EZH2 is the catalytic subunit of the PRC2 Polycomb complex and mediates transcriptional repression through its histone methyltransferase activity. EZH2 is upregulated in normal germinal center (GC) B cells and is implicated in lymphomagenesis. To explore the transcriptional programs controlled by EZH2, we performed chromatin immunoprecipitation (ChIP-onchip) in GC cells and found that it binds approximately 1800 promoters, often associated with DNA sequences similar to Droso-phila Polycomb response elements. While EZH2 targets overlapped extensively between GC B cells and embryonic stem cells, we also observed a large GC-specific EZH2 regulatory program. These genes are preferentially histone 3 lysine 27-trimethylated and repressed in GC B cells and include several key cell cycle-related tumor suppressor genes. Accordingly, siRNA-mediated down-regulation of EZH2 in diffuse large B-cell lymphoma ( IntroductionPolycomb proteins (PcG) are chromatin regulators with a crucial role in establishing and maintaining epigenetic memory during development and cellular differentiation. PcG is organized into 2 main sets of protein complexes: PRC1 and PRC2. EZH2 is a subunit of PRC2, 1 and its SET domain catalyzes trimethylation of H3K27, 1-3 a histone modification associated with transcriptional silencing. H3K27me3 helps recruit PRC1 to chromatin; it is thought that PRC1 is the effector of PcG-mediated silencing and long-term epigenetic memory. [4][5][6] It has been observed that H3K27me3 and DNA methylation, a distinct epigenetic mark, are associated with different sets of genes in murine and human embryonic stem cells (hESCs); 7,8 moreover DNA methylation and H3K27me3 are mutually exclusive at the imprinted Rasgrf1 locus. 9 However, this pattern of mutual exclusion between the 2 epigenetic mechanisms appears to be disrupted in cancer cells, where many hypermethylated promoters have been shown to also be H3K27-trimethylated. 10 From a functional point of view, mice deficient in PcG complexes show developmental abnormalities and embryonic lethality. 11 Within the B-cell lineage, it was shown that EZH2 is highly expressed in lymphoid progenitors, and EZH2 deficiency induces defects in early lymphopoiesis. 12 EZH2 declines in resting B cells but is then massively up-regulated when activated B cells form germinal centers (GCs), wherein they undergo rapid proliferation and immunoglobulin affinity maturation. 13 The latter observations suggest an important role for EZH2 in GC B-cell proliferation and a possible contribution to diffuse large B-cell lymphomas (DLBCLs), which are derived from GC B cells. The potential importance of EZH2 in lymphomagenesis is further supported by the discovery of a missense mutation in the EZH2 SET domain in a sizeable fraction of DLBCLs, especially those featuring the GC B-cell gene expression signature. 14 More generally, EZH2 is overexpressed in several other types of cancer (eg, in metastatic prostate cancer, 15 breast cancer, 16 and mantle cell lymphoma 17 ).The mechanisms by which EZH2-mediated...

SummarySem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability

The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits. The rules governing subunit assembly and placement are not understood. We show that a different kind of proteasome forms in yeast when the Pre9/a3 subunit is deleted. Purified pre9D proteasomes show a two-fold enrichment for the Pre6/a4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring. Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges. When Arabidopsis PAD1/a4 is expressed in yeast, it complements not only pre6D but also pre6D pre9D mutants; therefore, the plant a4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9D cells, suggesting an advantage for Pre9 over Pre6 incorporation at the a3 position that facilitates correct assembly.

A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6 ), and 912 (rdn4 ) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.