Argonaute (Ago) proteins mediate silencing of nucleic acid targets by small RNAs. In fission yeast, Ago1, Tas3 and Chp1 assemble into a RITS complex, which silences transcription near centromeres. Here we describe a repetitive motif within Tas3, termed the 'Argonaute hook', that is conserved from yeast to humans and binds Ago proteins through their PIWI domains in vitro and in vivo. Site-directed mutation of key residues in the motif disrupts Ago binding and heterochromatic silencing in vivo. Unexpectedly, a PIWI domain pocket that binds the 5' end of the short interfering RNA guide strand is required for direct binding of the Ago hook. Moreover, wild-type but not mutant Ago hook peptides derepress microRNA-mediated translational silencing of a target messenger RNA. Proteins containing the conserved Ago hook may thus be important regulatory components of effector complexes in RNA interference.

The FACT complex is a conserved cofactor for RNA polymerase II elongation through nucleosomes. FACT bears histone chaperone activity and contributes to chromatin integrity. However, the molecular mechanisms behind FACT function remain elusive. Here we report biochemical, structural, and mutational analyses that identify the peptidase homology domain of the Schizosaccharomyces pombe FACT large subunit Spt16 (Spt16-N) as a binding module for histones H3 and H4. The 2.1-Å crystal structure of Spt16-N reveals an aminopeptidase P fold whose enzymatic activity has been lost. Instead, the highly conserved fold directly binds histones H3-H4 through a tight interaction with their globular core domains, as well as with their N-terminal tails. Mutations within a conserved surface pocket in Spt16-N or posttranslational modification of the histone H4 tail reduce interaction in vitro, whereas the globular domains of H3-H4 and the H3 tail bind distinct Spt16-N surfaces. Our analysis suggests that the N-terminal domain of Spt16 may add to the known H2A-H2B chaperone activity of FACT by including a H3-H4 tail and H3-H4 core binding function mediated by the N terminus of Spt16. We suggest that these interactions may aid FACT-mediated nucleosome reorganization events.histone chaperone ͉ histone modifications ͉ protein evolution ͉ site-directed mutagenesis ͉ transcription N ucleosomes create a natural barrier to RNA polymerase II (Pol II) progression. Transcription of histone-wrapped DNA thus requires factors that promote nucleosome remodeling, such as the histone chaperone FACT (facilitates chromatin transcription), which in human cells purifies as a heterodimer of Spt16 and SSRP1 (1, 2).FACT is a highly conserved complex (2-5). In fungi, SSRP1 is largely encoded by POB3, whereas NHP6 encodes the archetypal HMG-box of SSRP1. The gene for the Spt16 subunit is essential in Saccharomyces cerevisiae and Schizosaccharomyces pombe, probably reflecting important chromatin-related functions in transcription, replication, and DNA repair (4, 6-9). Genetic screens identified Spt16 as a factor whose mutation restores the expression of a Ty1 transposon-silenced reporter gene by promoting its cryptic transcription, known as a suppressor of Ty1 phenotype, [Spt Ϫ ] (4, 7, 10, 11). Several POB3 alleles genes display [Spt Ϫ ] phenotypes (12), indicating that the maintenance of correct chromatin structure involves Pol II cofactors such as FACT (13). Indeed, FACT acts as a coactivator of transcriptional initiation and elongation (14), and many Spt16 alleles display genetic interactions with basal transcription factors. Furthermore, FACT subunits biochemically interact with the Pol II elongation complex Paf1 (15), bind the coding region of transcribed Pol II genes, and are recruited to inducible genes upon activation (16-19).FACT's biological roles in transcription (and replication) may stem from its histone chaperone activity (20,21). Histone chaperones stimulate reactions involving the transfer of histones (22), thereby mediating chromatin reorganiz...

Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular ‘nucleation sites' by RNA interference (RNAi), ensuring the mitotic stability of centromere-bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6HP1 are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6HP1 operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.