Drosophila Argonaute-1 is critical for transcriptional cosuppression and heterochromatin formation

Argonaute-1 (Ago-1) plays a crucial role in gene regulation and genome stability via biogenesis of small non-coding RNAs. Two “Argonaute” family genes, piwi and Ago-2 in Drosophila are involved in multiple silencing mechanisms in the nucleus, transgene cosuppression, long-distant chromosome interaction, nuclear organization and heterochromatin formation. To investigate whether Ago-1 also plays a similar role, we have generated a series of Ago-1 mutations by excising P element, inserted in the Ago-1 promoter (Ago-1k08121). AGO-1 protein is distributed uniformly in the nucleus and cytosol in early embryos but accumulated predominantly in the cytoplasm during the gastrulation stage. Repeat induced silencing produced by the mini-white (mw) array and transcriptional cosuppression of non-homologous transgenes Adh-w/w-Adh was disrupted by Ago-1 mutation. These effects of Ago-1 are distict from its role in microRNA processing because Dicer-1, a critical enzyme for miRNA biogenesis, has no role on the above silencing. Reduction of AGO-1 protein dislodged the POLYCOMB, EZ (enhancer of zeste) and H3me3K27 binding at the cosuppressed Adh-w transgene insertion sites suggesting its role in Polycomb dependent cosuppression. An overall reduction of methylated histone H3me2K9 and H3me3K27 from the polytene nuclei precisely from the mw promoters was also found that leads to concomitant changes in the chromatin structure. These results suggest a prominent role of Ago-1 in chromatin organization and transgene silencing and demonstrate a critical link between transcriptional transgene cosuppression, heterochromatin formation and chromatin organization. We propose Drosophila Ago-1 as a multifunctional RNAi component that interconnects at least two unrelated events, chromatin organization in the nucleus and microRNA processing in the cytoplasm, which may be extended to the other systems

Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila

<p>Abstract</p> <p>Background</p> <p>Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in <it>Schizosaccharomyces pombe </it>have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on <it>Drosophila </it>heterochromatin structure.</p> <p>Results</p> <p>The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (<it>dcr-2, ago1, ago2, piwi, Lip [D], aub </it>and <it>hls</it>). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of <it>white-mottled4h </it>position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using <it>Drosophila </it>embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites.</p> <p>Conclusion</p> <p>Our experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in <it>Drosophila</it>. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in <it>Drosophila</it>.</p

Telomeric Trans-Silencing in Drosophila melanogaster: Tissue Specificity, Development and Functional Interactions between Non-Homologous Telomeres

BACKGROUND: The study of P element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, "TAS") has the capacity to repress in trans, in the female germline, a homologous P-lacZ transgene located in euchromatin. TSE can show variegation in ovaries, displays a maternal effect as well as an epigenetic transmission through meiosis and involves heterochromatin and RNA silencing pathways. PRINCIPAL FINDINGS: Here, we analyze phenotypic and genetic properties of TSE. We report that TSE does not occur in the soma at the adult stage, but appears restricted to the female germline. It is detectable during development at the third instar larvae where it presents the same tissue specificity and maternal effect as in adults. Transgenes located in TAS at the telomeres of the main chromosomes can be silencers which in each case show the maternal effect. Silencers located at non-homologous telomeres functionally interact since they stimulate each other via the maternally-transmitted component. All germinally-expressed euchromatic transgenes tested, located on all major chromosomes, were found to be repressed by a telomeric silencer: thus we detected no TSE escaper. The presence of the euchromatic target transgene is not necessary to establish the maternal inheritance of TSE, responsible for its epigenetic behavior. A single telomeric silencer locus can simultaneously repress two P-lacZ targets located on different chromosomal arms. CONCLUSIONS AND SIGNIFICANCE: Therefore TSE appears to be a widespread phenomenon which can involve different telomeres and work across the genome. It can explain the P cytotype establishment by telomeric P elements in natural Drosophila populations

HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila

Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways

Genome-Wide Identification of Alternatively Spliced mRNA Targets of Specific RNA-Binding Proteins

BACKGROUND: Alternative splicing plays an important role in generating molecular and functional diversity in multi-cellular organisms. RNA binding proteins play crucial roles in modulating splice site choice. The majority of known binding sites for regulatory proteins are short, degenerate consensus sequences that occur frequently throughout the genome. This poses an important challenge to distinguish between functionally relevant sequences and a vast array of those occurring by chance. METHODOLOGY/PRINCIPAL FINDINGS: Here we have used a computational approach that combines a series of biological constraints to identify uridine-rich sequence motifs that are present within relevant biological contexts and thus are potential targets of the Drosophila master sex-switch protein Sex-lethal (SXL). This strategy led to the identification of one novel target. Moreover, our systematic analysis provides a starting point for the molecular and functional characterization of an additional target, which is dependent on SXL activity, either directly or indirectly, for regulation in a germline-specific manner. CONCLUSIONS/SIGNIFICANCE: This approach has successfully identified previously known, new, and potential SXL targets. Our analysis suggests that only a subset of potential SXL sites are regulated by SXL. Finally, this approach should be directly relevant to the large majority of splicing regulatory proteins for which bonafide targets are unknown

Retrotransposon-Induced Heterochromatin Spreading in the Mouse Revealed by Insertional Polymorphisms

The “arms race” relationship between transposable elements (TEs) and their host has promoted a series of epigenetic silencing mechanisms directed against TEs. Retrotransposons, a class of TEs, are often located in repressed regions and are thought to induce heterochromatin formation and spreading. However, direct evidence for TE–induced local heterochromatin in mammals is surprisingly scarce. To examine this phenomenon, we chose two mouse embryonic stem (ES) cell lines that possess insertionally polymorphic retrotransposons (IAP, ETn/MusD, and LINE elements) at specific loci in one cell line but not the other. Employing ChIP-seq data for these cell lines, we show that IAP elements robustly induce H3K9me3 and H4K20me3 marks in flanking genomic DNA. In contrast, such heterochromatin is not induced by LINE copies and only by a minority of polymorphic ETn/MusD copies. DNA methylation is independent of the presence of IAP copies, since it is present in flanking regions of both full and empty sites. Finally, such spreading into genes appears to be rare, since the transcriptional start sites of very few genes are less than one Kb from an IAP. However, the B3galtl gene is subject to transcriptional silencing via IAP-induced heterochromatin. Hence, although rare, IAP-induced local heterochromatin spreading into nearby genes may influence expression and, in turn, host fitness

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing

Abstract Background The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/− hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. Results Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/− hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/− hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. Conclusions Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing