In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC) to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA), which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and possibly in other apicomplexans. This study establishes Toxoplasma as a unique model system for studying the evolution and molecular mechanisms of RNA silencing among eukaryotes.

Posttranslational histone modifications modulate chromatin-templated processes in various biological systems. H4K20 methylation is considered to have an evolutionarily ancient role in DNA repair and genome integrity, while its function in heterochromatin function and gene expression is thought to have arisen later during evolution. Here, we identify and characterize H4K20 methylases of the Set8 family in Plasmodium and Toxoplasma, two medically important members of the protozoan phylum Apicomplexa. Remarkably, parasite Set8-related proteins display H4K20 mono-, di-, and trimethylase activities, in striking contrast to the monomethylase-restricted human Set8. Structurally, few residues forming the substrate-specific channel dictate enzyme methylation multiplicity. These enzymes are cell cycle regulated and focally enriched at pericentric and telomeric heterochromatin in both parasites. Collectively, our findings provide new insights into the evolution of Set8-mediated biochemical pathways, suggesting that the heterochromatic function of the marker is not restricted to metazoans. Thus, these lower eukaryotes have developed a diverse panel of biological stages through their high capacity to differentiate, and epigenetics only begins to emerge as a strong determinant of their biology.The fundamental unit of chromatin, termed the nucleosome, is subject to a dizzying array of posttranslational modifications, which work alone or in combination to constitute a histone code that regulates chromatin structure and function (18). Among these modifications, lysine methylations index chromatin regions, facilitating epigenetic organization of eukaryotic genomes. In contrast to other histone-modifying enzymes, histone lysine (K) methyltransferases (HKMTs) are enzymes devoted to the methylation of highly specific lysine residues which either promote gene activation (H3K4, H3K79, and H3K36) or generate a repressed chromatin state (H3K9, H3K27, and H4K20). With just one exception (Dot1p), these enzymes belong to the SET family. The SET domain, which initially took its name from the Drosophila genes Su(var)3-9, enhancer of zeste, and trithorax, is crucial for the catalytic activity of HKMTs (3, 9, 24). While lysine methylation mainly occurs on histone H3 tails, the only lysine residue of histone H4 shown to be methylated in vivo is lysine 20. Methylation of H4K20 is involved in a diverse array of nuclear processes, including gene silencing (12, 26), pericentric heterochromatin formation (37), mitotic regulation in metazoans (20, 21), and DNA damage checkpoint control in the cell cycle of Schizosaccharomyces pombe (35).A number of different SET proteins have been identified as being able to methylate H4K20, including metazoan Set8 (also named Pr-Set7) (12, 26), Drosophila ASH1 (2), murine NSD1 (30), mammalian SUV4-20H1/2 (37), and its S. pombe ortholog SET9 (35). The modification is evidently absent in several simple eukaryotes, such as Saccharomyces cerevisiae and the ciliated protozoan Tetrahymena thermophila (26). H4K20 methylati...

The alkyloid compound ellipticine derived from the berrywood tree is a topoisomerase II poison that is used in ovarian and breast cancer treatment. In this study, we report the identification of ellipticine derivatives and their tetracyclic angular benzopyridoindole analogues as novel ATP-competitive inhibitors of the protein kinase CK2. In vitro and in vivo assays showed that these compounds have a good pharmacologic profile, causing a marked inhibition of CK2 activity associated with cell cycle arrest and apoptosis in human cancer cells. Further, in vivo assays demonstrate antitumor activity in a mouse xenograft model of human glioblastoma. Finally, crystal structures of CK2-inhibitor complex provide structural insights on the molecular basis of CK2 inhibition. Our work lays the foundation for development of clinically useful CK2 inhibitors derived from a well-studied scaffold with suitable pharmacokinetics parameters. Cancer Res; 70(23); 9865-74. Ó2010 AACR.