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.     

Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila.

CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.     

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.     

Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.     

Global hypomethylation of the genome in XX embryonic stem cells

Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.     

Targets and dynamics of promoter DNA methylation during early mouse development

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.     

Deficiency of Mbd2 suppresses intestinal tumorigenesis

Gene silencing through de novo methylation of CpG island promoters contributes to cancer. We find that Mbd2, which recruits co-repressor complexes to methylated DNA, is essential for efficient tumorigenesis in the mouse intestine. As Mbd2-deficient mice are viable and fertile, their resistance to intestinal cancer may be of therapeutic relevance.     

A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation

RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.     

Dnmt3a is essential for hematopoietic stem cell differentiation

Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.     

De novo assembly and genotyping of variants using colored de Bruijn graphs

Detecting genetic variants that are highly divergent from a reference sequence remains a major challenge in genome sequencing. We introduce de novo assembly algorithms using colored de Bruijn graphs for detecting and genotyping simple and complex genetic variants in an individual or population. We provide an efficient software implementation, Cortex, the first de novo assembler capable of assembling multiple eukaryotic genomes simultaneously. Four applications of Cortex are presented. First, we detect and validate both simple and complex structural variations in a high-coverage human genome. Second, we identify more than 3 Mb of sequence absent from the human reference genome, in pooled low-coverage population sequence data from the 1000 Genomes Project. Third, we show how population information from ten chimpanzees enables accurate variant calls without a reference sequence. Last, we estimate classical human leukocyte antigen (HLA) genotypes at HLA-B, the most variable gene in the human genome.     

Regulation of DNA methylation of Rasgrf1.

In mammals, DNA is methylated at cytosines within CpG dinucleotides. Properly regulated methylation is crucial for normal development. Inappropriate methylation may contribute to tumorigenesis by silencing tumor-suppressor genes or by activating growth-stimulating genes. Although many genes have been identified that acquire methylation and whose expression is methylation-sensitive, little is known about how DNA methylation is controlled. We have identified a DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. In mice, the imprinted Rasgrf1 locus is methylated on the paternal allele within a differentially methylated domain (DMD) 30 kbp 5' of the promoter. Expression is exclusively from the paternal allele in neonatal brain. Methylation is regulated by a repeated sequence, consisting of a 41-mer repeated 40 times, found immediately 3' of the DMD. This sequence is present in organisms in which Rasgrf1 is imprinted. In addition, DMD methylation is required for imprinted Rasgrf1 expression. Together the DMD and repeat element constitute a binary switch that regulates imprinting at the locus.     

Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss

DNA methyltransferase 1 (DNMT1) is crucial for maintenance of methylation, gene regulation and chromatin stability. DNA mismatch repair, cell cycle regulation in post-mitotic neurons and neurogenesis are influenced by DNA methylation. Here we show that mutations in DNMT1 cause both central and peripheral neurodegeneration in one form of hereditary sensory and autonomic neuropathy with dementia and hearing loss. Exome sequencing led to the identification of DNMT1 mutation c.1484A>G (p.Tyr495Cys) in two American kindreds and one Japanese kindred and a triple nucleotide change, c.1470-1472TCC>ATA (p.Asp490Glu-Pro491Tyr), in one European kindred. All mutations are within the targeting-sequence domain of DNMT1. These mutations cause premature degradation of mutant proteins, reduced methyltransferase activity and impaired heterochromatin binding during the G2 cell cycle phase leading to global hypomethylation and site-specific hypermethylation. Our study shows that DNMT1 mutations cause the aberrant methylation implicated in complex pathogenesis. The discovered DNMT1 mutations provide a new framework for the study of neurodegenerative diseases.     

Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.     

Trans allele methylation and paramutation-like effects in mice

In mammals, imprinted genes have parent-of-origin-specific patterns of DNA methylation that cause allele-specific expression. At Rasgrf1 (encoding RAS protein-specific guanine nucleotide-releasing factor 1), a repeated DNA element is needed to establish methylation and expression of the active paternal allele. At Igf2r (encoding insulin-like growth factor 2 receptor), a sequence called region 2 is needed for methylation of the active maternal allele. Here we show that replacing the Rasgrf1 repeats on the paternal allele with region 2 allows both methylation and expression of the paternal copy of Rasgrf1, indicating that sequences that control methylation can function ectopically. Paternal transmission of the mutated allele also induced methylation and expression in trans of the normally unmethylated and silent wild-type maternal allele. Once activated, the wild-type maternal Rasgrf1 allele maintained its activated state in the next generation independently of the paternal allele. These results recapitulate in mice several features in common with paramutation described in plants.