Merav Hecht

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.

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.

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.

DNA replication timing of the human β-globin domain is controlled by histone modification at the origin

The human beta-globin genes constitute a large chromosomal domain that is developmentally regulated. In nonerythroid cells, these genes replicate late in S phase, while in erythroid cells, replication is early. The replication origin is packaged with acetylated histones in erythroid cells, yet is associated with deacetylated histones in nonerythroid cells. Recruitment of histone acetylases to this origin brings about a transcription-independent shift to early replication in lymphocytes. In contrast, tethering of a histone deacetylase in erythroblasts causes a shift to late replication. These results suggest that histone modification at the origin serves as a binary switch for controlling replication timing.

Establishment of methylation patterns in ES cells

After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at implantation. We have identified a demethylation pathway in mouse embryonic cells that uses hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this demethylation system is not necessary for generating the overall bimodal methylation pattern but does appear to be involved in resetting methylation patterns during somatic-cell reprogramming.

Gender-specific postnatal demethylation and establishment of epigenetic memory

DNA methylation patterns are set up in a relatively fixed programmed manner during normal embryonic development and are then stably maintained. Using genome-wide analysis, we discovered a postnatal pathway involving gender-specific demethylation that occurs exclusively in the male liver. This demodification is programmed to take place at tissue-specific enhancer sequences, and our data show that the methylation state at these loci is associated with and appears to play a role in the transcriptional regulation of nearby genes. This process is mediated by the secretion of testosterone at the time of sexual maturity, but the resulting methylation profile is stable and therefore can serve as an epigenetic memory even in the absence of this inducer. These findings add a new dimension to our understanding of the role of DNA methylation in vivo and provide the foundations for deciphering how environment can impact on the epigenetic regulation of genes in general.

Genome-wide de novo methylation in epithelial ovarian cancer.

Background: DNA methylation regulates gene expression during development. The methylation pattern is established at the time of implantation. CpG islands are genome regions usually protected from methylation; however, selected islands are methylated later. Many undergo methylation in cancer, causing epigenetic gene silencing. Aberrant methylation occurs early in tumorigenesis, in a specific pattern, inhibiting differentiation. Although methylation of specific genes in ovarian tumors has been demonstrated in numerous studies, they represent only a fraction of all methylated genes in tumorigenesis. Objectives: To explore the hypermethylation design in ovarian cancer compared with the methylation profile of normal ovaries, on a genome-wide scale, thus shedding light on the role of gene silencing in ovarian carcinogenesis. Identifying genes that undergo de novo methylation in ovarian cancer may assist in creating biomarkers for disease diagnosis, prognosis, and treatment responsiveness. Methods: DNA was collected from human epithelial ovarian cancers and normal ovaries. Methylation was detected by immunoprecipitation using 5-methyl-cytosine-antibodies. DNA was hybridized to a CpG island microarray containing 237,220 gene promoter probes. Results were analyzed by hybridization intensity, validated by bisulfite analysis. Results: A total of 367 CpG islands were specifically methylated in cancer cells. There was enrichment of methylated genes in functional categories related to cell differentiation and proliferation inhibition. It seems that their silencing enables tumor proliferation. Conclusions: This study provides new perspectives on methylation in ovarian carcinoma, genome-wide. It illustrates how methylation of CpG islands causes silencing of genes that have a role in cell differentiation and functioning. It creates potential biomarkers for diagnosis, prognosis, and treatment responsiveness.

Aberrant DNA Methylation in ES Cells

Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.

Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type–specific methylation in enhancers

Significance We have studied the dynamics of DNA methylation in pancreatic α- and β-cells and reached surprising insights into the establishment of islet cell identity. Different islet cell types share lack of methylation in cell-type–specific gene promoters, while DNA methylation differences between islet cell types are concentrated in enhancer regions. The findings support the fundamental role of enhancer methylation in determining cell identity, and have implications for the understanding of islet cell plasticity in diabetes. DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic β-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3+ embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and β-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and β-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for β-cell reprogramming in diabetes and diagnosis of β-cell death using methylation patterns of circulating DNA.

Programming asynchronous replication in stem cells

Many regions of the genome replicate asynchronously and are expressed monoallelically. It is thought that asynchronous replication may be involved in choosing one allele over the other, but little is known about how these patterns are established during development. We show that, unlike somatic cells, which replicate in a clonal manner, embryonic and adult stem cells are programmed to undergo switching, such that daughter cells with an early-replicating paternal allele are derived from mother cells that have a late-replicating paternal allele. Furthermore, using ground-state embryonic stem (ES) cells, we demonstrate that in the initial transition to asynchronous replication, it is always the paternal allele that is chosen to replicate early, suggesting that primary allelic choice is directed by preset gametic DNA markers. Taken together, these studies help define a basic general strategy for establishing allelic discrimination and generating allelic diversity throughout the organism.