-Ras (Fig 3A) and radiation can induce FAS in association with aSMAse Translocation of FAS and aSMAse to membrane domain lipid rafts affects lipid-raft composition Hydrolysis of sphingomyeline by aSMAse produces ceramide This in situ produced ceramide displaces cholesterol from cholesterol rafts transforming them into ceramide rafts [91], which predominantly transmit death signals [128–130,172–179] All the components in the figures are not drawn to the same scale For example, lipid rafts in membranes are drawn as circles many fold larger than the membrane leaflet H-Ras conversion to an activated, GTP-loaded state GTP hydrolysis releases the raft-resident H-Ras [83,90–93] eliciting downstream signals for DNA methylation-mediated repression of cell-cycle control and pro-apoptotic genes (for example, FAS) [1–4] Activation of the Ras–MAPK pathway stimulates histone H3, S10 and S28 phosphorylation and nucleosome remodeling and gene expression ⁄ repression by acetylation-dependent and -independent mechanisms [165– 168] The location of the serine 10 residue in close proximity to other modifiable amino acids in the histone H3 tail suggests a possible interaction between phosphorylation of serine 10 and methylation and ⁄ or acetylation of lysine and lysine 14 [165,168] Visualization with indirect immunofluorescence shows most foci of phosphorylated H3 S28 did not co-localize with foci of H3 phosphorylated on S10 or S10 and K14, suggesting that these phosphorylation events act independently [166,167] Lipid rafts are not just modulators of the epigenetic response to chemotherapy [169–175], but also targets of chemotherapeutic drugs known to block DNA methylation [27,28] Radiation therapy also modulates both the size and composition of lipid rafts [129,130,176] and induces DNA demethylation-mediated expression of genes [177,178] The inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is shown experimentally to be brought about by altering the lipid order of rafts in HT29 colon cancer cells [169] Small drugs, including edelfosine, perifosine, ether lipid ET-18-OCH(3) and aplidine alter cytoskeleton-mediated FAS and FASL concentrations in lipid rafts These rafts form apoptosis-promoting clusters in cancer chemotherapy [170–175] Thus, there is a relationship between lipid rafts, Ras–MAPK signaling and the response to chemotherapeutic agents Activation of Ras in lipid rafts triggers DNA methylation and the silencing of repair and apoptotic genes The FAS gene is repressed by DNA methylation transduced by H-Ras and K-Ras signaling that is facilitated by lipid rafts, on the one hand [98,144] (Fig 3A), and radiation ⁄ chemotherapy can cause demethylation-induced FAS expression, on the other hand (Fig 3B) Eventually, demethylation also results in activation of the gene encoding acid sphingomyelinase (aSMAase) Translocation of the increased levels of aSMAase to membrane lipid rafts [128,129] lead to the transformation of the proliferative cholesterol raft to a death-inducing ceramide raft and is associated with FAS–DISC internalization and eventual cell death [91] It is therefore important whether radiation ⁄ druginduced changes in lipid raft composition transmit the altered Ras-MAPK signal that causes eventual FAS demethylation and expression, or whether radiation ⁄ drug-induced DNA repair-based FAS expression, post-translation FAS modification and simultaneous translocation with aSMAase to raft domains cause changes of lipid raft composition (compare Fig 3A,B) One therapeutic implication of the complex relationship among lipid rafts, RAS signaling and epigenetic modulation is that it might be possible to identify new strategies to combine inhibitors of Ras-mediated epigenetic silencing of cell cycle and damage repair genes and chemotherapeutic agents [179–191] By understanding the pathway leading from membrane lipid rafts to DNA hypermethylation it would be possible to dissect points which could be targeted by pharmacological inhibitors For example, mouse embryonic stem cells have been shown to be hypersensitive to apoptosis triggered by DNA damaging agents due to the high activity of E2F1-regulated mismatch repair [190,191], FEBS Journal 275 (2008) 5217–5235 ª 2008 The Authors Journal compilation ª 2008 FEBS 5227 DNA methylation-mediated nucleosome dynamics S K Patra and M Szyf suggesting that induction of repair enzymes would lead to hypersensitization to chemotherapeutic agents Conclusion and perspectives We have discussed data supporting DNA methylationmediated chromatin dynamics and described how one of the signals for reversible DNA methylation is transmitted by Ras oncoproteins We also developed the hypothesis that the Ras signal for DNA methylation emanates from the membrane and is coordinated by lipid rafts One or more components of the potent cell killing machinery, including FAS, FASL, FADD and RASSF1 genes are often repressed by DNA methylation in carcinogenesis in response to activation of the Ras signaling pathway The silencing of repair genes by methylation has consequences for the genetic integrity of cells, as well as for the responsiveness of cells to chemotherapeutic agents There is a bilateral relationship between genetic lesions and epigenetic aberrations Epigenetic silencing of repair genes such as MGMT could lead to elevated levels of Ras mutations, and Ras-activating mutations turn on downstream signaling, resulting in epigenetic silencing There is also a bilateral relationship between chemotherapeutic agents and epigenetic states Chemotherapeutic drugs can cause demethylation and activation of repair genes, whereas methylation or demethylation would alter responsivity to chemotherapeutic agents In contrast to genetic alteration, epigenetic marks are reversible and thus the epigenetic consequences of Ras-mediated FAS modulations could be targeted therapeutically The signals for aberrant DNA methylation by H-Ras and K-Ras 4A and 4B may be transmitted through different locations in membranes We suggest that future studies with Ras-regulated chromatin dynamics and DNA modification should focus on the following issues: (a) dissection of lipid raft-dependent and -independent RAS signaling for DNA methylation; (b) determine the relationship between chromatin activation and nucleosome opening in response to radiation and chemotherapeutic drugs, and signals from plasma membrane microdomains to the nucleus; and (c) search for gene-specific extinguishers of Ras-triggered aberrant DNA demethylation and hypermethylation Acknowledgements This review is dedicated (by SKP) to Dr MK Pal, retired professor of biochemistry, University of Kalyani and Dr D Chattopadhyay, professor of biochemistry, molecular biology and biotechnology, University of Calcutta, India Work from the laboratory of MS is 5228 supported by grants from the National Cancer Institute of Canada and the Canadian Institute of Health Research MS is a fellow of the Canadian Institute for Advanced research We apologise for many other important contributions that we have not been able to include and 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