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Cell Growth & Differentiation Vol. 13, 149-162, April 2002
© 2002 American Association for Cancer Research


Review

Methylation Matters: Modeling a Manageable Genome1

Jared M. Ordway and Tom Curran2

Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105


    Introduction
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 
"Chromatin can’t be important otherwise bacteria would have it." —Comment made at a transcription meeting.

Transcriptioncontrol was once an understandable topic. The prevailing view was that transcription factors sought out and bound to specific DNA sequences, thereby introducing activators or repressors to particular target genes. Although these interactions could erect elaborate castles on DNA, it was possible to consider these edifices as a kind of simplistic "Lego model." For more than a decade, transcription regulation was presented in cartoon representations of ever-increasing Technicolor glory with DNA drawn in a straight line. Slowly, the line began to bend as concepts dealing with the structural packaging of DNA were considered. Today, an accumulated wealth of data has placed chromatin structure in a pre-eminent position in the field of transcription regulation.

It used to be that discussion of chromatin was relegated to the postbanquet morning session at conferences. Attended by just the die-hards and the remnants of the night before who had not yet made it back to their rooms, the talks were often replete with rigorous science, and they sparked intense discussion. From the field of epigenetic regulation, DNA methylation was among the first topics to emerge onto center stage and to be featured in plenary talks, with histone acetylation close behind. The transcription factor "Lego" models adopted new components, and a loose coalition was formed between the transcription and chromatin fields. Now, it is quite respectable to discuss the importance of chromatin structure, DNA methylation, and histone modification to transcriptional control in many biological contexts and particularly in cancer. Therefore, it is worth some effort to consider the relative contributions and to examine the cooperative interactions among all components of the gene regulation machinery. This is particularly pertinent if we hope to intervene in the mechanisms that control gene expression to correct the errors that result in oncogenic transformation. Here, we will review recent progress toward understanding the role of DNA methylation in epigenetic regulation of gene expression, the interactions between DNA methylation and other epigenetic systems that modulate chromatin structure, and the relevance of these topics to cancer.


    The DNA (Cytosine-5) Methyltransferases: Plotting the Methylation Landscape
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 
In mammals, a major form of DNA modification involves methylation of the C5 position of cytosines within CpG dinucleotides (1 , 2) . Several studies have reported the presence of 5mC3 at non-CpG sequences (2, 3, 4, 5, 6, 7, 8) , and functional roles for these modifications have been proposed recently (3) . However, for the purpose of this review, we will focus exclusively on the processes and functional consequences of CpG methylation.

The distribution of CpG dinucleotides in mammalian genomes is not random. Within coding regions, CpG occurs at a low frequency (~1 CpG/100 bp), and these are predominantly methylated on both strands. However, the promoter and 5' transcribed sequences of many genes include a region in which CpG occurs at or near the expected random frequency (~1 CpG/10 bp). These "CpG islands" tend to be undermethylated, with the exceptions of CpG islands that are associated with transcriptionally silent alleles of imprinted genes and silent genes on the inactive X chromosome (reviewed in Ref. 4 ). Despite the higher frequency of CpG methylation at these loci, CpG islands associated with imprinted and X-inactivated genes account for <10% of total genomic 5mC. The bulk of 5mC in the genome (~70%) resides within CpG-rich transposons scattered among extragenic and intronic sequences (5 , 6) .

The first eukaryotic DNA methyltransferase to be cloned, DNA (cytosine-5) methyltransferase-1 (Dnmt1), was identified based on its biological activity (7) . The Dnmt1 genomic locus contains three known transcription start sites. A somatic cell-specific promoter is activated shortly after implantation, and it drives expression of a transcript including a somatic cell-specific exon (1s). Translation initiation within exon 1s results in the full-length 1620 amino acid protein (Dnmt1s; Refs. 8 , 9 ). An upstream oocyte-specific promoter drives expression of an alternative transcript including an oocyte-specific 5' exon (1o), resulting in translation initiation within exon 4 (8) . The Dnmt1o protein contains the methyltransferase catalytic domain but lacks the extreme NH2-terminal 118 amino acids of Dnmt1s. The timing of expression and intracellular trafficking of these forms have been reviewed recently (10) .

The COOH-terminal region of Dnmt1 contains a series of motifs characteristic of the known DNA (cytosine-5) methyltransferases from bacteria to humans (7 , 11 , 12) . These regions cooperate to form binding sites for the reaction substrates, S-adenosyl-L-methionine (AdoMet) and DNA, and the catalytic domain responsible for transfer of the methyl donor group from AdoMet to C5 of the CpG dinucleotide. The precise mechanism of this enzymatic reaction has been reviewed elsewhere (10) . Accumulating data have demonstrated that the NH2-terminal region of Dnmt1 comprises several important functional domains. These include a nuclear localization signal (13) , a region that targets the protein to replication foci during S phase (14 , 15) , sequences that partially reduce the de novo methyltransferase activity of the catalytic domain, and a cysteine-rich zinc-binding domain (16 , 17) . As discussed below, the NH2-terminal domain also includes sites of interaction with various proteins involved in modulation of chromatin structure and gene regulation.

The enzymatic properties of Dnmt1 have been studied extensively using in vitro biochemical assays as well as in vivo genetic approaches. Although Dnmt1 can transfer a methyl group to symmetrically unmethylated CpG dinucleotides in vitro, it preferentially methylates hemimethylated target sequences. The degree of this preference ranges from 5- to 50-fold, depending on the specific study (9 , 18, 19, 20, 21, 22, 23) , and little if any target sequence specificity has been revealed outside the CpG dinucleotide itself (9) . These findings suggest that Dnmt1 functions in the maintenance of CpG methylation by methylating the daughter strand CpG after replication of symmetrically methylated loci.

Genetic studies in mice revealed that partial loss of Dnmt1 function results in embryonic lethality. However, homozygous mutant ES cells are viable, and they exhibit no obvious growth or morphological abnormalities (24) . Although these cells exhibit substantial demethylation of endogenous retroviral DNA, they retain ~30% of the normal level of genomic 5mC. Similar results were obtained with a complete loss-of-function dnmt1 allele (25) . Although genomic 5mC content is reduced to levels significantly lower than those of ES cells expressing a partial loss-of-function dnmt1 mutant, Dnmt1-null ES cells exhibit a low level of CpG methylation. Furthermore, they retain the ability to de novo methylate newly integrated retroviral DNA. These studies confirmed the existence of additional DNA (cytosine-5) methyltransferases and, together with previous in vitro data, suggested that Dnmt1 functions to maintain rather than to establish patterns of CpG methylation.

Support for an exclusively maintenance function of Dnmt1 came from studies in which mammalian Dnmt was expressed in Drosophila (26) , a species with only trace amounts of genomic cytosine methylation (27 , 28) . Whereas expression of a mammalian de novo DNA methyltransferase (described below) increased cytosine methylation, expression of exogenous Dnmt1 did not. Co-expression of both enzymes resulted in a 31% increase in genomic 5mC content relative to flies expressing the de novo methyltransferase alone, possibly attributable to maintenance methyltransferase activity of Dnmt1 on hemimethylated substrates. These data indicate that Dnmt1 has no de novo activity in vivo. However, these studies were carried out in cells that do not normally methylate DNA at a level comparable with that in mammalian cells. Thus, Drosophila cells may lack additional cofactors that direct proper de novo and maintenance cytosine methylation. A recent in vitro kinetic study suggested that the zinc-binding domain of Dnmt1 preferentially interacts with symmetrically methylated DNA, and addition of symmetrically methylated DNA stimulates zinc-dependent de novo methyltransferase activity (16) . Thus, in mammalian cells, Dnmt1 may catalyze the spread of CpG methylation from regions with pre-existing methylated CpG dinucleotides into nearby unmethylated regions. This provides a plausible alternative explanation for the lack of Dnmt1 de novo activity in Drosophila and the observation that there is a cooperative effect of coexpression of Dnmt1 and a de novo DNA methyltransferase. Regardless, the findings outlined above demonstrate that Dnmt1 represents the major mammalian enzyme responsible for maintenance of CpG methylation and that it is complemented by one or more other enzymes capable of de novo methylation.

Additional studies of cells with partial or complete loss of Dnmt1 function revealed abnormalities in transcriptional silencing of specific imprinted alleles (29) , the Xist allele on the active X chromosome (30) , and endogenous retroviral elements (31) . Dnmt1 activity may also play a role in modulating rates of mutations (32 , 33) . The mechanisms by which Dnmt1 and presumably DNA methylation affect mutagenesis may be complex. For example, in one study, loss of Dnmt1 activity led to elevated rates of endogenous and exogenous gene deletion by mitotic recombination or chromosomal loss (34) , whereas in another, Dnmt1 deficiency led to decreased rates of missense mutations and loss of randomly integrated marker genes (35) .

The identification of Dnmt3a and Dnmt3b confirmed the existence of a family of mammalian DNA (cytosine-5) methyltransferases. These genes were identified in expressed sequence tag databases by their sequence similarity with the catalytic domain of Dnmt1. However, the Dnmt3 proteins have no homology to Dnmt1 outside this region (21) . The Dnmt3a locus encodes a single protein, whereas three alternatively spliced Dnmt3b isoforms have been detected (21 , 36) . The Dnmt3 proteins contain a cysteine-rich domain related to the plant homeodomain present in many chromatin-associated proteins. This region is most similar to the plant homeodomain-like domain of ATRX (37) , a member of the SNF2 family of helicase/ATPases (38 , 39) . Interestingly, mutations in ATRX cause X-linked {alpha}-thalassemia mental retardation (ATRX) syndrome, which is associated with both hyper- and hypomethylation abnormalities of specific repetitive elements (40) .

The properties of the Dnmt3 family implicated these proteins as the long-awaited de novo DNA (cytosine-5) methyltransferases. Unlike Dnmt1, neither Dnmt3a nor Dnmt3b shows a preference for hemimethylated DNA target sites in vitro (21 , 36) . Furthermore, the expression patterns of the Dnmt3 genes correlate with the timing of developmental de novo methylation. Although Dnmt1 is expressed ubiquitously in somatic cells, the Dnmt3 genes are expressed at a high level in undifferentiated embryonic stem cells but at low levels in differentiated somatic tissues (21) . The de novo methyltransferase functions of Dnmt3a and Dnmt3b have been confirmed by studies in genetically modified mice. Okano et al. (41) produced embryonic stem cell lines with homozygous null mutations in Dnmt3a and Dnmt3b, separately and in combination. Both single knock-out lines retained the ability to methylate foreign retroviral DNA, whereas the double knock-out cells completely lacked this activity, demonstrating both the requirement and the redundancy of Dnmt3a and Dnmt3b for de novo methyltransferase activity. Despite their overlapping patterns of expression and their largely redundant functions, the effects of independent loss of the two enzymes demonstrate that the two enzymes have distinguishable functions. Mice deficient in Dnmt3a survive to term, but they become runted and die at ~4 weeks of age. However, Dnmt3b-/- embryos develop normally before embryonic day (E) 9.5, but they die prior to term. Embryos lacking both enzymes show abnormal morphology by E8.5, and they die by E11.5. Genomic methylation abnormalities in these embryos further demonstrate both overlapping and specific functions of the Dnmt3 family. For example, C-type retroviral DNA and intracisternal A particle repeats are unaffected (Dnmt3a-/-) or only slightly undermethylated (Dnmt3b-/-) in single knock-out embryos, but they are substantially undermethylated in double knock-out embryos. However, the overall methylation level in double knock-out embryos remains higher than the level in embryos lacking Dnmt1. Centromeric minor satellite repeats are significantly demethylated in Dnmt3b-/- cells but unaffected in Dnmt3a-/- cells, suggesting that this class of sequence is methylated specifically by Dnmt3b.

The identification of mutations responsible for ICF (immunodeficiency, centromeric region instability, and facial anomalies) syndrome provided a natural demonstration of target specificity for Dnmt3b (41, 42, 43) . This genetic disorder is characterized cytogenetically by marked hypomethylation of specific classical satellite repeats (44 , 45) , elongation of juxtacentromeric heterochromatin in lymphocytes, and various structural abnormalities involving chromosomes 1, 9, and 16 (46) . Therefore, although none of the known DNA (cytosine-5) methyltransferases show target specificity in vitro, mechanisms exist to direct their activity to appropriate genomic loci in vivo. This is currently an exciting area of research, and as discussed below, the recent identification of key molecules is beginning to shed light on critical pathways that regulate DNA methylation.


    The Methyl-binding Proteins: Linking DNA Methylation and Chromatin Structure
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 
The correlation between DNA methylation and transcriptional inactivity is well established. However, a causative role for CpG methylation in repression of transcription has often been a subject for debate. Although many silenced genes are associated with dense CpG methylation, this epigenetic mark could be a downstream consequence of transcriptional inactivity rather than an active participant in the process of repression. However, the identification of a family of proteins that bind to DNA containing methylated CpG dinucleotides established a causative link between CpG methylation and repression of transcription. The members of this family have been comprehensively reviewed (47, 48, 49, 50) . Therefore, we will briefly discuss the general characteristics of the mammalian family members.

The presence of methyl-CpG-binding proteins in human cell extracts was demonstrated nearly two decades ago (51) . MeCP2 was the first individual component of these complexes to be purified and biochemically characterized (52 , 53) . This protein contains an NH2-terminal MBD (54) and a COOH-terminal TRD (55) . It associates with chromatin (53) and localizes to methyl-CpG-rich sequences in vivo (56) . In vitro, the intact protein or the MBD alone selectively binds to DNA containing symmetrically methylated CpG dinucleotides with an affinity directly proportional to methyl-CpG density (52 , 57) . Likewise, the TRD represses transcription independently of other MeCP2 sequences (54 , 58, 59, 60) . Binding of the Sin3a corepressor to the TRD recruits HDAC into the complex (58 , 59) . These findings provide a conceptual framework for a chain of events by which DNA methylation actively promotes transcriptional silencing. According to the model, MeCP2 binds to chromosomal regions containing methylated CpG dinucleotides. A histone deacetylase corepressor complex is then recruited by the binding of Sin3a/HDAC to MeCP2. Histone deacetylation, in turn, results in condensation of chromatin leading to a local chromatin structure that is refractory to initiation of transcription (reviewed in Ref. 50 ). Consistent with this model, in vitro transcription repression by MeCP2 is sensitive to histone deacetylase inhibitors (58 , 59) . However, the complexity of interactions among members of both the methyltransferase and MBD families suggests that this pathway may be one of several intersecting pathways leading to methylation-dependent transcription repression. In fact, there is evidence for HDAC-independent mechanisms of transcription repression by MBD proteins, further extending the potential impact of DNA methylation on gene expression (47 , 60, 61, 62) . Furthermore, in colorectal carcinoma and in leukemic cell lines, hypermethylated, transcriptionally silent genes can be reactivated by simultaneous treatment with the HDAC inhibitor TSA and the demethylating agent 5-Aza-dC but not by TSA alone (63) . These findings suggest either that DNA methylation has additional repressive effects that are independent of histone deacetylation, or that unknown TSA-insensitive HDACs are also involved.

Additional MBD-containing proteins have been identified through an in silico approach to cloning (64 , 65) . Five MBD family members have now been identified. MBD1 has an NH2-terminal MBD and a COOH-terminal TRD. In addition, full-length MBD1 contains three CXXC motifs similar to the motif present in Dnmt1 (64) . MBD1 binds preferentially to densely methylated DNA in vitro, and it represses transcription in a HDAC-dependent manner in transfected cells (64 , 66 , 67) . Consistent with its in vitro DNA binding characteristics, overexpressed green fluorescent protein-tagged MBD1 localizes to densely methylated major satellite DNA in mouse cells (65) , and it is concentrated at methylated pericentromeric regions of chromosome 1 in human cells (68) . Endogenous MBD1 is detected along euchromatic regions in human diploid metaphase chromosome spreads, but it concentrates at centromeric heterochromatic regions of chromosomes 1, 9, 15, and 16, as well as regions of densely methylated spacer DNA sequences interspersed among rRNA genes. Furthermore, the intensity of MBD1 staining is generally inversely proportional to the staining intensity of acetylated histone H4 (66) . In human cells, MBD1 mRNA is expressed as five alternatively spliced forms that encode isoforms differing in their COOH-terminal and CXXC regions (65 , 68) . Although the functional consequences of these alternative forms are unclear, inclusion of all three CXXC motifs results in an MBD1 isoform capable of repressing transcription independently of DNA methylation in transfected cells (67 , 68) .

MBD2 includes partially overlapping MBD and TRD domains (69) . The MBD binds to DNA with a single methylated CpG in vitro (65 , 70) , and an MBD2-GFP fusion protein binds to major satellite DNA in transfected mouse cells (65) . Consistent with an HDAC-dependent model of gene repression, the TRD exhibits TSA-sensitive transcription repression activity in reporter assays (62) . Ng et al. (62) identified MBD2 as the methyl-binding component of the MeCP1 complex, a methyl-CpG-binding activity that is distinguishable from MeCP2 in that MeCP1 requires more densely methylated DNA for binding (71) . MeCP1 was recently suggested to comprise a chromatin remodeling ATPase (NuRD) complex with associated HDAC activity (62 , 72) . Furthermore, MBD2 has been reported to be associated with Sin3a (69) . It is likely that the variation in reported factors and complexes associated with MBD2 reflects several distinct yet overlapping cell context-dependent functions of this family of proteins.

MBD3 has extensive sequence similarity to MBD2 (65) . It is expressed as several splice variants, some of which disrupt the MBD (65 , 73) . The protein has been identified as a component of the Mi-2/NuRD transcriptional corepressor complex that includes Mi-2 ATPase, HDAC, and other proteins (70 , 73 , 74) . However, in vitro, mammalian MBD3 has little if any methyl-CpG-binding activity, likely because of amino acid substitutions within the MBD (65 , 70) . Therefore, unlike the case for the Xenopus orthologue of MBD3 which contains a MBD with methyl-CpG-binding activity (70) , it is unlikely that mammalian MBD3 plays a role in methylation-dependent transcription repression.

Finally, MBD4 includes a MBD similar to that of MeCP2, although the COOH-terminal domain is homologous to bacterial DNA repair enzymes (65) . Although MBD4 is capable of binding to methyl-CpG sites, it has a higher affinity for 5mCpG-TpG mismatched sites (75) , and the DNA repair domain provides DNA N-glycosylase activity at G-T mismatches (75 , 76) . Therefore, MBD4 is ideally suited to function in the repair of point mutations that result from spontaneous deamination of 5-methylcytosine to thymine. In addition, MBD4 (also known as MED1) binds to the MLH1 DNA mismatch repair protein in vivo. Expression of a MBD4 mutant lacking the MBD induces microsatellite instability in cell lines, implicating MBD4 in this form of DNA repair as well (77) . These data suggest that MBD4 may serve as a strand discrimination factor for MLH1, directing mismatch repair activity to the newly synthesized strand. However, in an in vitro assay, nuclear extracts containing MBD4 perform mismatch repair independently of target CpG methylation status (78) .


    Chromatin Cross-Talk
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 
Collectively, the studies reviewed above have contributed to a basic understanding of the enzymes that establish and maintain CpG methylation, as well as mechanisms by which these epigenetic signals are interpreted. However, much remains to be sorted out, and recent findings have added additional layers of complexity to the pathways involved. A productive approach toward understanding the regulation of DNA methylation has been the search for proteins that interact with the methyltransferases (reviewed in Ref. 79 ). Because Dnmt1 was the first mammalian (cytosine-5) methyltransferase to be identified, it remains the most extensively studied to date. PCNA, a DNA polymerase processivity factor required for DNA replication (80) , binds to an NH2-terminal region of Dnmt1 (15) . PCNA is recruited to sites of replicating DNA by CAF-1 p150, a factor responsible for assembly of nucleosomes onto replicating DNA (81) . The interaction of PCNA with Dnmt1 therefore provides an attractive mechanism by which Dnmt1 is directed to sites of newly replicating DNA to maintain full methylation status after replication (82) .

In addition to the indirect recruitment of HDACs via MBD proteins, Dnmt1 interacts directly with HDAC1 and HDAC2 (83, 84, 85) . This interaction may provide an additional association between DNA methylation and chromatin condensation by bringing the factors required for both into proximity. The association of HDAC with MBD proteins may then serve a maintenance-repressive role by keeping histones in a deacetylated state independently of Dnmt1. As discussed below, factors involved in histone modification and chromatin remodeling may also establish a local chromatin structure that provides the DNA methylation machinery access to DNA targets.

Rountree et al. (85) identified a novel protein, DMAP1, that binds to the extreme NH2-terminus of Dnmt1. When fused to a generic DNA binding domain, DMAP1 functions as an HDAC-independent transcriptional repressor, possibly accounting for at least some of the HDAC-independent repression ability of Dnmt1 (83 , 85) . Dnmt1 has also been shown to exist in complex with the pRb tumor suppressor and the pRb-associated E2F-1 transcriptional activator in vivo (84) . In nondividing cells, pRb binds to E2F-1 and represses transactivation of genes involved in cell cycle progression (86) . Additionally, pRb binds to HDAC (87) . The interaction of Dnmt1 with pRb may enhance its repressive effects by targeted methylation of E2F-1 binding sites or by recruitment of histone deacetylases to these loci.

These discoveries demonstrate that Dnmt1 is linked to several pathways associated with transcription repression. Ongoing studies of the other DNA (cytosine-5) methyltransferase family members will certainly provide additional insights into the complex relationship between these enzymes and the regulation of gene expression. In fact, Fuks et al. (88) recently demonstrated that Dnmt3a binds RP58, a transcriptional repressor associated with heterochromatin and with promoters of various tissue-specific genes, potentially providing a mechanism for sequence-specific targeting of the enzyme (89) . Dnmt3a also directly interacts with HDAC1 (88) .

Although the interactions of DNA (cytosine-5) methyltransferases with sequence-specific DNA binding factors may target the activity of these enzymes to some degree, the identified interactions do not explain how global DNA methylation patterns are precisely established. DNA (cytosine-5) methyltransferases must establish and maintain the methylation status of appropriate sequences (repetitive elements, imprinted alleles, and others) while other critical CpG-rich sequences remain unmethylated (CpG islands of transcribed genes). Recent studies have begun to shed light on these mechanisms, revealing additional levels of complexity and interdependence associated with DNA methylation and chromatin structure.

Although the effect of CpG methylation on transcription is mediated largely by histone-modifying factors, it is now apparent that chromatin remodeling factors are also involved in regulation of the global methylation pattern. In Arabidopsis thaliana, the ddm1 (decrease in DNA methylation 1) mutant exhibits a 70% reduction of genomic cytosine methylation involving mostly repetitive sequences (90) . Decreased methylation of low-copy sequences occurs over multiple generations (91) . DDM1 is a member of the SNF2-like ATPase/helicase family of proteins that catalyze ATP-dependent disruption of histone-DNA interactions (92) . A model was proposed in which nucleosome remodeling is essential for the establishment and maintenance of DNA methylation, possibly by allowing the methylation machinery access to DNA targets (Fig. 1)Citation . An analogous system was subsequently identified in mammals. Homozygous disruption of lymphoid-specific helicase, Lsh (also known as PASG, proliferation-associated SNF2-like gene), results in perinatal lethality in mice (93) and ~50% reduction in global cytosine methylation content affecting repetitive elements and CpG islands associated with select imprinted alleles (94) . Lsh, which is ubiquitously expressed primarily during S-phase, is closely related to the SNF2 subfamily. It contains ATPase and helicase domains similar to those of Arabidopsis DDM1 and other proteins in yeast, mice, and humans (reviewed in Ref. 95 ).



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Fig. 1. Various factors affect DNA methylation-associated transcription repression. A, SNF2-like ATPases (i.e., Lsh) loosen DNA-histone contacts. Histone methyltransferases (i.e., Suv39H1) methylate lysine 9 of histone H3 (stars). B, these modifications may lead to a chromatin configuration that is permissive for CpG methylation. C, sequence-specific DNA-binding factors may target DNA (cytosine-5) methyltransferases to appropriate loci. Examples of potential Dnmt1 targeting factors include pRb and PML-RAR. Dnmt1 directly binds to HDACs, which are also complexed with MBD proteins such as MeCP2. Dnmt1 associates with DMAP1, a putative HDAC-independent transcription repressor. D, DNA (cytosine-5) methyltransferases catalyze CpG methylation (filled circles), creating binding sites for MBD proteins. E, MBD proteins may remain associated with the methylated loci, allowing chromatin condensation independently of DNA (cytosine-5) methyltransferases.

 
Recent studies of DNA methylation in Neurospora crassa may also provide insight into the regulation and functional role of DNA methylation in mammals. In a genetic screen for mutants with decreased genomic cytosine methylation, Kouzminova and Selker (96) identified dim-2, a DNA methyltransferase responsible for both de novo and maintenance cytosine methylation. A subsequent insertional mutagenesis strategy fortuitously generated a mutation in an independent gene, dim-5, that is also essential for cytosine methylation. Surprisingly, this gene encodes a protein methyltransferase that specifically methylates lysine 9 of histone H3 (H3-Lys9; Ref. 97 ). The protein is homologous to the H3-Lys9 methyltransferases of Saccharomyces pombe (Clr4), Drosophila, and humans (Suv39h; Ref. 98 ). Consistent with the requirement of H3-Lys9 methylation for DNA cytosine methylation, expression of histone H3 mutants with amino acid substitutions of Lys9 results in decreased genomic cytosine methylation in a wild-type strain (97) . Adding yet another level of regulation to the system, methylation of histone H3-Lys9 is itself an epigenetic mark of heterochromatin. The mechanism involves binding of heterochromatin protein 1 (HP1, reviewed in Refs. 99 , 100 ) to Lys9-methylated histone H3, resulting in chromatin remodeling and gene silencing (101, 102, 103, 104) . If there is also a requirement for histone H3-Lys9 methylation for DNA methylation in mammalian cells, this suggests that an interplay of DNA and protein modifications may mediate the establishment and maintenance of DNA methylation as well as the epigenetic modulation of transcription repression (Fig. 1)Citation . The ultimate target of these events is the nucleosome. For example, ATP-dependent nucleosome remodeling factors (i.e., Lsh) may be required to loosen DNA-histone contacts, facilitating access of DNA (cytosine-5) methyltransferases to target DNA sequences. Simultaneously, histone methyltransferases (i.e., Suv93H1) may promote establishment of nucleoprotein complexes that recruit DNA (cytosine-5) methyltransferases to target loci or remodel chromatin into conformations permissive for DNA methylation. Alternatively, these processes may be linked to protection against DNA demethylation rather than the establishment or maintenance of DNA methylation. Finally, the epigenetic territorial markers, CpG and H3-Lys9 methylation, also recruit factors that further modify histones, resulting in locally condensed, transcriptionally silent heterochromatin.

The studies summarized above suggest that disruptions within chromatin modification pathways affect highly repetitive, constitutive heterochromatin. However, precise epigenetic patterns must also be established at low-copy loci. For example, CpG methylation and histone H3-Lys9 methylation associate with the facultative heterochromatin of the inactive X chromosome and promoters of specific X-inactivated genes (105 , 106) . Furthermore, reminiscent of its potential targeting function for Dnmt1, pRb has recently been shown to bind to both Suv39H1 and HP1, directing their activities to the cyclin E promoter (107) . Finally, genetic inactivation of an unconventional member of the Dnmt family revealed a role in targeting de novo methylation. Dnmt3L has extensive homology to Dnmt3a and Dnmt3b, but it lacks a functional DNA methyltransferase catalytic domain (108) . Bourc’his et al. (109) recently produced mice lacking functional Dnmt3L. This mutation behaves as a maternal effect lethal. Homozygous mutant males are viable, but sterile. Homozygous females are fertile, yet their heterozygous offspring die before term. Surprisingly, these mutant embryos exhibit a very specific DNA methylation abnormality. They lack the ability to properly methylate maternally repressed imprinted genes (Snrpn and Peg1), yet they maintain proper allele-specific methylation of the paternally methylated H19 gene. Therefore, Dnmt3L is required for the de novo establishment, but not maintenance, of specific genomic imprints. It is likely that Dnmt3L functions not as a methyltransferase but instead as a regulatory cofactor that directs the activity of other methyltransferase(s) to appropriate targets.

These findings raise many additional questions concerning the interdependence of pathways leading to chromatin remodeling and regulation of transcription. Such elaborate regulatory networks present many potential points of deregulation (Fig. 2)Citation . Further elucidation of the mechanisms involved will likely increase our understanding of the process and consequences of CpG methylation, and they may uncover currently unrecognized avenues leading to tumorigenesis.



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Fig. 2. Relationships among the CpG methylation and chromatin remodeling machinery. Lightning bolts, potential points of deregulation that could alter CpG methylation patterns and gene expression in tumors. See text for details.

 

    DNA Methylation Abnormalities in Tumors
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 
At a simplistic level, tumorigenesis arises as a consequence of two related events, increased activity of factors that promote cell proliferation and decreased activity of factors that suppress unchecked proliferation. These basic scenarios demonstrate the involvement of both activating and inactivating mechanisms in tumorigenesis. However, additional abnormalities must accompany (or even supercede) changes in proliferative capacity for a transformed cell to cause malignancy. For example, transformed cells acquire the ability to invade surrounding tissue and travel long distances to establish themselves in new environments. They remodel local vasculature to feed aggressively growing cells, they circumvent intrinsic surveillance cell death mechanisms, and they impair DNA repair systems. Therefore, cancer involves not only aberrant proliferation but also subversion of mechanisms involved in the regulation of cell-cell and cell-matrix attachment, growth factor signaling, apoptosis, and recombination. In terms of inactivation of tumor suppressor genes, Knudson’s "two-hit" hypothesis is a cornerstone concept. DNA mutations and chromosomal loss or rearrangements have traditionally received the most attention. However, given the number of pathways altered during creation of an environment permissive for tumorigenesis, processes linked to DNA methylation, provide additional potential mechanisms leading to heritable genomic changes that promote cancer.

The discovery of global CpG methylation abnormalities in tumors led to the initial implication of a role of DNA methylation in cancer (110 , 111) . Tumor cells may harbor simultaneous hypermethylation of specific CpG islands and global hypomethylation of widespread transposon elements. Potentially, both events play active roles in tumorigenesis. Although precise mechanisms have not yet been demonstrated, DNA methylation may protect the genome by inhibiting homologous recombination between highly repetitive sequences (112) . Therefore, loss of DNA methylation at these loci may increase the frequency of inappropriate recombination leading to chromosomal abnormalities prevalent in tumors. This hypothesis is supported by the finding that ES cells lacking functional Dnmt1 have a 10-fold higher mutation rate involving gene rearrangements than wild-type ES cells (34) . Because cytosine methylation increases the frequency of C-to-T point mutations attributable to deamination of 5mC to uracil, CpG methylation may play a passive role in promoting point mutations. For example, a high frequency of p53 mutations occur at presumably methylated exonic CpG sites (113 , 114) . Regarding hypermethylation of specific CpG islands, several transcriptionally silent genes exhibit dense CpG island methylation in tumors and tumor cell lines, suggesting that these events either initiate transcription silencing, or they participate in maintaining genes in a repressed state. As reviewed by Baylin and Herman (115) , nearly half of the tumor suppressor genes carrying germ-line mutations in familial cancers have been shown to be inactivated in association with CpG island hypermethylation in sporadic cancers. These include VHL, p16INK4a, pRb, ARF/INK4a, and several others. Equally telling, the list to date includes genes whose protein products participate in many processes required to create a microenvironment suitable for tumorigenesis and metastasis. Among these are genes that function in suppression of invasion (E-cadherin, mts-1, and others), inhibition of angiogenesis (Thrombospondin-1, TIMP3), apoptosis (DAPK1, Fas) and DNA protection or repair (O6-MGMT, hMLH1, GSTP1, and BRCA-1). Genes silenced in association with hypermethylation in various tumor types have been reviewed (79 , 115, 116, 117) . This group likely represents only a fraction of the aberrant methylation events potentially important in cancer. Furthermore, the current list probably reflects an ascertainment bias toward genes with previously demonstrated roles in tumorigenesis, because of the candidate gene approach traditionally used for identification of methylation abnormalities. Consequently, unbiased approaches for identification of methylation changes associated with cancer have been established recently.

Methylated CpG island amplification identifies differentially methylated loci based on their ability to be amplified by PCR subsequent to digestion by methylation-sensitive restriction enzymes (118) . Parallel methylated CpG island amplification of tumor and normal DNA, followed by representational difference analysis (119) , can identify a pool of genomic fragments differentially methylated in the tumor samples. Toyota et al. (120) screened 50 colorectal cancers and 15 colonic adenomas for methylation changes at 30 candidate loci identified by MCA. The authors found that the majority of CpG island hypermethylation events occurred at loci incrementally hypermethylated in normal colonic tissue during the aging process, demonstrating a concordance between tumor-associated DNA hypermethylation and methylation changes that occur during the normal aging process. Additionally, a tumor subgroup emerged that displayed a CpG island methylator phenotype (CIMP), potentially equivalent to genomic instability in terms of effects on gene expression. The hypermethylation profile of CIMP+ colorectal tumors included loci unique to tumor tissues and distinguished the majority of sporadic colon cancers with microsatellite instability related to hMLH1 hypermethylation. Further analyses of CIMP+ and CIMP- colorectal tumors demonstrated that methylation status correlated with the mutation status of p53 and K-RAS, suggesting direct links between genetic and epigenetic pathways in tumorigenesis (121) .

Differential methylation hybridization (DMH) (122) combines methylated CpG island amplification with hybridization of amplicons from tumor and normal controls to an arrayed library of genomic CpG island fragments (57) . In a differential methylation hybridization study of primary breast tumors, cluster analyses revealed an association between widespread CpG island methylation patterns and histological tumor classification whereby poorly differentiated tumors generally displayed more extensive hypermethylation than moderately or well-differentiated tumors (123) .

These studies suggest that elucidation of genome-wide "DNA methylation signatures" of normal, preneoplastic, and tumor tissues is essential for understanding the role of DNA methylation in cancer. A technique based on restriction landmark genomic scanning (RLGS) (124) has recently produced promising results toward these goals (125, 126, 127, 128) . Methylation-sensitive restriction landmark genomic scanning involves digestion of genomic DNA with methylation-sensitive enzymes, followed by resolution of differentially digested fragments by two-dimensional gel electrophoresis. Comparison of gel migration profiles obtained from normal and tumor samples allows identification of aberrantly methylated loci. This technique was used to analyze the methylation status of 1184 CpG islands in 98 primary human tumors (125) . The authors found that on average, ~600 CpG islands are aberrantly methylated in tumors. As suggested by studies of substantially smaller sets of CpG island loci, the results uncovered global nonrandom patterns of methylation in tumors, with specific methylation events associated with particular tumor types.

Within the past few years, microarray-based approaches have allowed simultaneous analysis of mRNA expression levels representing thousands of genes. Likewise, array-based strategies are being adapted for high-throughput analysis of DNA methylation patterns (129 , 130) . Although these technologies are in their infancy, the implications are evident. One can envision a future in which global signatures of the epigenome, transcriptome, and proteome are mapped for various conditions, providing unprecedented tools for cancer diagnostics, prognostics, and therapeutics.


    DNA Hypermethylation and Cancer: Cause or Effect?
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 
The initial proposal that DNA methylation plays a direct role in transcriptional regulation was met with skepticism. Similarly, the concept that aberrant DNA methylation plays a direct role in tumorigenesis was not immediately embraced by the field. It is now clear that DNA methylation can actively participate in transcriptional repression in several ways. Furthermore, hypermethylation of CpG islands associated with transcriptionally silent genes is featured in many tumors. However, questions remain regarding the causative role of CpG island hypermethylation in tumorigenesis. Because tumor cells represent the final state of a complex process leading to cancer, these questions are often difficult to answer. Several observations suggest that DNA methylation abnormalities represent more than simple markers of transformation. For example, in particular familial or sporadic cancers, hypermethylation is the sole detectable explanation for complete loss of expression and activity of pRb, VHL, BRCA1, or p16INK4a tumor suppressor genes (reviewed in Refs. 79 , 115 , )). Using a high-throughput candidate gene approach based on methylation-specific PCR, Esteller et al. (131) found that tumors from patients with inherited cancers in which one allele of a particular tumor suppressor gene was mutated and the remaining allele was lost, the retained mutant allele was never hypermethylated. However, in tumors in which both the mutated and the wild-type allele were retained, DNA hypermethylation served as a frequent "second hit" to inactivate the functional allele. These findings demonstrate a powerful selective force for methylation-associated inactivation independent of genetic mutation (131) . Other examples suggest that DNA hypermethylation can actually promote genetic instability. The DNA repair gene hMLH1 is frequently hypermethylated in sporadic colorectal carcinomas with microsatellite instability. Treatment of cell lines derived from these tumors with the demethylating agent 5-Aza-dC results in re-expression of hMLH1 and partial restoration of mismatch repair activity (132) . Finally, CpG methylation changes can occur very early in tumorigenesis. Hypermethylation of p16INK4a in lung cancer has been detected in preneoplastic cells, the degree of which is directly proportional to disease progression (133, 134, 135) .

Although these findings establish a strong correlation between tumorigenesis and hypermethylation of specific tumor suppressor genes, confirmation of a causative relationship awaits demonstration of the precise mechanisms of aberrant DNA methylation and analysis of their functional consequences throughout the process of cellular transformation. One potential mechanism is up-regulated expression of the DNA (cytosine-5) methyltransferase themselves. Kautiainen and Jones (136) demonstrated increased DNA methyltransferase activity in various tumorigenic cell lines relative to nontumorigenic cells. Subsequently, increased DNA methyltransferase activity was shown to coincide with increased expression of DNMT1 in human neoplastic cells and tumor tissues, and the magnitude of expression increased with progressive stages of disease (137 , 138) . Forced overexpression of a mouse Dnmt1 cDNA results in increased genomic DNA methylation and induction of cellular transformation and tumorigenic potential of NIH 3T3 cells (139) . In fibroblasts transformed by overexpression of human DNMT1, de novo methylation of particular CpG islands occurs within ~70 population doublings (140) . More recently, elevated expression of the DNMT3b (141) and decreased expression of MeCP2 and MBD2 (142) in tumors have been reported. Furthermore, in some colorectal carcinomas with microsatellite instability, frameshift mutations arising from small insertions or deletions within an (A)10 tract in the coding region of MBD4 have been detected (143 , 144) . The frameshift mutations could lead to expression of a truncated form of MBD4 that includes the MBD but lacks the DNA repair domain. This could potentially enhance mutagenesis in tumors with microsatellite instability because of a dominant-negative effect of the truncated protein (143) or complete inactivation of MBD4 function by loss of heterozygosity (144) .

Evidence for a functional role of Dnmt1 in tumorigenesis has also been obtained using genetically modified mice. The Min mouse, a valuable model of intestinal cancer, is heterozygous for a germ-line mutation in the adenomatous polyposis coli (Apc) gene. Human APC is mutated (145) or associated with promoter hypermethylation (146) in the majority of colon cancers, a disease in which promoter hypermethylation of several genes has been reported. On the B6 mouse genetic background, heterozygosity for the Min allele predisposes to development of hundreds of intestinal adenomas (147) . Laird et al. (148) demonstrated that crossing B6-Min/+ mice to mice with one mutated Dnmt1 allele (129/SvJ-Dnmt1S/+) results in a dramatic decrease in the frequency of intestinal adenomas. Early treatment of these mice with 5-Aza-dC resulted in further reduction of tumor incidence by nearly 60-fold (148) . By crossing B6-Min/+ mice to the homogeneous B6-Dnmt1N/+ strain, Cormier and Dove (149) found that the effect of Dnmt1 deficiency on tumor incidence and growth is independent of the status of p53 or modifier of Min 1 (Mom1), two loci that confer strong resistance to Min-induced intestinal tumorigenesis. Interestingly, although Dnmt1 deficiency and Mom1 affect tumorigenesis independently, together they act synergistically to reduce tumor incidence by >40-fold, and they completely prevent tumor development in nearly half of the mice studied (149) .

Mechanistically, overexpression of Dnmt1 has been associated with transforming oncogenes including ras (150 , 151) , SV40 large T-antigen (152) , and fos (153) , suggesting that increased expression of Dnmt1 may play a role in cellular transformation. In these model systems, up-regulation of Dnmt1 expression and activity appears to be necessary for complete cellular transformation because antisense inhibition of Dnmt1 expression leads to restoration of nontransformed cellular morphology and growth properties (152, 153, 154, 155) , and it decreases tumorigenic growth of transformed cells in syngeneic mice (154 , 155) . Additionally, treatment of fos-transformed fibroblasts with TSA results in reversion to a more normal cellular morphology, implicating chromatin remodeling in aspects of the transformed phenotype (153) . Importantly, steady-state Dnmt1 mRNA levels vary during the cell cycle, increasing in parallel with proliferation (156) . Therefore, elevation of Dnmt1 levels has been proposed to simply reflect the increased proliferative capacity of transformed cells (10 , 157 , 158) . However, studies of the role of Dnmt1 in cellular transformation by fos demonstrated that increased expression and activity of Dnmt1 can be uncoupled from cell cycle regulation. Enforced expression of fos in cultured fibroblasts results in morphological cellular transformation independently of cell proliferation (159) . In growth-arrested fibroblasts, ectopic induction of c-fos expression results in morphological transformation, increased expression of the endogenous Dnmt1 gene, elevated DNA methyltransferase activity, and increased genomic 5mC content (153) . When ectopic expression of c-fos is repressed, these cells revert to a nontransformed morphology, Dnmt1 expression and activity decrease, and genomic 5mC content returns to basal levels. Intriguingly, these results demonstrate that genomic 5mC content can be both increased and decreased independently of cell division, implying that an active demethylation process functions during reversion of fos transformation. Indeed, an isoform of the MBD2 protein has been reported to possess 5mC demethylating activity in vitro (160) . This finding implies that MBD2 may possess bimodal functions involving both methylation-dependent transcriptional repression and modulation of precise DNA methylation patterns. However, the demethylating ability of MBD2 has been disputed (62 , 70) .

These results demonstrate a potential proliferation-independent role for increased Dnmt1 activity during oncogenic transformation. However, because transformation requires continued oncogene expression, the relevant defect may lie in sustained activity of DNA (cytosine-5) methyltransferases within an inappropriate cellular context rather than in the absolute level of Dnmt1. Consistent with this possibility, DNA methyltransferase activity decreases during G0-G1 arrest of normal bladder fibroblasts, yet bladder tumor cells maintain a higher level of methylation activity throughout the period of growth arrest (161) . Furthermore, the Dnmt1 isoform expressed in tumors has not been shown to independently induce cellular transformation or tumorigenesis. The studies described above in which ectopic expression of Dnmt1 induced cellular transformation and tumorigenesis used an NH2-terminally truncated form of Dnmt1 (Dnmt1o) corresponding to the oocyte-specific version of the enzyme (139 , 140 , 153) . In our hands, overexpression of the full-length somatic isoform of Dnmt1 (Dnmt1s) induces cell death rather than cellular transformation.4 Yet, during cellular transformation by ras, SV40 large T-antigen or fos, the endogenous somatic Dnmt1 isoform is necessary for full cellular transformation (152, 153, 154, 155) and tumorigenic potential (154 , 155) . Therefore, it appears that deregulation of Dnmt1 activity participates in transformation only in cells in which oncogenic pathways have been activated. This implies that the mechanisms involving Dnmt1 (including but not restricted to DNA methylation) must interact with additional cellular processes to participate in transformation. The fact that the oocyte-specific form of Dnmt1, which lacks an NH2-terminal region that participates in various protein-protein interactions, can induce transformation and tumorigenesis may direct attention toward converging pathways linking Dnmt1 to mechanisms of tumorigenesis.

A recent study of the mechanisms of transcription repression by PML-RAR suggests a link between DNA (cytosine-5) methyltransferases and an oncogenic transcription factor fusion that is independent of the level of Dnmt1 expression (162) . In >90% of APL cases, a reciprocal chromosomal translocation involving PML and the RA receptor RAR{alpha} leads to expression of an oncogenic PML-RAR fusion transcription factor (163 , 164) . PML-RAR functions as a repressor of RA target gene transcription through a mechanism involving recruitment of an HDAC complex, resulting in a block in the differentiation of APL blasts (165) . Di Croce et al. (162) found that induction of PML-RAR results in increased CpG methylation in the 5' region of the RA receptor RARß2, a gene repressed by PML-RAR. Physical interactions between PML-RAR and either Dnmt1 or Dnmt3a were detected by coimmunoprecipitation in an inducible PML-RAR expression cell line and in APL-derived cells. In the presence of PML-RAR, Dnmt1 and Dnmt3a are recruited to the RARß2 promoter, and the proteins colocalize in transfected cells. Furthermore, RA and 5-Aza-dC act synergistically to reduce RARß2 CpG methylation and reactivate RARß2 expression in APL-derived cells (162) . These results suggest that repression of gene expression by oncogenic PML-RAR involves recruitment of Dnmts to target loci as well as direct association with HDAC complexes. The additional interactions among Dnmts, MBDs, and HDACs assembled at these loci may cooperate to ensure transcriptional repression of putative tumor suppressor genes.


    Conclusions
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 
Although the relationships between DNA methylation, chromatin remodeling, and transcription repression have now been established convincingly, conflicting observations still fuel skepticism regarding the role of DNA methylation in cancer. Overexpression of DNA (cytosine-5) methyltransferases is not invariably detected in tumor tissues in which DNA methylation abnormalities exist. Tumor cells exhibit both DNA hyper- and hypomethylation abnormalities, making mechanisms based on increased methyltransferase activity difficult to reconcile. Furthermore, somatic inactivation of DNMT1 by homologous recombination in human colorectal carcinoma cells leads to a dramatic decrease in DNA (cytosine-5) methyltransferase activity; yet, these cells maintain the hypermethylated status of the p16INK4a tumor suppressor gene, demonstrating that Dnmt1 is not necessary for maintenance of this DNA methylation abnormality (166) . Regarding these points, it is important to keep in mind that the properties of tumor cells represent an end-state of a process, and they do not necessarily reflect the combination of abnormalities that participate in tumor development. Therefore, DNA (cytosine-5) methyltransferase levels and global methylation patterns in tumor cells do not necessarily reflect their progressive abnormalities that contributed to the evolution of the tumor. As reviewed above, CpG methylation patterns are influenced by numerous cooperating pathways, including de novo and maintenance methylation, demethylation, and factors that may direct or prevent methylation of appropriate and inappropriate targets, respectively. Cancer is a disease involving both genetic and epigenetic abnormalities. Numerous cellular events promote mutagenic "hits" within gene targets that initiate or promote tumorigenesis. Likewise, abnormalities within the complex pathways that are linked to DNA methylation can lead to several simultaneous "hits" within the epigenome, altering expression of numerous genes by modulating chromatin structure (Fig. 2)Citation . The current explosion of data relating to the complex pathways that target, maintain, and interpret epigenetic information encoded by DNA methylation promises a more comprehensive understanding of the process of tumorigenesis. Bacteria may not have chromatin, but they do not get cancer either.


    Acknowledgments
 
We thank David Benhayon, Hiromichi Kimura, and Stysia Romer for critical review of the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by NIH Cancer Center Support CORE Grant P30 CA21765, NIH Training in the Biology of Cancer Grant T32 CA09346-20 (to J. M. O.), NIH Grant RO1 CA84139 (to T. C.), and the American Lebanese Syrian Associated Charities. Back

2 To whom requests for reprints should be addressed, at Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105. E-mail: fos1{at}aol.com Back

3 The abbreviations used are: 5mC, 5-methyl-cytosine; MBD, methyl-CpG-binding domain; TRD, transcription repression domain; HDAC, histone deacetylase; TSA, trichostatin A; 5-Aza-dC, 5-aza-2'-deoxycytidine; pRb, retinoblastoma protein; APL, acute promyelocytic leukemia; RA, retinoic acid; RAR, RA receptor; PML, promyelocytic leukemia. Back

4 Unpublished observations. Back

Received for publication 3/12/02. Accepted for publication 3/19/02.


    References
 TOP
 Introduction
 The DNA (Cytosine-5)...
 The Methyl-binding Proteins:...
 Chromatin Cross-Talk
 DNA Methylation Abnormalities in...
 DNA Hypermethylation and Cancer:...
 Conclusions
 References
 

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