The Polycomb group protein EZH2 directly controls DNA methylation

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.     

Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer

Specific chromosomal deletions sometimes associated with tumours such as retinoblastoma (chromosome 13q14) and Wilm's tumour (chromosome 11p13) have led to the hypothesis that recessive genes may be involved in tumorigenesis. This hypothesis is supported by demonstration of allele loss specific for these regions using polymorphic DNA markers and by the isolation of a complementary DNA clone for the retinoblastoma gene. A cytogenetic deletion in chromosome 3 (p14-p23) was reported in small-cell lung cancer (SCLC) by Whang-Peng et al. At least one homologue of chromosome 3 was affected in the majority of SCLC tumours; however, the multiple chromosomal changes seen presented the possibility that chromosome 3 was rearranged, not deleted. We used polymorphic DNA probes for chromosome 3p and compared tumour and constitutional genotypes of nine SCLC patients. Our data show loss of alleles of chromosome 3p markers in tumour DNA of all nine patients supporting the hypothesis that this region contributes to tumorigenesis in SCLC.     

SV40 enhancer and large-T antigen are instrumental in development of choroid plexus tumours in transgenic mice

We have shown recently that choroid plexus tumours frequently develop in transgenic mice which have developed from fertilized eggs injected with DNA molecules containing both simian virus 40 (SV40) early-region genes and metallothionein (MT) fusion genes, and several lines of mice have now been established in which all of the offspring that inherit the foreign DNA succumb to these tumours at 3-5 months of age (ref. 1 and our unpublished data). Several other tissues, notably thymus and kidney, occasionally also show pathological changes. SV40 large-T antigen protein and messenger RNA are always present in affected tissues at much greater concentrations than in unaffected tissues, suggesting that SV40 early-region genes are preferentially activated in choroid plexus, thymus and kidney and that this activation frequently leads to tumorigenesis in the choroid plexus. To determine which regions of the original constructs are important for this tumorigenesis, we have now tested several derivatives and report here that the large-T antigen is sufficient, that the MT fusion gene is dispensable and that the SV40 enhancer (72-base-pair repeat region) has an important role in directing tumours to the choroid plexus. Deletion of the SV40 enhancer region alone commonly leads to peripheral neuropathy, as well as liver and pancreatic tumours, which are the subject of the accompanying paper. Evidence is presented that these pathologies may result from an enhancing effect of the MT sequences on large-T antigen genes, made possible by removal of the otherwise dominant SV40 enhancer.     

Novel mutations target distinct subgroups of medulloblastoma

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.     

Vernalization requires epigenetic silencing of FLC by histone methylation

To ensure flowering in favourable conditions, many plants flower only after an extended period of cold, namely winter. In Arabidopsis, the acceleration of flowering by prolonged cold, a process called vernalization, involves downregulation of the protein FLC, which would otherwise prevent flowering. This lowered FLC expression is maintained through subsequent development by the activity of VERNALIZATION (VRN) genes. VRN1 encodes a DNA-binding protein whereas VRN2 encodes a homologue of one of the Polycomb group proteins, which maintain the silencing of genes during animal development. Here we show that vernalization causes changes in histone methylation in discrete domains within the FLC locus, increasing dimethylation of lysines 9 and 27 on histone H3. Such modifications identify silenced chromatin states in Drosophila and human cells. Dimethylation of H3 K27 was lost only in vrn2 mutants, but dimethylation of H3 K9 was absent from both vrn1 and vrn2, consistent with VRN1 functioning downstream of VRN2. The epigenetic memory of winter is thus mediated by a 'histone code' that specifies a silent chromatin state conserved between animals and plants.     

Defects in mismatch repair promote telomerase-independent proliferation

Mismatch repair has a central role in maintaining genomic stability by repairing DNA replication errors and inhibiting recombination between non-identical (homeologous) sequences. Defects in mismatch repair have been linked to certain human cancers, including hereditary non-polyposis colorectal cancer (HNPCC) and sporadic tumours. A crucial requirement for tumour cell proliferation is the maintenance of telomere length, and most tumours achieve this by reactivating telomerase. In both yeast and human cells, however, telomerase-independent telomere maintenance can occur as a result of recombination-dependent exchanges between often imperfectly matched telomeric sequences. Here we show that loss of mismatch-repair function promotes cellular proliferation in the absence of telomerase. Defects in mismatch repair, including mutations that correspond to the same amino-acid changes recovered from HNPCC tumours, enhance telomerase-independent survival in both Saccharomyces cerevisiae and a related budding yeast with a degree of telomere sequence homology that is similar to human telomeres. These results indicate that enhanced telomeric recombination in human cells with mismatch-repair defects may contribute to cell immortalization and hence tumorigenesis.     

In vivo binding pattern of a trans-regulator of homoeotic genes in Drosophila melanogaster

The specification and maintenance of the metameric pattern in Drosophila melanogaster is regulated by complicated gene interactions. The differential expression of the homoeotic genes of the Antennapedia complex (ANT-C) and bithorax complex (BX-C), which determine segmental identities, is partly controlled by cross-regulatory interactions of loci within the two clusters and partly by trans-acting factors located outside the two complexes. One of the trans-regulatory genes, Polycomb (Pc), acts as a repressor of the ANT-C and BX-C. Mutations of Polycomb result in a complete depression of the homoeotic genes, leading to abdominal transformations of all body segments. Polycomb is part of a large class of trans-regulatory genes (Pc-group), estimated to comprise up to 40 loci. We have raised antibodies against the Polycomb protein, and, using an improved immunostaining technique, showed that the Polycomb protein binds to 60 discrete sites along the polytene chromosomes of salivary glands. These sites comprise the ANT-C and the BX-C as well as several locations of Pc-group genes. This is the first clear evidence for a direct interaction of Polycomb with homoeotic loci and other Pc-group genes.     

Identification of nuclear proteins encoded by viral and cellular myc oncogenes

The myelocytomatosis viruses are a family of replication-defective avian retroviruses that cause a variety of tumours in chickens and transform both fibroblasts and macrophages in culture through the activity of their oncogene v-myc. A closely related gene (c-myc) is found in vertebrate animals and is thought to be the progenitor of v-myc. Changes in the expression and perhaps the structure of c-myc have been implicated in the genesis of avian, murine and human tumours (for a review, see ref. 15). Elucidation of the mechanisms by which v-myc and c-myc might elicit tumorigenesis requires identification of the proteins encoded by these genes. To this end, we have expressed a portion of v-myc in a bacterial host and used the resulting protein to raise antisera that react with myc proteins. We report here that v-myc and c-myc encode closely related proteins with molecular weights (MWs) of approximately 58,000. Integration of retroviral DNA near or within c-myc in avian lymphomas apparently enhances expression of the gene. Here we have used cells from one such tumour to identify the protein encoded by c-myc and find that the coding domain for the gene is probably intact.     

A Polycomb-based switch underlying quantitative epigenetic memory

The conserved Polycomb repressive complex 2 (PRC2) generates trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with stable epigenetic silencing. Much is known about PRC2-induced silencing but key questions remain concerning its nucleation and stability. Vernalization, the perception and memory of winter in plants, is a classic epigenetic process that, in Arabidopsis, involves PRC2-based silencing of the floral repressor FLC. The slow dynamics of vernalization, taking place over weeks in the cold, generate a level of stable silencing of FLC in the subsequent warm that depends quantitatively on the length of the prior cold. These features make vernalization an ideal experimental system to investigate both the maintenance of epigenetic states and the switching between them. Here, using mathematical modelling, chromatin immunoprecipitation and an FLC:GUS reporter assay, we show that the quantitative nature of vernalization is generated by H3K27me3-mediated FLC silencing in the warm in a subpopulation of cells whose number depends on the length of the prior cold. During the cold, H3K27me3 levels progressively increase at a tightly localized nucleation region within FLC. At the end of the cold, numerical simulations predict that such a nucleation region is capable of switching the bistable epigenetic state of an individual locus, with the probability of overall FLC coverage by silencing H3K27me3 marks depending on the length of cold exposure. Thus, the model predicts a bistable pattern of FLC gene expression in individual cells, a prediction we verify using the FLC:GUS reporter system. Our proposed switching mechanism, involving the local nucleation of an opposing histone modification, is likely to be widely relevant in epigenetic reprogramming.     

Polycomb group genes as the key regulators in gene silencing

The Polycomb group （PcG） genes repress gene expression mainly through chromatin modifications and regulation of chromatin structure. At present, at/east four protein complexes of PcG proteins are identified, including Polycomb repressive complex 1 （PRC1）, Polycomb repressive complex 2 （PRC2）, PHO-repressive complex （PhoRC） and Polycomb repressive deubiquitinase （PR-DUB）. In this review, the recent discoveries of the composition of the above complexes, as well as their roles in regulating histone modifications and gene silencing are discussed. We mainly focus on the composition of PRC1 and PRC2 complex and recruitment of PcG to target genes and mechanisms of PRC1 and PRC2-mediated gene silencing. Although much progress has been made in understanding gene silencing mediated by PcG proteins, we also discuss several important questions that still remained unanswered, such as the inheritance of histone modifications during cell division.     

14-3-3sigma controls mitotic translation to facilitate cytokinesis

14-3-3 proteins are crucial in a wide variety of cellular responses including cell cycle progression, DNA damage checkpoints and apoptosis. One particular 14-3-3 isoform, sigma, is a p53-responsive gene, the function of which is frequently lost in human tumours, including breast and prostate cancers as a result of either hypermethylation of the 14-3-3sigma promoter or induction of an oestrogen-responsive ubiquitin ligase that specifically targets 14-3-3sigma for proteasomal degradation. Loss of 14-3-3sigma protein occurs not only within the tumours themselves but also in the surrounding pre-dysplastic tissue (so-called field cancerization), indicating that 14-3-3sigma might have an important tumour suppressor function that becomes lost early in the process of tumour evolution. The molecular basis for the tumour suppressor function of 14-3-3sigma is unknown. Here we report a previously unknown function for 14-3-3sigma as a regulator of mitotic translation through its direct mitosis-specific binding to a variety of translation/initiation factors, including eukaryotic initiation factor 4B in a stoichiometric manner. Cells lacking 14-3-3sigma, in marked contrast to normal cells, cannot suppress cap-dependent translation and do not stimulate cap-independent translation during and immediately after mitosis. This defective switch in the mechanism of translation results in reduced mitotic-specific expression of the endogenous internal ribosomal entry site (IRES)-dependent form of the cyclin-dependent kinase Cdk11 (p58 PITSLRE), leading to impaired cytokinesis, loss of Polo-like kinase-1 at the midbody, and the accumulation of binucleate cells. The aberrant mitotic phenotype of 14-3-3sigma-depleted cells can be rescued by forced expression of p58 PITSLRE or by extinguishing cap-dependent translation and increasing cap-independent translation during mitosis by using rapamycin. Our findings show how aberrant mitotic translation in the absence of 14-3-3sigma impairs mitotic exit to generate binucleate cells and provides a potential explanation of how 14-3-3sigma-deficient cells may progress on the path to aneuploidy and tumorigenesis.     

Tumour maintenance is mediated by eNOS

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.