Epigenetic status of human embryonic stem cells

We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.     

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.     

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.     

Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random. We also identified Idb4 as a putative tumor-suppressor gene that is methylated in most mouse and human leukemias but in only a minority of other human cancers.     

Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells

Cytosine methylation is required for mammalian development and is often perturbed in human cancer. To determine how this epigenetic modification is distributed in the genomes of primary and transformed cells, we used an immunocapturing approach followed by DNA microarray analysis to generate methylation profiles of all human chromosomes at 80-kb resolution and for a large set of CpG islands. In primary cells we identified broad genomic regions of differential methylation with higher levels in gene-rich neighborhoods. Female and male cells had indistinguishable profiles for autosomes but differences on the X chromosome. The inactive X chromosome (Xi) was hypermethylated at only a subset of gene-rich regions and, unexpectedly, overall hypomethylated relative to its active counterpart. The chromosomal methylation profile of transformed cells was similar to that of primary cells. Nevertheless, we detected large genomic segments with hypomethylation in the transformed cell residing in gene-poor areas. Furthermore, analysis of 6,000 CpG islands showed that only a small set of promoters was methylated differentially, suggesting that aberrant methylation of CpG island promoters in malignancy might be less frequent than previously hypothesized.     

Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications. The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.     

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.     

Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia

Abnormal epigenetic regulation has been implicated in oncogenesis. We report here the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). We discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOXB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed Arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases. Our work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases.     

Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes

Imprinted genes are clustered in domains, and their allelic repression is mediated by imprinting control regions. These imprinting control regions are marked by DNA methylation, which is essential to maintain imprinting in the embryo. To explore how imprinting is regulated in placenta, we studied the Kcnq1 domain on mouse distal chromosome 7. This large domain is controlled by an intronic imprinting control region and comprises multiple genes that are imprinted in placenta, without the involvement of promoter DNA methylation. We found that the paternal repression along the domain involves acquisition of trimethylation at Lys27 and dimethylation at Lys9 of histone H3. Eed-Ezh2 Polycomb complexes are recruited to the paternal chromosome and potentially regulate its repressive histone methylation. Studies on embryonic stem cells and early embryos support our proposal that chromatin repression is established early in development and is maintained in the placenta. In the embryo, however, imprinting is stably maintained only at genes that have promoter DNA methylation. These data underscore the importance of histone methylation in placental imprinting and identify mechanistic similarities with X-chromosome inactivation in extraembryonic tissues, suggesting that the two epigenetic mechanisms are evolutionarily linked.     

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.     

Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes.

Studies of histone methylation have shown that H3 can be methylated at lysine 4 (Lys4) or lysine 9 (Lys9). Whereas H3-Lys4 methylation has been correlated with active gene expression, H3-Lys9 methylation has been linked to gene silencing and assembly of heterochromatin in mouse and Schizosaccharomyces pombe. The chromodomain of mouse HP1 (and Swi6 in S. pombe) binds H3 methylated at Lys9, and methylation at this site is thought to mark and promote heterochromatin assembly. We have used a well-studied model of mammalian epigenetic silencing, the human inactive X chromosome, to show that enrichment for H3 methylated at Lys9 is also a distinguishing mark of facultative heterochromatin. In contrast, H3 methylated at Lys4 is depleted in the inactive X chromosome, except in three 'hot spots' of enrichment along its length. Chromatin immunoprecipitation analyses further show that Lys9 methylation is associated with promoters of inactive genes, whereas Lys4 methylation is associated with active genes on the X chromosome. These data demonstrate that differential methylation at two distinct sites of the H3 amino terminus correlates with contrasting gene activities and may be part of a 'histone code' involved in establishing and maintaining facultative heterochromatin.     

Integrated genomic and epigenomic analyses pinpoint biallelic gene inactivation in tumors

Aberrant methylation of CpG islands and genomic deletion are two predominant mechanisms of gene inactivation in tumorigenesis, but the extent to which they interact is largely unknown. The lack of an integrated approach to study these mechanisms has limited the understanding of tumor genomes and cancer genes. Restriction landmark genomic scanning (RLGS; ref. 1) is useful for global analysis of aberrant methylation of CpG islands, but has not been amenable to alignment with deletion maps because the identity of most RLGS fragments is unknown. Here, we determined the nucleotide sequence and exact chromosomal position of RLGS fragments throughout the genome using the whole chromosome of origin of the fragments and in silico restriction digestion of the human genome sequence. To study the interaction of these gene-inactivation mechanisms in primary brain tumors, we integrated RLGS-based methylation analysis with high-resolution deletion maps from microarray-based comparative genomic hybridization (array CGH; ref. 3). Certain subsets of gene-associated CpG islands were preferentially affected by convergent methylation and deletion, including genes that exhibit tumor-suppressor activity, such as CISH1 (encoding SOCS1; ref. 4), as well as genes such as COE3 that have been missed by traditional non-integrated approaches. Our results show that most aberrant methylation events are focal and independent of deletions, and the rare convergence of these mechanisms can pinpoint biallelic gene inactivation without the use of positional cloning.     

Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation

Epigenetic silencing in cancer cells is mediated by at least two distinct histone modifications, polycomb-based histone H3 lysine 27 trimethylation (H3K27triM) and H3K9 dimethylation. The relationship between DNA hypermethylation and these histone modifications is not completely understood. Using chromatin immunoprecipitation microarrays (ChIP-chip) in prostate cancer cells compared to normal prostate, we found that up to 5% of promoters (16% CpG islands and 84% non-CpG islands) were enriched with H3K27triM. These genes were silenced specifically in prostate cancer, and those CpG islands affected showed low levels of DNA methylation. Downregulation of the EZH2 histone methyltransferase restored expression of the H3K27triM target genes alone or in synergy with histone deacetylase inhibition, without affecting promoter DNA methylation, and with no effect on the expression of genes silenced by DNA hypermethylation. These data establish EZH2-mediated H3K27triM as a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation.     

Methylation of CpG sites of two X-linked genes coincides with X-inactivation in the female mouse embryo but not in the germ line.

To further our understanding of initiation and imprinting of X-chromosome inactivation, we have examined methylation of specific CpG sites of X-linked Pgk-1 and G6pd genes throughout female mouse development. Methylation occurs around the time of inactivation and earlier for Pgk-1, which is closer to the X-inactivation centre. In female primordial germ cells, the inactive X chromosome escapes methylation; this may underly the reversibility of inactivation at meiosis. Similarly, the genes are unmethylated on the inactive X chromosome in sperm; hence, the imprint specifying preferential X-inactivation in extra-embryonic tissues must reside elsewhere.     

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.