The specialized ribonuclease Dicer initiates RNA interference by cleaving double-stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.

Histone modification marks play an important role in many chromatin processes1,2. During DNA replication, both heterochromatin and euchromatin are disrupted ahead of the replication fork and then reassembled into their original epigenetic states behind the fork3,4. How the histone marks are faithfully inherited during each generation is still poorly understood. In fission yeast RNA interference (RNAi)-mediated histone methylation is cell-cycle regulated. Centromere repeats are transiently transcribed at S phase and processed into small interference RNAs (siRNAs) by RITS and RDRC complexes5-7. The small RNAs, in concert with silencing factors, including Dos1/Clr8, Dos2/Clr7, Rik1 and Lid2, promote heterochromatic H3K9 methylation by a histone methyltransferase, Clr48-13. H3K9 methylation serves as a binding site for Swi6, a structural and functional homolog of metazoan Heterochromatin Protein 1 (HP1)14. Here we characterize a silencing complex, which contains Dos2, Rik1, Mms19, and Cdc20 (DNA polymerase epsilon). The complex regulates RNA Pol II activity in heterochromatin, and is required for DNA replication and heterochromatin assembly. Our findings provide a molecular link between DNA replication and histone methylation, shedding light on how epigenetic marks are transmitted during each cell cycle.

SUMMARY In most eukaryotes, histone methylation patterns regulate chromatin architecture and function: methylation of histone H3 lysine-9 (H3K9) demarcates heterochromatin while H3K4 methylation demarcates euchromatin. We show here that the S. pombe JmjC-domain protein Lid2 is a trimethyl H3K4 demethylase responsible for H3K4 hypomethylation in heterochromatin. Lid2 interacts with the histone lysine-9 methyltransferase, Clr4, through the Dos1/Clr8-Rik1 complex, which also functions in the RNA interference pathway. Disruption of the JmjC-domain alone results in severe heterochromatin defects and depletion of siRNA, while overexpressing Lid2 enhances heterochromatin silencing. The physical and functional link between H3K4 demethylation and H3K9 methylation suggests that the two reactions act in a coordinated manner. Surprisingly, cross-regulation of H3K4 and H3K9 methylation in euchromatin also requires Lid2. We provide evidence suggesting that Lid2 enzymatic activity in euchromatin is regulated through dynamic interplay with other histone modification enzymes. Our findings provide a mechanistic insight into the coordination of H3K4 and H3K9 methylation.

Dos1 and Dos2 are required for the formation of heterochromatin in fission yeast. We hypothesize that the physical interaction between Dos1 and Rik1 represents a role in regulating activity of the Rik1/Clr4 complex. Dos2 contributes to this role by regulating Dos1 localization. Our findings suggest a mechanism for recruitment of Clr4 in the RNAi-dependent heterochromatin pathway, in which Dos1 and Dos2 are essential.

The centromere is a specific chromosomal locus that organizes the assembly of the kinetochore. It plays a fundamental role in accurate chromosome segregation. In most eukaryotic organisms, each chromosome contains a single centromere the position and function of which are epigenetically specified. Occasionally, centromeres form at ectopic loci, which can be detrimental to the cell. However, the mechanisms that protect the cell against ectopic centromeres (neocentromeres) remain poorly understood. Centromere protein-A (CENP-A), a centromere-specific histone 3 (H3) variant, is found in all centromeres and is indispensable for centromere function. Here we report that the overexpression of CENP-A Cnp1 in fission yeast results in the assembly of CENP-A Cnp1 at noncentromeric chromatin during mitosis and meiosis. The noncentromeric CENP-A preferentially assembles near heterochromatin and is capable of recruiting kinetochore components. Consistent with this, cells overexpressing CENP-A Cnp1 exhibit severe chromosome missegregation and spindle microtubule disorganization. In addition, pulse induction of CENP-A Cnp1 overexpression reveals that ectopic CENP-A chromatin can persist for multiple generations. Intriguingly, ectopic assembly of CENP-A cnp1 is suppressed by overexpression of histone H3 or H4. Finally, we demonstrate that deletion of the N-terminal domain of CENP-A cnp1 results in an increase in the number of ectopic CENP-A sites and provide evidence that the N-terminal domain of CENP-A prevents CENP-A assembly at ectopic loci via the ubiquitindependent proteolysis. These studies expand our current understanding of how noncentromeric chromatin is protected from mistakenly assembling CENP-A.T HE centromere is a specific chromosomal locus that organizes the assembly of the kinetochore. It is vital for the proper segregation of chromosomes during mitosis and meiosis (Allshire and Karpen 2008;Henikoff and Furuyama 2010). Most eukaryotic chromosomes contain a single centromere that is faithfully inherited at the same position within the chromosome through generations. In "point" centromeres of Saccharomyces cerevisiae, a specific 125-bp DNA sequence is both necessary and sufficient to specify centromere position (Clarke and Carbon 1980;Cottarel et al. 1989). However, most other eukaryotes contain regional centromeres, which are more complex and usually consist of large blocks of repetitive DNA sequences (Pluta et al. 1995;Henikoff et al. 2001). Epigenetic mechanisms appear to play a dominant role in the formation and inheritance of regional centromeres (Allshire and Karpen 2008;Henikoff and Furuyama 2010).Centromere protein-A (CENP-A), a centromere-specific histone 3 (H3) variant, has been proposed to act as the epigenetic mark for centromere positioning (Palmer et al. 1991;Henikoff and Furuyama 2010;Burrack and Berman 2012;Muller and Almouzni 2014). CENP-A partially replaces canonical histone H3 at the centromere and provides the structural and functional foundation for the assembly of the kinetochore (Blac...