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Soon after its discovery 75 years ago, heterochromatin, a dense chromosomal material, was found to silence genes. But its importance in regulating gene expression was controversial. Long thought to be inert, heterochromatin is now known to give rise to small RNAs, which, by means of RNA interference, direct the modification of proteins and DNA in heterochromatic repeats and transposable elements. Heterochromatin has thus emerged as a key factor in epigenetic regulation of gene expression, chromosome behaviour and evolution.

Mobile small RNA in plants provides cell-to-cell communication that affects gene expression during development. It has been known for almost 100 years that when a plant virus infects a leaf, mobile signals are transmitted through vessels in the stem to other leaves to confer resistance to subsequent infection. More recently, the silencing of exogenous transgenes has been shown to involve a mobile signal ( 1 ). Although RNA molecules have been implicated in systemic plant cell-to-cell communication, the nature of mobile RNA that silences gene expression has not been clear ( 2 ). Now, four studies—including those by Molnar et al. ( 3 ) and Dunoyer et al. ( 4 ) on pages 872 and 912 of this issue—report that small interfering RNA (siRNA) and microRNA (miRNA) are mobile signals that control gene expression during plant development.

Marking a histone for life DNA replication disrupts both heterochromatin and euchromatin structure, yet the histone marks within chromatin are still transmitted accurately from generation to generation. Li et al . describe a molecular link between DNA replication and histone methylation that sheds light on how epigenetic marks are transmitted during the cell cycle. In the fission yeast Schizosaccharomyces pombe , the methylation of histone H3K9 is regulated by the cell cycle and involves RNA interference. A complex containing two silencing factors, Dos2 and Rik1, together with transcription regulatory factor Mms19 and the catalytic subunit of DNA polymerase ɛ, is required for coordinated DNA replication and heterochromatin assembly, and affects the activity of RNA polymerase II within heterochromatin. Histone modification marks have an important role in many chromatin processes 1 , 2 . During DNA replication, both heterochromatin and euchromatin are disrupted ahead of the replication fork and are then reassembled into their original epigenetic states behind the fork 3 , 4 . How histone marks are accurately inherited from generation to generation is still poorly understood. In fission yeast ( Schizosaccharomyces pombe ), RNA interference (RNAi)-mediated histone methylation is cell cycle regulated. Centromeric repeats are transiently transcribed in the S phase of the cell cycle and are processed into short interfering RNAs (siRNAs) by the complexes RITS (RNA-induced initiation of transcriptional gene silencing) and RDRC (RNA-directed RNA polymerase complex) 5 , 6 , 7 . The small RNAs together with silencing factors—including Dos1 (also known as Clr8 and Raf1), Dos2 (also known as Clr7 and Raf2), Rik1 and Lid2—promote heterochromatic methylation of histone H3 at lysine 9 (H3K9) by a histone methyltransferase, Clr4 (refs 8–13 ). The methylation of H3K9 provides a binding site for Swi6, a structural and functional homologue of metazoan heterochromatin protein 1 (HP1) 14 . Here we characterize a silencing complex in fission yeast that contains Dos2, Rik1, Mms19 and Cdc20 (the catalytic subunit of DNA polymerase-ε). This complex regulates RNA polymerase II (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.

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Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.

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We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4and 10-5per F1plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

The bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts. A third translocation mechanism in chloroplasts [the proton concentration difference (ΔpH) pathway] was previously thought to be unique. The hcf106 mutation of maize disrupts the localization of proteins transported through this ΔpH pathway in isolated chloroplasts. The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the ΔpH pathway has been conserved since the endosymbiotic origin of chloroplasts. Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.

Gene silencing depends on the higher-order structure of heterochromatin as well as repressive biochemical modifications. In eukaryotes, the two states of chromatin were first distinguished cytologically according to their different levels of condensation. Euchromatin has an open conformation that correlates with an active state of gene expression, whereas the transcriptionally silent heterochromatin is tightly packaged. On page 1448 of this issue, Moissiard et al. describe the identification of proteins that are specifically required for the condensed structure of heterochromatin in flowering plants ( 1 ). And on page 1445 of this issue, Qian et al. ( 2 ) identify a plant protein that binds to biochemically modified DNA and itself modifies histones to generate a chromatin state that allows active DNA demethylation (and gene expression).