Ilana Keshet

Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer

Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.

DNA methylation affects the formation of active chromatin

To study the mechanism of gene repression by DNA methylation, M13 gene constructs were methylated to completion and inserted into mouse L cells by DNA-mediated gene transfer. All unmethylated sequences, regardless of their source, integrated into the DNA in a potentially active DNAase I-sensitive conformation. Total CpG methylation prevented the formation of this structure and rendered these sequences DNAase I-insensitive over the entire methylated domain. Whereas unmethylated DNA demonstrated additional conformational features of active genes, such as DNAase I hypersensitivity and restriction endonuclease-sensitive segments, these markers were not present when methylated DNA was used for transfection. The use of micrococcal nuclease to probe for active or inactive supranucleosome particles also showed that DNA methylation directs DNA into an inactive type of structure. The results suggest that DNA methylation may exert its effect on gene transcription by altering both specific and nonspecific interactions between DNA and nuclear proteins.

Evidence for an instructive mechanism of de novo methylation in cancer cells

DNA methylation has a role in the regulation of gene expression during normal mammalian development but can also mediate epigenetic silencing of CpG island genes in cancer and other diseases. Many individual genes (including tumor suppressors) have been shown to undergo de novo methylation in specific tumor types, but the biological logic inherent in this process is not understood. To decipher this mechanism, we have adopted a new approach for detecting CpG island DNA methylation that can be used together with microarray technology. Genome-wide analysis by this technique demonstrated that tumor-specific methylated genes belong to distinct functional categories, have common sequence motifs in their promoters and are found in clusters on chromosomes. In addition, many are already repressed in normal cells. These results are consistent with the hypothesis that cancer-related de novo methylation may come about through an instructive mechanism.

The role of DNA methylation in setting up chromatin structure during development

DNA methylation inhibits gene expression in animal cells, probably by affecting chromatin structure. Biochemical studies suggest that this process may be mediated by methyl-specific binding proteins that recruit enzymatic machinery capable of locally altering histone modification. To test whether DNA methylation actually has a role in the assembly of chromatin during normal development, we used cell transfection and a transgene construct genetically programmed to be either methylated or unmethylated in all cell types of the mouse. Chromatin immunoprecipitation (ChIP) analysis shows that the presence of DNA methylation brings about the deacetylation of histone H4 and methylation of Lys9 of histone H3 (H3 Lys9) and prevents methylation of Lys4 of histone H3 (H3 Lys4), thus generating a structure identical to that of methylated sequences in the genome. These results indicate that the methylation pattern established in early embryogenesis is profoundly important in setting up the structural profile of the genome.

DNA methylation models histone acetylation

One of the main determinants of chromatin structure is histone acetylation. Local chromosomal acetylation can be regulated dynamically, both through the involvement of transactivating factors with intrinsic histone acetylase activity, and through the recruitment of deacetylase complexes that repress gene expression. Histone acetylation status is transiently modified from one state to another in response to physiological changes operating in the cell. It is not yet known, however, how the basic histone acetylation profiles on tissue-specific and housekeeping gene sequences are established during normal development. Here we show that DNA methylation takes part in this process by inducing decreased levels of chromatin acetylation.

DNA Demethylation In Vitro: Involvement of RNA

An in vitro system for studying DNA demethylation has been established using extracts from tissue culture cells. This reaction, which is unusually resistant to proteinase K, takes place through the removal of a 5-methylcytosine nucleotide unit from the DNA substrate and its conversion to an RNase-sensitive form. It is likely that this represents the in vivo mechanism, as well, since extracts from L8 myoblasts specifically demethylate an alpha-actin gene, while extracts from F9 teratocarcinoma cells specifically demodify the Aprt CpG island. After pretreatment with proteinase K, these extracts demethylate both genes equally, suggesting that gene specificity may be controlled by protein factors.

Establishment of transcriptional competence in early and late S phase

In animal cells, the process of DNA replication takes place in a programmed manner, with each gene region designated to replicate at a fixed time slot in S phase. Housekeeping genes undergo replication in the first half of S phase in all cell types, whereas the replication of many tissue specific genes is developmentally controlled, being late in most tissues but early in the tissue of expression. Here we employ nuclear DNA injection as an experimental system to test whether this phenomenon is due to differences in the ability to set up transcriptional competence during S phase. Our results show that, regardless of sequence, exogenous genes are a better template for transcription when injected into nuclei of cells in early as opposed to late S phase, and this expression state, once initiated, is preserved after cell division. DNA injected in late S phase is apparently repressed because it is packaged into chromatin containing deacetylated histones, and the same is true for late replicating chromosomal DNA. These findings suggest a mechanistic connection between replication timing and gene expression that might help to explain how epigenetic states can be maintained in vivo.

Role of DNA Methylation in Stable Gene Repression

A large fraction of the animal genome is maintained in a transcriptionally repressed state throughout development. By generating viable Dnmt1-/- mouse cells we have been able to study the effect of DNA methylation on both gene expression and chromatin structure. Our results confirm that the underlying methylation pattern has a profound effect on histone acetylation and is the major effector of me-H3(K4) in the animal genome. We demonstrate that many methylated genes are subject to additional repression mechanisms that also impact on histone acetylation, and the data suggest that late replication timing may play an important role in this process.

An upstream repressor element plays a role in Igf2 imprinting

The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans‐ acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo.

CTCF Elements Direct Allele-Specific Undermethylation at the Imprinted H19 Locus

The H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation, and replication timing. This ICR becomes de novo methylated during late spermatogenesis in the male but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development and in all tissues of the mouse. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated, this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo.