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.     

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.     

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.     

Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations

Chromatin immunoprecipitation (ChIP) defines the genomic distribution of proteins and their modifications but is limited by the cell numbers required (ideally >10(7)). Here we describe a protocol that uses carrier chromatin and PCR, 'carrier' ChIP (CChIP), to permit analysis of as few as 100 cells. We assayed histone modifications at key regulator genes (such as Nanog, Pou5f1 (also known as Oct4) and Cdx2) by CChIP in mouse embryonic stem (ES) cells and in inner cell mass (ICM) and trophectoderm of cultured blastocysts. Activating and silencing modifications (H4 acetylation and H3K9 methylation) mark active and silent promoters as predicted, and we find close correlation between values derived from CChIP (1,000 ES cells) and conventional ChIP (5 x 10(7) ES cells). Studies on genes silenced in both ICM and ES cells (Cdx2, Cfc1, Hhex and Nkx2-2, also known as Nkx) show that the intensity of silencing marks is relatively diminished in ES cells, indicating a possible relaxation of some components of silencing on adaptation to culture.     

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.     

Epigenetic stem cell signature in cancer

Embryonic stem cells rely on Polycomb group proteins to reversibly repress genes required for differentiation. We report that stem cell Polycomb group targets are up to 12-fold more likely to have cancer-specific promoter DNA hypermethylation than non-targets, supporting a stem cell origin of cancer in which reversible gene repression is replaced by permanent silencing, locking the cell into a perpetual state of self-renewal and thereby predisposing to subsequent malignant transformation.     

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 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.     

Dnmt3a silences hematopoietic stem cell self-renewal

DNA methylation is an epigenetic mark stably directing gene expression throughout development. A new study uncovers a role for the DNA methyltransferase Dnmt3a in silencing self-renewal genes in hematopoietic stem cells (HSCs) to permit efficient hematopoietic differentiation.     

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.     

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.     

Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome

The overgrowth- and tumor-associated Beckwith-Wiedemann syndrome results from dysregulation of imprinted genes on chromosome 11p15.5. Here we show that inherited microdeletions in the H19 differentially methylated region (DMR) that abolish two CTCF target sites cause this disease. Maternal transmission of the deletions results in hypermethylation of the H19 DMR, biallelic IGF2 expression, H19 silencing and Beckwith-Wiedemann syndrome, indicative of loss of function of the IGF2-H19 imprinting control element.