The impact of chromatin in human cancer: linking DNA methylation to gene silencing
Esteban Ballestar and
Manel Esteller,1
Cancer Epigenetics Laboratory, Molecular Pathology Program, Centro Nacional de Investigaciones Oncologicas, 28029 Madrid, Spain
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Abstract
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For decades, chromatin was considered to be an inert structure whose only role was the compacting and confining of DNA inside the eukaryotic nucleus. However, tremendous progress in this field over the last 10 years has dramatically elevated chromatin to a key position in the control of gene activity. Its role in mediating the transformation of a normal cell into a malignant state is particularly interesting. On one side of this story there is the discovery that aberrant methylation patterns in an increasing number of tumour suppressor and DNA repair genes determine carcinogenetic transformation; while on the other side, there is the existence of a series of methyl-DNA binding activities that recruit co-repressor complexes and modify the structure of the chromatin to produce a transcriptionally silenced state. Although this field has seen rapid progress in recent years, detailed mechanisms by which this machinery modifies chromatin structure to its appropriate state and the specific targeting of repressor complexes have yet to be resolved. In this review we present the models of how repressor complexes may modify chromatin structure and mediate silencing of tumour suppressor and DNA repair genes.
Abbreviations: DMT, DNA methyltransferases; HATS, histone acetyltransferases; HDACs, histone deacetylases; MBD, methyl-CpG binding domain.
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Introduction
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DNA methylation involvement in cancer has become one of the hottest topics in cancer research. A major breakthrough in the field within the last 5 years has been the recognition of the key role of chromatin as a mediator between DNA methylation and transcriptional silencing of genes relevant to cancer. The spectacular progress over the past decade in research in the areas of chromatin and DNA methylation has prepared the ground for the meeting of two traditionally separate fields.
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Progress in the field of chromatin research: from an inert structure to an active entity
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For decades, chromatin roles were limited to DNA compaction and subsequent gene repression. In the 1970s, a combination of physical and molecular biology techniques revealed that chromatin consists of a repetitive nucleoprotein complex, the nucleosome (1). This particle comprises a histone octamer, with two copies of each of the histones H2A, H2B, H3 and H4, wrapped by 147 bp of DNA. In the octamer, histones H3 and H4 are assembled in a tetramer, which is flanked by two H2AH2B dimers. A variable length of DNA completes the second turn around the histone octamer and interacts with a fifth histone, named H1. After the discovery of the nucleosome, subsequent structural studies refined our knowledge of its structure. The chromatin, a monotonous array of nucleosomes, according to early models, seemed to be a static structure in which little room was left for regulatory functions of gene activity. Once the repetitive nature of the chromatin had been defined and its structural details delineated, interest in chromatin decreased. Much of the effort on transcriptional regulation during the 1980s focused on the further definition of cis-acting elements and trans-acting factors involved in the transcription process (see ref. 2 for review). A remarkable exception to the little progress made in chromatin research during those years was the recognition of the existence of strict nucleosome positioning around eukaryotic genes, which suggested that histones might have specific effects on the transcription process. The early 1990s witnessed the high-resolution description of the histone octamer, the protein component of the nucleosome (3). The new data showed the existence of an architectural motif, the histone fold, which is shared by all core histones, and is responsible for their dimerization within the octamer. Furthermore, the histone fold was also found in several regulatory proteins. Their assembly into nucleosomal structures may confer specialized functions on individual chromosomal domains (4). In addition, the existence of histone variants, encoded by multiple genes, contributes to a unique nucleosomal architecture, and this heterogeneity can be exploited to regulate a wide range of nuclear functions (4). Another important source of heterogeneity in chromatin is provided by the occurrence of histone modifications at their protruding N-termini. Although histone acetylation has been known since the mid 1960s (5) and its relationship with transcriptional activation was long suspected, its exact consequences have remained unclear for years. This has partly been because attempts to isolate histone acetyltransferases (HATs) and histone deacetylases (HDACs) failed for almost three decades. Acetylation, the major post-translational modification of histones, occurs at the lysine residues of their highly conserved N-terminal tails. This modification reduces the positive net charge of the histones and was originally thought to weaken histoneDNA contacts facilitating accessibility to transcriptional factors (6). Although it is not clear that acetylation affects intranucleosomal contacts, the discovery that histone acetylation does alleviate the repressive effects of chromatin enhanced the interest in the role of nucleosomes in gene control. During the mid 1990s HATs and HDACs started to be isolated and cloned (7). HATs and HDACs were shown to be components of large co-activator and co-repressor complexes. The isolation of HAT and HDAC complexes shed light on the mechanisms by which chromatin is modified in order to yield a transcriptionally active or inactive structure. In parallel, other histone post-translational modifications, such as phosphorylation and methylation have been studied extensively in recent years. It has been proposed recently that histone modifications may constitute a pattern for interaction with specific factors. This model is referred to as the histone code hypothesis (8). Transcriptional regulation by HATs and HADCs acts in concert with ATP-dependent remodelling factors, large molecular machines with the dedicated function of disrupting chromatin structure and facilitating transcription (9). These ATPase subunits, classified currently in the SWISNF, the Mi-2 and the ISWI families, generate super helical torsion that results in the sliding of nucleosomes along the helical path of DNA.
Although this presentation is just a brief summary of major achievements in the chromatin field, the main general discovery is the key role played by chromatin in regulating transcriptional activity. Eukaryotic cells require a fine-tuning of gene expression and chromatin provides an interphase that controls the activity of transcriptional factors. Remodelling activities and histone-modifying enzymes act at specific sites through the association with subunits that directly or indirectly have sequence specificity.
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DNA hypermethylation, gene silencing and cancer
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In parallel with the advances in the chromatin field, considerable efforts have been expended in the area of DNA methylation during recent decades. Cytosine methylation at CpG dinucleotides is the most common modification of eukaryotic genomes. In vertebrates, methylation occurs globally throughout the genome, with the exception of CpG islands. These are CG-rich regions of DNA, stretching for an average of ~1 kb, coincident with the promoters of ~60% of human RNA polymerase II-transcribed genes. Early analysis of the role of methylation using tissue-specific genes introduced into mammalian cells led to a general consensus that DNA methylation results in the formation of nuclease-resistant chromatin and subsequent repression of gene activity (10). Over the past 20 years, an increasing number of cancers have been found to be associated with aberrant patterns of methylation, in particular, global genomic hypomethylation (11,12) and localized hypermethylation in normally unmethylated CpG islands (13,14). Since the establishment of the relationship between DNA methylation defects and cancer, the list of hypermethylated genes in human cancers has grown considerably, formed mainly by the inclusion of tumour suppresor and DNA repair genes, such as hMLH1, p16INK4a, BRCA1, p14ARF, GSTP1, MGMT, APC, E-cadherin, LKB1/STK11, Rb, VHL, etc. (1522). In fact, an exquisite pattern of CpG island promoter hypermethylation exists according to the tumour type (23). In parallel, different changes in the chromatin of hypermethylated promoters have been observed. Table I
summarizes reports on chromatin changes associated with hypermethylated promoters in human cancer. Whether DNA methylation is a consequence or a cause of cancer is a long-standing issue. The early occurrence of DNA methylation in several cancers (15,19) and the genesis of mutations in key genes induced by the methylation of the DNA repair genes hMLH1 and MGMT (24) suggests that DNA methylation is responsible for tumoral transformation.
The relevance of CpG methylation in cancer comes from the high frequency and nature of the genes involved in the process and the similarity of the effects compared with those of mutations in the coding regions of these genes. Although the mechanisms that determine aberrant methylation in cancer are unclear, DNA methyltransferases probably have a role in this process. The best studied is DNA methyltransferase 1 (DNMT1), which appears to be required for maintenance of the methylation patterns. In mammals, there are two additional active DNA methyltransferases, DNMT3a and DNMT3b, which act in de novo DNA methylation.
As mentioned above, DNA hypermethylation of tumour suppressor and DNA repair genes leads to their silencing and subsequent oncogenic transformation (13,25). This effect is similar to the loss of function that occurs by mutation. However, there are fundamental differences between genetic and epigenetic defects (see ref. 14). In the former instance, the loss of function occurs at a fixed level. In contrast, gene silencing due to CpG island hypermethylation depends on the density of methylation and it has been shown that this density can increase over time. On the other hand, promoter hypermethylation is potentially reversible, in contrast to mutations, thus providing an additional potential source of pharmacological therapy. Advances in the design of specific and potent drugs to prevent or overcome the effects of DNA methylation require the investigation of the mechanisms by which gene silencing is established.
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Methyl-DNA binding proteins: establishing a bridge between two worlds
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As already mentioned, some of the first evidence about the key role of chromatin in the establishment of a silenced state upon DNA methylation was the discovery that methylation results in the formation of a nuclease-resistant chromatin state (10). A decrease in nuclease accessibility to DNA is easily interpreted in terms of an increase in compaction of chromatin.
Once the correlation between DNA methylation and gene repression had been recognized, two models were proposed to explain how nuclear factors would interpret the information encoded by DNA methylation. One of the models proposed that DNA methylation of cytosines at the gene promoters of genes would interfere with the binding of trans-acting factors. The other model proposed the existence of factors that specifically bind methylated DNA. The search for proteins with differential binding properties for methylated and unmethylated DNA initially yielded two activities that were named MeCP1 and MeCP2, respectively, a 400800 kDa complex and a 55 kDa single polypeptide (26). Subsequent deletion studies of MeCP2 led to the definition of a minimal portion that confers the ability to bind methylated DNA, the methyl-CpG binding domain (MBD) (27). MeCP2 was shown to repress transcription and, similar deletion studies allowed the delineation of a minimal transcriptional repression domain (TRD) (28). In 1998, Bird et al. (29) and Wolffe et al. (30) independently proved the association of MeCP2 and HDAC activity to repress transcription. In parallel, database searches revealed the novel MBD-containing proteins, namely MBD1, MBD2, MBD3 and MBD4 (31). Except MBD4, which is involved in repairing DNA mismatches, the remaining members of the MBD family have proved to be part of similar HDAC-containing complexes. The current model, illustrated in Figure 1
, conceives of MBD proteins recruiting HDAC activities to methylated promoters, which, in turn, deacetylate histones, leading to a repressed state.

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Fig. 1. Model of molecular mechanism of MBD-mediated repression of methylated DNA. Histone octamers are represented by grey circles. DNA is represented as a dark line. Acetylated histone tails are protruding lines from octamers, whereas deacetylated histone tails are not shown. Methyl-CpG dinucleotides are represented by black circles.
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MBD1 is part of a still undefined 200400 kDa complex and is able to repress transcription in an HDAC-dependent manner. MBD2 is a component of the MeCP1 complex (32) and also represses transcription via HDAC. The entire sequence of MBD3 is highly similar to that of MBD2. It is part of the Mi-2NURD complex which, besides a histone deacetylase, contains a chromatin remodelling enzyme, Mi-2 (33). Although as yet undemonstrated, MBD3 is likely to repress transcription in a similar way to MBD1 and MBD2.
All the MBD family members specifically interact with methylated DNA, except MBD3. In the case of MBD3 this ability appears to vary with species (34). In particular, human MBD3 does not seem to interact directly with methylated DNA. It has been shown that the MBD3-containing complex, i.e. the Mi-2NURD complex, is targeted to methylated DNA through an association with MBD2. As mentioned above, among the members of the MBD family, MBD2 and MBD3 are the only two that share homology outside the MBD.
MBD proteins provide the connection between DNA methylation and transcriptional silencing through the modification of chromatin. Three out of the four MBD complexes involved in gene regulation recruit HDACs. Mi-2NURD complex also contains an ATPase-dependent activity, Mi-2.
A current hypothesis is that different MBD-containing co-repressor complexes are selectively associated with different methylated CpG islands. The specific association of the MBD complex with diverse loci has been demonstrated in a few cases, although, determinants for specific targeting remain unknown. MBD proteins do not seem to be sequence-specific. Several mechanisms have been proposed to explain specific targeting, including the density and distribution of methylcytosine, and the chromatin organization (35). Also, the involvement of sequence-specific transcription factors in the selective recruitment of MBD-containing complexes by interaction with any of their subunits is likely to occur. For instance, Drosophila Mi-2 interacts with the Hunchback transcriptional repressor, which binds directly to regulatory sequences of HOX genes (36). Also, the zinc-finger protein Ikaros interacts with Mi-2 in erythroid cells (37).
To date, the most common technique for investigating the specific targeting of MBDs to promoters is the chromatin immunoprecipitation (ChIP) assay. This technique is based on the use of specific antibodies against a particular protein, in this case an MBD, to immunoprecipitate formaldehyde cross-linked chromatin, followed by PCR amplification of precipitated DNA fragments. Although the technique is very useful, it has two limitations: artefacts due to the cross-linking and the limited resolution of the technique. The development of novel techniques may facilitate the identification of MBD associated with the entire sequence of particular promoters. By using the ChIP assay, Magdinier and Wolffe (38) have shown that MBD2 is selectively recruited by the silenced locus p14ARF/p16INK4a in human neoplasia. These results contrast with those reported by Nguyen et al. (39) who have demonstrated recently the involvement of MeCP2 in the silencing of the same gene. Although this discrepancy has not been fully explained, cross-reactivity of different antibodies can lead to conflicting results.
In addition to MBD proteins, the list of factors with potential relevance to the link between DNA methylation and transcriptional activity is growing. Another key connection between DNA methylation and chromatin has been established by the finding that DNMT1 associates with HDAC1 (40) and is part of a complex that also contains the RB tumour suppressor gene product and E2F. Robertson et al. (41) showed that DNMT1 cooperates to repress transcription from promoters containing E2F binding sites. These results established a link between DNA methylation and gene-specific transcriptional repression. It has been suggested that loss of function of Rb, a frequent event in several types of tumours, may result in improper regulation of this complex, resulting in mislocalization of DNMT1 and the production of aberrant methylation patterns. Recent work has also shown that certain MBD-containing proteins interact in vivo with DNMT1, which itself has been found in complexes with HDAC (42). DNMT1 is also known to associate with HDAC2 and DMAP1 (for DNMT1-associated protein), a novel protein (43). DNMT1 may not only maintain DNA methylation, but also directly target, in a heritable manner, transcriptionally repressive chromatin to the genome during DNA replication. Similarly to DNMT1, DNMT3a associates with HDAC1 and RP58, a DNA binding transcriptional repressor found at transcriptionally silent heterochromatin (44).
In addition to the well-defined MBD family of proteins, there has been described recently a novel mammalian methyl-DNA binding activity that requires at least two symmetrical methyl-CpG dinucleotides in its recognition sequence, preferably within the sequence CGCG. A key component of this activity is Kaiso (45), a protein with POZ and zinc-finger domains that is known to associate with p120 catenin. Kaiso behaves as a methylation-dependent transcriptional repressor and is a constituent of the MeCP1 complex. The data suggest that zinc-finger motifs are responsible for DNA binding, and may therefore target repression to specific methylated regions of the genome. As Kaiso associates with p120 catenin, it may link events at the cell surface with DNA methylation-dependent gene silencing.
As mentioned above, an alternative model to the existence of factors that specifically recognize methylcytosine and mediate transcriptional repression, is the interference of methyl groups for the binding of transcriptional factors that prefer unmethylated sequences. In fact both models are compatible. Recent reports support the interference model (46), in which in vivo footprinting experiments show that methylation prevents proteinDNA interactions in the
-globin CpG island. In support of the interference model, a transcriptional activator named CGBP has also been identified (47), which is a CpG binding protein. This factor specifically binds unmethylated CpGs. This protein contains three cysteine-rich domains, one of which exhibits homology with the CXXC motif identified in DNA methyltransferase, human trithorax and MBD1. hCGBP is ubiquitously expressed and is crucial for early embryonic development (48). The current data indicate that hCGBP is a transcriptional activator that recognizes unmethylated CpG dinucleotides, suggesting that it plays a role in modulating the expression of genes located within CpG islands. Although our knowledge of the functional role of CGBP is still limited, this protein may be relevant to cancer arising from CpG island hypermethylation.
CGBP represents an example of transcriptional factors sensitive to the methylation status of DNA. One could also view CGBP as an example of chromatin-independent mechanisms by which CpG island hypermethylation could affect gene expression. In fact, both chromatin-dependent and -independent mechanisms seem to play roles in DNA methylation-driven gene inactivation (for a summary see Table II
). It has been shown that MeCP2 can also repress transcription in a chromatin-independent manner. Kaludov and Wolffe (49) have found that MeCP2 selectively inhibits transcription of methylated DNA in the absence of chromatin. These authors observe that the TRD of MeCP2 associates with transcription factor IIB.
Although methyl groups may interfere with the binding of transcriptional factors, most of the studied cases of gene inactivation by DNA hypermethylation in cancer involve changes in chromatin. For this reason it is especially interesting to study mechanisms of gene silencing that involve methyl-DNA binding proteins.
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Mechanisms of chromatin modification in hypermethylated promoters
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A number of studies have shown that aberrant methylation is associated with changes in the chromatin structure, in particular, nucleosome position patterning (50,51) and histone acetylation levels (52,53). In fact, these two changes represent the effects of the two best-characterized chromatin-modifying machineries, i.e. HAT and HDACs and ATPase-dependent remodelling enzymes.
MeCP2, MBD1, MBD2, MBD3, DNMT1 and DNMT3a link DNA hypermethylation to local histone deacetylation. This localized histone deacetylation in hypermethylated promoters has been reported in several cases. For instance, BRCA1 aberrant methylation is associated with histone hypoacetylation and chromatin condensation (54). The details of the mechanism by which histone acetylation or deacetylation, respectively, produces a transcriptionally active or inactive state remains unclear, and two models coexist. One of these models is based on the electrostatic change associated with lysine acetylationdeacetylation, which may alter the interaction between the histone tails and DNA resulting in an increase or decrease of accessibility to transcription factors (6). Although there is no strong evidence that histone acetylation results in gross changes of nucleosome structure, recent evidence indicates that histone acetylation opens higher order chromatin structure (55). Alternatively, histone acetylationdeacetylation combinations may be interpreted as symbols of the histone code (8), which result in the interaction with trans-acting factors. It has been suggested that DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, where CpG hypermethylation has a dominant effect on the maintenance of a silent state at these loci. This is supported by the fact that certain hypermethylated genes cannot be transcriptionally reactivated with only TSA, an inhibitor of HDAC activity (56). In contrast, minimal de-methylation caused by the presence of a low dose of the drug 5-aza-2'deoxycytidine results in slight re-expression of the gene. When both HDAC inhibitors and de-methylating agents are used, the treatment results in robust re-expression of each gene.
The fact that histone deacetylation inhibition is not the only requirement for reactivating the expression of hypermethylated genes suggests that additional chromatin-independent mechanisms are involved in DNA methylation-driven gene silencing. As mentioned above, MeCP2 has a direct effect on repressing transcription, and specific interaction with elements of the transcriptional machinery has been reported (49).
In addition, reports of changes in nucleosome positioning suggest the activity of nucleosome remodelling activities. For instance, aberrant methylation at the MGMT CpG island has been associated with the loss of nucleosome-like positioning (51). This type of effect should be the result of nucleosome remodelling activity. At least, the MBD3-containing Mi-2NURD complex contains an ATPase remodelling subunit, whose activity may be of key importance in the establishment of a silenced transcriptional state. In vitro studies by Guschin et al. (57) suggest that the nucleosomal ATPase Mi-2 alters chromatin through sliding, similar to the mechanism used by other ATPases such as ISWI. Mi-2 complex activity would generate a transcriptionally repressed state.
It has also been suggested that histone acetylation status may also serve as a code that ATP-dependent chromatin remodelling factors translate. Thus, chromatin remodelling events may be targeted via the action of histone-modifying enzymes. Although some ATP-dependent remodelling activities, such as SWISNF are not sensitive to histone acetylation status, histone N-terminal tails are required for multiple rounds of nucleosomal remodelling (58). Mi-2 ATPase also seems to be insensitive to acetylation status and histone tail presence, although nucleosomal structure is required for its activity. It is also possible that different MBD-containing complexes are responsible for chromatin remodelling at different locations along the promoter (Figure 2
). As a CpG island can extend up to several thousands of base pairs, different MBD complexes may play different roles in the remodelling of a particular promoter at different portions of its sequence. Therefore, different MBD complexes may use different mechanisms and act synergistically to change chromatin structure and produce an appropiate transcriptional state.

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Fig. 2. Model showing different chromatin events that take place in hypermethylated promoters. An array of nucleosomes is shown. Histone octamers, DNA, acetylated and deacetylated histone tails are represented as in Figure 1 . The result of ATPase remodelling activity is shown with a w line.
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The Mi-2NURD complex would be involved in the remodelling of hypermethylated promoters through the involvement of MBD3 or MBD2. It has been reported recently that MeCP1, besides its histone deacetylase activity, is also involved in nucleosome remodelling (59). Additionally, other ATPase-dependent remodelling activities may be involved in nucleosome remodelling through interactions with other MBD-containing complexes or the recognition of particular histone acetylation status.
In Figure 3
, a summary of the different complexes and factors involved in DNA hypermethylation-dependent repression is shown. The bottom part of the figure contains factors that selectively interact with hypermethylated DNA whereas the top part corresponds to factors that bind hypomethylated DNA.

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Fig. 3. Factors involved in DNA methylation-driven repression. The upper half includes CGBP and specific transcription factors that bind exclusively unmethylated DNA and HATs that activate gene expression. The lower half includes HDAC-associated DNMTs and MBD-containing repressor complexes that associate to methylated DNA.
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Involvement of other proteins in MBD-mediated gene repression
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The specific association of MBD-containing co-repressor complexes to genes may occur through interaction of subunits of the complex and sequence-specific transcriptional factors. As commented above, Drosophila Mi-2 interacts with the Hunchback transcriptional repressor (36), which binds directly to HOX genes. Studies on a Drosophila MBD-like protein showed specific association with discrete loci, even though this homologue of mammalian MBD proteins does not interact with methylated DNA (60). In this case, immunolocalization of the Drosophila MBD-like protein on polytene chromosomes coincided with genes regulated by ecdysone, suggesting that a component of this MBD-containing complex may interact with the ecdysone receptor or its partner Ultraspiracle.
Progress in the understanding of the mechanisms of MBD-mediated repression and their involvement in promoter inactivation by hypermethylation thus depends on finding new interactions between MBD complexes and activators, repressors, hormone receptors, etc. These proteins provide specificity to the targeting of MBD complexes to each promoter.
For instance, it has been reported recently that MeCP2 directly binds c-Ski and N-CoR (61). C-Ski/Sno act as co-repressors and directly bind to other co-repressors like N-CoR/SMRT and mSin3A. The binding of MeCP2 to Ski is a requirement for transcriptional repression. There are several lines of evidence that suggest that MIZF, a novel zinc-finger protein, interacts with MBD2 (62). The presence of zinc fingers in MIZF suggests an association with specific genes. MIZF may be involved in the targeting of MeCP1 or NURD to discrete loci. The association of E2F to DNMT1 is responsible for the specific targeting of HDAC activity to promoters with E2F binding sites. Research efforts need to focus on identifying interactions between sequence-specific determinants and repressor complexes at every relevant CpG island-containing promoter.
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Future prospects
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Progress in our understanding of inactivation by hypermethylation of tumour suppressor and DNA repair genes in human cancer has been determined by the advances made in the field of chromatin research. Investigation of other post-translational modifications of histones, such as methylation and phosphorylation, mechanisms of nucleosome remodelling and targeting will lead to the discovery of further connections between DNA methylation and chromatin. For instance, SUV39H1 is a histone methylase that selectively methylates histone H3, generating a binding site for HP1 proteins that mediate silencing at heterochromatin sites (63,64). Nielsen et al. (65) have demonstrated recently that Rb associates with SUV39H1 in vivo. These new results suggest that Rb is involved in H3 methylation and silencing by HP1 proteins at specific sites. Interestingly enough, Suv39h-deficient mice display chromosomal instabilities that are associated with an increased tumour risk (66). Recent reports demonstrate that DNA methylation in Neurospora crassa depends on a histone H3 methyltransferase (67). This new connection may be of importance in providing additional sources of gene inactivation by DNA methylation. The understanding of the mechanisms by which chromatin connects DNA methylation to gene silencing is fundamental to the design of drugs that specifically reactivate the silenced tumour suppressor genes.
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Notes
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1 To whom correspondence should be addressed Email: mesteller{at}cnio.es 
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Acknowledgments
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Supported by I+D+I SAF 2001-0059 grant and Ramon y Cajal program of the Ministerio de Ciencia y Tecnologia and The International Rett Syndrome Association.
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Received December 21, 2001;
revised February 12, 2002;
accepted February 12, 2002.