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.

The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Here we show that Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, we observe specific reactivation of the Myc-repressed p21Cip1 gene, whereas the expression of Myc-activated E-boxes genes is unchanged. In addition, we find that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA-binding factor Miz-1. Consistent with this, we observe that Myc and Dnmt3a form a ternary complex with Miz-1 and that this complex can corepress the p21Cip1 promoter. Finally, we show that DNA methylation is required for Myc-mediated repression of p21Cip1. Our data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell.

In plants, as in mammals, mutations in SNF2-like DNA helicases/ATPases were shown to affect not only chromatin structure but also global methylation patterns, suggesting a potential functional link between chromatin structure and epigenetic marks. The SNF2-like ATPase containing nucleosome remodeling and deacetylase corepressor complex (NuRD) is involved in gene transcriptional repression and chromatin remodeling. We have previously shown that the leukemogenic protein PML-RARa represses target genes through recruitment of DNA methytransferases and Polycomb complex. Here, we demonstrate a direct role of the NuRD complex in aberrant gene repression and transmission of epigenetic repressive marks in acute promyelocytic leukemia (APL). We show that PML-RARa binds and recruits NuRD to target genes, including to the tumor-suppressor gene RAR␤2. In turn, the NuRD complex facilitates Polycomb binding and histone methylation at lysine 27. Retinoic acid treatment, which is often used for patients at the early phase of the disease, reduced the promoter occupancy of the NuRD complex. Knockdown of the NuRD complex in leukemic cells not only prevented histone deacetylation and chromatin compaction but also impaired DNA and histone methylation, as well as stable silencing, thus favoring cellular differentiation. These results unveil an important role for NuRD in the establishment of altered epigenetic marks in APL, demonstrating an essential link between chromatin structure and epigenetics in leukemogenesis that could be exploited for therapeutic intervention.

It has been previously shown that S100A2 is down-regulated in tumor cells and can be considered a tumor suppressor. We have recently shown that this down-regulation can be observed particularly in epithelial tissue, where S100A2 expression decreases remarkably in tumors as compared with normal specimens. In the present paper we investigate whether S100A2 could play a tumor-suppressor role in certain epithelial tissues by acting at the cell migration level. To this end, we made use of five in vitro human head and neck squamous cell carcinoma lines in which we characterized S100A2 expression at both RNA and protein level. To characterize the influence of S100A2 on cell kinetic and cell motility features, we used two complementary approaches involving specific antisense oligonucleotides and the addition of S100A2 to the culture media. The different expression analyses gave a coherent demonstration of the fact that the FADU and the RPMI-2650 cell lines exhibit high and low levels of S100A2 expression, respectively. Antisense oligonucleotides (in FADU) and extracellular treatments (in RPMI) showed that, for these two models, S100A2 had a clear inhibitory influence on cell motility while modifying the cell kinetic parameters only slightly. These effects seem to be related, at least in part, to a modification in the polymerization/depolymerization dynamics of the actin microfilamentary cytoskeleton. Furthermore, we found evidence of the presence of the receptor for advanced glycation end-products (RAGE) in RPMI cells, which may act as a receptor for extracellular S100A2. The present study therefore presents experimentally based evidence showing that S100A2 could play a tumor-suppressor role in certain epithelial tissues by restraining cell migration features, at least in the case of head and neck squamous cell carcinomas.