Complete loss of H3K9 methylation dissolves mouse heterochromatin organization.

Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all six SET domain lysine methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEF cells no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is a compelling analysis of H3K9 methylation-deficient mammalian chromatin and reveals a definitive function for H3K9 methylation in protecting heterochromatin organization and genome integrity.

PRSS50 is a testis protease responsible for proper sperm tail formation and function.

Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenoteratozoospermia. We have identified protease serine 50 (PRSS50) as having a crucial role in sperm development, because Prss50-null mice presented with impaired fertility and sperm tail abnormalities. PRSS50 could also be involved in centrosome function because these mice showed a threefold increase in acephalic sperm (head-tail junction defect), sperm with multiple heads (spermatid division defect) and sperm with multiple tails, including novel two conjoined sperm (complete or partial parts of several flagellum on the same plasma membrane). Our data support that, in the testis, as in tumorigenesis, PRSS50 activates NFκB target genes, such as the centromere protein leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1), which is required for heterochromatin maintenance. Prss50-null testes have increased IκκB, and reduced LRWD1 and histone expression. Low levels of de-repressed histone markers, such as H3K9me3, in the Prss50-null mouse testis may cause increases in post-meiosis proteins, such as AKAP4, affecting sperm formation. We provide important insights into the complex mechanisms of sperm development, the importance of testis proteases in fertility and a novel mechanism for MMAF.

The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast.

Cumulative evidence suggests that non-proteolytic functions of the proteasome are involved in transcriptional regulation, mRNA export, and ubiquitin-dependent histone modification and thereby modulate the intracellular levels of regulatory proteins implicated in controlling key cellular functions. To date, the non-proteolytic roles of the proteasome have been mainly investigated in euchromatin; their effects on heterochromatin are largely unknown. Here, using fission yeast as a model, we randomly mutagenized the subunits of the 19S proteasome subcomplex and sought to uncover a direct role of the proteasome in heterochromatin regulation. We identified a mutant allele, rpt4-1, that disrupts a non-proteolytic function of the proteasome, also known as a non-proteolytic allele. Experiments performed using rpt4-1 cells revealed that the proteasome is involved in the regulation of heterochromatin spreading to prevent its uncontrolled invasion into neighboring euchromatin regions. Intriguingly, the phenotype of the non-proteolytic rpt4-1 mutant resembled that of epe1Δ cells, which lack the Epe1 protein that counteracts heterochromatin spreading. Both mutants exhibited variegated gene-silencing phenotypes across yeast colonies, spreading of heterochromatin, bypassing of the requirement for RNAi in heterochromatin formation at the outer repeat region (otr), and up-regulation of RNA polymerase II. Further analysis revealed Mst2, another factor that antagonizes heterochromatin spreading, may function redundantly with Rpt4. These observations suggest that the 19S proteasome may be involved in modulating the activities of Epe1 and Mst2. In conclusion, our findings indicate that the proteasome appears to have a heterochromatin-regulating function that is independent of its canonical function in proteolysis.

The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1.

Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

Histone demethylase JARID1C inactivation triggers genomic instability in sporadic renal cancer.

Mutations in genes encoding chromatin-remodeling proteins are often identified in a variety of cancers. For example, the histone demethylase JARID1C is frequently inactivated in patients with clear cell renal cell carcinoma (ccRCC); however, it is largely unknown how JARID1C dysfunction promotes cancer. Here, we determined that JARID1C binds broadly to chromatin domains characterized by the trimethylation of lysine 9 (H3K9me3), which is a histone mark enriched in heterochromatin. Moreover, we found that JARID1C localizes on heterochromatin, is required for heterochromatin replication, and forms a complex with established players of heterochromatin assembly, including SUV39H1 and HP1α, as well as with proteins not previously associated with heterochromatin assembly, such as the cullin 4 (CUL4) complex adaptor protein DDB1. Transcription on heterochromatin is tightly suppressed to safeguard the genome, and in ccRCC cells, JARID1C inactivation led to the unrestrained expression of heterochromatic noncoding RNAs (ncRNAs) that in turn triggered genomic instability. Moreover, ccRCC patients harboring JARID1C mutations exhibited aberrant ncRNA expression and increased genomic rearrangements compared with ccRCC patients with tumors endowed with other genetic lesions. Together, these data suggest that inactivation of JARID1C in renal cancer leads to heterochromatin disruption, genomic rearrangement, and aggressive ccRCCs. Moreover, our results shed light on a mechanism that underlies genomic instability in sporadic cancers.

Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae.

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin.

Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼ 90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Histone acetylation: from code to web and router via intrinsically disordered regions.

Structural changes of chromatin, which consists of nucleosomes and nucleosome-associated factors, lead to functional changes that are important determinants of eukaryotic gene regulation. These structural changes are regulated by modifications of histones and DNA, both of which are components of nucleosomes, as well as by replacement of histone variants and the actions of noncoding RNAs. In studies of chromatin modifications, a great deal of attention has been paid to histone acetylation. Progress in understanding this subject has been extensive, including i) elucidation of the relationship of histone acetylation and gene activity; ii) the first isolation of a histonemodifying enzyme; iii) the first identification of a factor that recognizes a modified site; iv) elucidation of the mechanism by which histone modification leads to structural changes in nucleosomes; and v) elucidation of the mechanism of border formation between euchromatin and heterochromatin. Histone acetylation is considered to be fundamental in several fields, including studies of a) the role of chromatin and epigenetics in higher-order biochemical systems such as transcription, DNA replication, and repair; b) biological phenomena such as cell proliferation and differentiation; and c) cancer and aging, potentially leading to clinical applications. In this review, I will discuss the histone code hypothesis, at one time believed to represent a unified theory regarding the functions of histone modification. In addition, I will describe the "modification web theory, " by which the problems in the histone code hypothesis can be overcome, as well as the "signal router theory, " which explains the mechanisms of formation, development, and evolution of the modification web from a structural viewpoint. Lastly, I will illustrate how these novel theories partially explain the robustness of biological systems against various perturbations, and elucidate the strategy that a cell employs to avoid fatal fragility.

Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling.

DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle-dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

FANCJ couples replication past natural fork barriers with maintenance of chromatin structure.

Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer-predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner.

Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation.

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein-DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.

H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases.

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.

DNA methyltransferase 3b preferentially associates with condensed chromatin.

In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a 'reader' of higher-order chromatin structure leading to gene silencing through DNA methylation.

Las terapias epigenéticas, más allá de los biológicos en el tratamiento de la artritis reumatoide / Epigenetic therapies, a step beyond biologics for rheumatoid arthritis

La artritis reumatoide ha experimentado en la última década una revolución terapéutica, derivada del conocimiento de los procesos patogénicos y favorecida por el desarrollo de la tecnología necesaria para distribuir tratamientos moleculares. Las nuevas terapias permiten diferenciar subtipos de pacientes según la respuesta clínica y además mejoran nuestra comprensión de la enfermedad. Ello hace vaticinar la llegada de nuevas generaciones de moléculas para un tratamiento individualizado. Uno de los campos hacia donde se dirigen las investigaciones es la epigenética. Los mecanismos de regulación epigenéticos son interruptores que deciden cuándo y cómo expresar determinados genes en cada célula. Actuando como vigilantes de una expresión génica inapropiada, protegen al organismo del desarrollo de tumores. La principal ventaja de los tratamientos epigenéticos podría ser su selectividad por las células que muestran patrones epigenéticos alterados, por lo que el reto es identificar estas alteraciones entre los pacientes con artritis reumatoide. Aunque debe establecerse su perfil de seguridad, parece probable el uso de terapias epigenéticas en las enfermedades autoinmunes (AU)

Over the last decade, the management of rheumatoid arthritis has evolved as a result of both the understanding of disease-related processes and the availability of the necessary high-throughput technology to provide patients with molecule-based therapies. New therapies allow the classification of patients into subsets as regards clinical response, at the same time adding to our knowledge of rheumatoid arthritis pathogenesis. New generations of molecules will likely soon be ready for “a la carte” treatment of patients. A promising field of research is epigenetics. Epigenetic regulatory mechanisms switch on and off the transcription of specific genes in individual cells. Acting as observers on non-adequate gene expression, these mechanisms yield protection against the development of tumours. The major achievement of epigenetic therapies could be their selective action on cells with altered epigenetic programs, and it is our challenge to recognize these alterations among patients with rheumatoid arthritis. Although safety concerns may arise, epigenetic drugs will likely be used to treat autoimmune diseases (AU)

Major and essential role for the DNA methylation mark in mouse embryogenesis and stable association of DNMT1 with newly replicated regions.

DNA methyltransferase 1 (DNMT1) plays an important role in the inheritance of genomic DNA methylation, which is coupled to the DNA replication process. Early embryonic lethality in DNMT1-null mutant (Dnmt1(c)) mice indicates that DNA methylation is essential for mammalian development. DNMT1, however, interacts with a number of transcriptional regulators and has a transcriptional repressor activity independent of its catalytic activity. To examine the roles of the catalytic activity of DNMT1 in vivo, we generated a Dnmt1(ps) allele that expresses a point-mutated protein that lacks catalytic activity (DNMT1-C1229S). Dnmt1(ps) mutant mice showed developmental arrest shortly after gastrulation, near-complete loss of DNA methylation, and an altered distribution of repressive chromatin markers in the nuclei; these phenotypes are quite similar to those of the Dnmt1(c) mutant. The mutant DNMT1 protein failed to associate with replication foci in Dnmt1(ps) cells. Reconstitution experiments and replication labeling in Dnmt1-/- Dnmt3a-/- Dnmt3b-/- (i.e., unmethylated) embryonic stem cells revealed that preexisting DNA methylation is a major determinant for the cell cycle-dependent localization of DNMT1. The C-terminal catalytic domain of DNMT1 inhibited its stable association with unmethylated chromatin. Our results reveal essential roles for the DNA methylation mark in mammalian development and in DNMT1 localization.

A two-way street: LSD1 regulates chromatin boundary formation in S. pombe and Drosophila.

Two recent studies in Molecular Cell (Lan et al., 2007; Rudolph et al., 2007) implicate histone demethylation by LSD1 in the regulation of boundaries between silenced and active chromatin domains in both fission yeast and flies, but by distinct mechanisms.

Polycomb protein Cbx4 promotes SUMO modification of de novo DNA methyltransferase Dnmt3a.

The 'de novo methyltransferase' Dnmt3a (DNA methyltransferase 3a) has been shown to mediate transcriptional repression. Post-translational modification of Dnmt3a by SUMOylation affects its ability to transcriptionally repress. However, very little is known about how the SUMOylation process is regulated. In the present study, we identified a PcG (Polycomb group) protein, Cbx4 (chromobox 4), as a specific interaction partner of Dnmt3a. Co-expression of Cbx4 and SUMO-1 (small ubiquitin-related modifier-1) along with Dnmt3a in transfected cells results in enhanced modification of Dnmt3a with SUMO-1. Purified Cbx4 also promotes SUMOylation of Dnmt3a in vitro. The modification occurs in the N-terminal regulatory region, including the PWWP (Pro-Trp-Trp-Pro) domain. Our results suggest that Cbx4 functions as a SUMO E3 ligase for Dnmt3a and it might be involved in the functional regulation of DNA methyltransferases by promoting their SUMO modification.

Histone phosphorylation and pericentromeric histone modifications in oocyte meiosis.

Epigenetic regulation of pericentromeric heterochromatin is crucial for proper interactions between kinetochores and spindle microtubules governing accurate chromosome segregation. Here, we first examined the dynamic distribution of phosphorylated serine 10 and 28 on H3 during mouse oocyte maturation and early embryo development using immunofluorescent staining and confocal microscopy. Our results revealed strong signals of phosphorylated H3/ser10 and 28 in the pericentromeric heterochromatin area and continuous persistent staining of the chromosome periphery, respectively. A panel of specific antibodies against various acetylated lysine, dimethylated lysine or phosphorylated serine residues on histone H3 or H4 were used to investigate the effects of Trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), on histone modifications of pericentromeric heterochromatin. Unexpectedly, TSA treatment was unable to alter the acetylation and methylation status of pericentromeric heterochromatin, however, it resulted in significant dephosphorylation of H3/ser10 at this site during mouse oocyte meiosis, which is likely to play a role in the TSA-induced defective chromosome segregation. Furthermore, by using ZM447439, an inhibitor of Aurora kinases, we revealed that Aurora kinases may participate in the regulation of histone phosphorylation during mouse oocyte maturation.

Heterochromatin proteins and the control of heterochromatic gene silencing in Arabidopsis.

The SU(VAR)3-9 protein family was first identified in animals as heterochromatin-associated proteins and found to control establishment of heterochromatic chromatin domains by histone H3 lysine 9 methylation. In Arabidopsis ten SU(VAR)3-9 homologous SUVH genes are found where SUVH1, SUVH2 and SUVH4 represent different subgroups of genes. Also the SUVH1, SUVH2 and SUVH4 proteins represent heterochromatin-associated proteins and display differential effects on control of heterochromatic histone methylation marks. In Arabidopsis the heterochromatin specific histone methylation marks are mono- and dimethyl H3K9, mono- and dimethyl H3K27 and monomethyl H4K20. In contrast to animal systems trimethyl H3K9, trimethyl H3K27 and di- and trimethyl H4K20 do not index chromocenter heterochromatin in Arabidopsis. SUVH2 shows a central role in control of heterochromatin formation and heterochromatic gene silencing in Arabidopsis. Loss-of-function of SUVH2 results in significant reduction of all heterochromatin-specific histone methylation marks and causes DNA hypomethylation at chromocenter heterochromatin. SUVH2 overexpression leads to ectopic heterochromatisation accompanied with significant growth defects. SUVH2 shows strong dosage-dependent effects on transcriptional gene silencing. In Arabidopsis different experimental systems connected with transcriptional gene silencing have been used for genetic dissection of molecular mechanisms controlling epigenetic processes. Molecular analysis of the genes identified by the isolated modifier mutants suggests that transcriptional gene silencing in plants is caused by heterochromatisation. A new efficient experimental system for the analysis of transcriptional gene silencing has been established with the help of LUCIFERASE transgene repeats. The different lines established show either complete or partial silencing of the luciferase transgene repeats. These lines have been successfully used either for mutant isolation or for functional analysis of SUVH proteins in control of heterochromatic gene silencing.

EGO-1, a putative RNA-dependent RNA polymerase, is required for heterochromatin assembly on unpaired dna during C. elegans meiosis.

During meiosis in C. elegans, unpaired chromosomes and chromosomal regions accumulate high levels of histone H3 lysine 9 dimethylation (H3K9me2), a modification associated with facultative heterochromatin assembly and the resulting transcriptional silencing. Meiotic silencing of unpaired DNA may be a widely conserved genome defense mechanism. The mechanisms of meiotic silencing remain unclear, although both transcriptional and posttranscriptional processes are implicated. Cellular RNA-dependent RNA polymerases (RdRPs) function in development and RNA-mediated silencing in many species and in heterochromatin assembly in S. pombe. There are four C. elegans RdRPs, including two with known germline functions. EGO-1 is required for fertility and robust germline RNAi. RRF-3 acts genetically to repress RNAi and is required for normal meiosis and spermatogenesis at elevated temperatures (S. L'Hernault, personal communication). Among C. elegans RdRPs, we find that only EGO-1 is required for H3K9me2 enrichment on unpaired chromosomal regions during meiosis. This H3K9me2 enrichment does not require Dicer or Drosha nuclease or any of several other proteins required for RNAi. ego-1 interacts genetically with him-17, another regulator of chromatin and meiosis, to promote germline development. We conclude that EGO-1 is an essential component of meiotic silencing in C. elegans.