Ekaterina Voronina

Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis

Despite the well-established role of heterochromatin in protecting chromosomal integrity during meiosis and mitosis, the contribution and extent of heterochromatic histone posttranslational modifications (PTMs) remain poorly defined. Here, we gained novel functional insight about heterochromatic PTMs by analyzing histone H3 purified from the heterochromatic germline micronucleus of the model organism Tetrahymena thermophila. Mass spectrometric sequencing of micronuclear H3 identified H3K23 trimethylation (H3K23me3), a previously uncharacterized PTM. H3K23me3 became particularly enriched during meiotic leptotene and zygotene in germline chromatin of Tetrahymena and C. elegans. Loss of H3K23me3 in Tetrahymena through deletion of the methyltransferase Ezl3p caused mislocalization of meiosis-induced DNA double-strand breaks (DSBs) to heterochromatin, and a decrease in progeny viability. These results show that an evolutionarily conserved developmental pathway regulates H3K23me3 during meiosis, and our studies in Tetrahymena suggest this pathway may function to protect heterochromatin from DSBs. DOI: http://dx.doi.org/10.7554/eLife.02996.001

The P granule component PGL-1 promotes the localization and silencing activity of the PUF protein FBF-2 in germline stem cells

In the C. elegans germline, maintenance of undifferentiated stem cells depends on the PUF family RNA-binding proteins FBF-1 and FBF-2. FBF-1 and FBF-2 are 89% identical and are required redundantly to silence the expression of mRNAs that promote meiosis. Here we show that, despite their extensive sequence similarity, FBF-1 and FBF-2 have different effects on target mRNAs. FBF-1 promotes the degradation and/or transport of meiotic mRNAs out of the stem cell region, whereas FBF-2 prevents translation. FBF-2 activity depends on the P granule component PGL-1. PGL-1 is required to localize FBF-2 to perinuclear P granules and for efficient binding of FBF-2 to its mRNA targets. We conclude that multiple regulatory mechanisms converge on meiotic RNAs to ensure silencing in germline stem cells. Our findings also support the view that P granules facilitate mRNA silencing by providing an environment in which translational repressors can encounter their mRNA targets immediately upon exit from the nucleus.

The diverse functions of germline P-granules in Caenorhabditis elegans

P‐granules are conserved cytoplasmic organelles, similar to nuage, that are present in Caenorhabditis elegans germ cells. Based on the prevailing sterility phenotype of the component mutants, P‐granules have been seen as regulators of germ cell development and function. Yet, specific germline defects resulting from P‐granule failure vary, depending on which component(s) are inactivated, at which stage of development, as well as on the presence of stress factors during animal culture. This review discusses the unifying themes in many P‐granule functions, with the main focus on their role as organizing centers nucleating RNA regulation in the germ cell cytoplasm. Mol. Reprod. Dev. 80:624–631, 2013.

Reader domain specificity and lysine demethylase-4 family function

The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitro demethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis.

Analysis of the Caenorhabditis elegans innate immune response to Coxiella burnetii.

The nematode Caenorhabditis elegans is well established as a system for characterization and discovery of molecular mechanisms mediating microbe-specific inducible innate immune responses to human pathogens. Coxiella burnetii is an obligate intracellular bacterium that causes a flu-like syndrome in humans (Q fever), as well as abortions in domesticated livestock, worldwide. Initially, when wild type C. elegans (N2 strain) was exposed to mCherry-expressing C. burnetii (CCB) a number of overt pathological manifestations resulted, including intestinal distension, deformed anal region and a decreased lifespan. However, nematodes fed autoclave-killed CCB did not exhibit these symptoms. Although vertebrates detect C. burnetii via TLRs, pathologies in tol-1(–) mutant nematodes were indistinguishable from N2, and indicate nematodes do not employ this orthologue for detection of C. burnetii. sek-1(–) MAP kinase mutant nematodes succumbed to infection faster, suggesting that this signaling pathway plays a role in immune activation, as previously shown for orthologues in vertebrates during a C. burnetii infection. C. elegans daf-2(–) mutants are hyper-immune and exhibited significantly reduced pathological consequences during challenge. Collectively, these results demonstrate the utility of C. elegans for studying the innate immune response against C. burnetii and could lead to discovery of novel methods for prevention and treatment of disease in humans and livestock.

Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos.

Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope (NE), where they facilitate exchange of molecules between the cytoplasm and the nucleoplasm. In most cell types, NPCs are evenly distributed around the NE. However, the mechanisms dictating NPC distribution are largely unknown. Here, we used the model organism Caenorhabditis elegans to identify genes that affect NPC distribution during early embryonic divisions. We found that down-regulation of the Sm proteins, which are core components of the spliceosome, but not down-regulation of other splicing factors, led to clustering of NPCs. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. We further found that these mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.

Dynein light chain DLC-1 promotes localization and function of the PUF protein FBF-2 in germline progenitor cells.

PUF family translational repressors are conserved developmental regulators, but the molecular function provided by the regions flanking the PUF RNA-binding domain is unknown. In C. elegans, the PUF proteins FBF-1 and FBF-2 support germline progenitor maintenance by repressing production of meiotic proteins and use distinct mechanisms to repress their target mRNAs. We identify dynein light chain DLC-1 as an important regulator of FBF-2 function. DLC-1 directly binds to FBF-2 outside of the RNA-binding domain and promotes FBF-2 localization and function. By contrast, DLC-1 does not interact with FBF-1 and does not contribute to FBF-1 activity. Surprisingly, we find that the contribution of DLC-1 to FBF-2 activity is independent of the dynein motor. Our findings suggest that PUF protein localization and activity are mediated by sequences flanking the RNA-binding domain that bind specific molecular partners. Furthermore, these results identify a new role for DLC-1 in post-transcriptional regulation of gene expression. Summary: The C. elegans PUF protein FBF-2, but not its homolog FBF-1, directly binds dynein light chain DLC-1, regulating FBF-2 localization and activity in a dynein motor-independent manner.

Splicing Machinery Facilitates Post-Transcriptional Regulation by FBFs and Other RNA-Binding Proteins in Caenorhabditis elegans Germline

Genetic interaction screens are an important approach for understanding complex regulatory networks governing development. We used a genetic interaction screen to identify cofactors of FBF-1 and FBF-2, RNA-binding proteins that regulate germline stem cell proliferation in Caenorhabditis elegans. We found that components of splicing machinery contribute to FBF activity as splicing factor knockdowns enhance sterility of fbf-1 and fbf-2 single mutants. This sterility phenocopied multiple aspects of loss of fbf function, suggesting that splicing factors contribute to stem cell maintenance. However, previous reports indicate that splicing factors instead promote the opposite cell fate, namely, differentiation. We explain this discrepancy by proposing that splicing factors facilitate overall RNA regulation in the germline. Indeed, we find that loss of splicing factors produces synthetic phenotypes with a mutation in another RNA regulator, FOG-1, but not with a mutation in a gene unrelated to posttranscriptional regulation (dhc-1). We conclude that inefficient pre-mRNA splicing may interfere with multiple posttranscriptional regulatory events, which has to be considered when interpreting results of genetic interaction screens.

Methylation of histone H3 at lysine 23 in meiotic heterochromatin

Heterochromatin and its associated histone modifications are important for repressing transcription and maintaining chromosomal integrity during meiosis and mitosis. The complex repertoire of histone modifications that decorate heterochromatin has yet to be fully characterized, in part because most eukaryotic cells have a single nucleus where distinct chromatin types are intermingled on contiguous stretches of chromosomes. To obtain highly-purified heterochromatin, we turned to the model organism Tetrahymena thermophila, which has a biochemically separable heterochromatic micronucleus. We characterized combinatorial histone modifications on heterochromatic H3 from Tetrahymena and identified species of H3 dually-modified by both H3K23me3 and H3K27me3 as a previously unreported binary ‘mark’ specific for heterochromatin. Furthermore, H3K23me3 levels dramatically increased during meiosis in Tetrahymena micronuclei, C. elegans, and mice, suggesting this histone ‘mark’ plays a conserved role in germline development. Lastly, disrupting the H3K23 methyltransferase in Tetrahymena caused a lag in meiotic progression. Together, our data suggests H3K23me3 is a conserved heterochromatic histone PTM strongly associated with meiosis, and misregulation of this modification may be linked to problems with reproductive fitness and development.

Caenorhabditis elegans DLC-1 associates with ribonucleoprotein complexes to promote mRNA regulation

Ribonucleoprotein complexes, which contain mRNAs and their regulator proteins, carry out post‐transcriptional control of gene expression. The function of many RNA‐binding proteins depends on their association with cofactors. Here, we use a genomic approach to identify transcripts associated with DLC‐1, a protein previously identified as a cofactor of two unrelated RNA‐binding proteins that act in the Caenorhabditis elegans germline. Among the 2732 potential DLC‐1 targets, most are germline mRNAs associated with oogenesis. Removal of DLC‐1 affects expression of its targets expressed in the oocytes, meg‐1 and meg‐3. We propose that DLC‐1 acts as a cofactor for multiple ribonucleoprotein complexes, including the ones regulating gene expression during oogenesis.