SummaryEukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression.

The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWIpiRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwimediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged PiwipiRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.[Keywords: H3K9 methylation; Piwi; transposon silencing; heterochromatin formation; piRNA pathway; transcriptional silencing] Supplemental material is available for this article. In fungi, plants, and animals, nuclear Argonaute proteins target nascent transcripts-RNAs that are still attached to their originating DNA locus via the transcribing RNA polymerase. This provides an opportunity for small RNA silencing pathways to connect to the various cellular chromatin-modifying activities for the sequence-specific formation of heterochromatin, typically at transposable element (TE) insertions (Castel and Martienssen 2013).Pioneering work in fission yeast has identified methylation of histone H3 at Lys9 (H3K9me2/3) and histone deacetylation as two major hallmarks that are required for small RNA-guided silencing and heterochromatin formation (Nakayama et al. 2001;Hall et al. 2002;Volpe et al. 2002;Yamada et al. 2005). A sequential order of events downstream from the recruitment of the Schizosaccharomyces pombe Ago1/siRNA complex to nascent RNA of centromeric repeats has been described. According to this, the Ago1/siRNA complex recruits the H3K9 methyltransferase Clr4 to chromatin, which results in H3K9 methylation (Noma et al. 2004;Verdel et al. 2004). This is believed to be a binding platform for the chromodomain-containing protein Swi6/HP1, which in turn recruits the major histone deacetylase and remodeling complex SHREC (Sugiyama et al. 2007). The final outcome of these events is the establishment of a nucleosome-dense region with low histone turnover, which effectively prevents the recruitment of RNA polymerase II (Pol II) to transcription initiation sites and hence transcription as such (e.g., Aygun et al. 2013).H3K9 methylation is also a hallmark of small RNAguided heterochromatin formation in plants, ciliates, and multicellular animals. In animal gonads, many TE insertions are methylated at...

The PIWI-interacting RNA (piRNA) pathway is a small RNA silencing system that keeps selfish genetic elements such as transposons under control in animal gonads. Several lines of evidence indicate that nuclear PIWI family proteins guide transcriptional silencing of their targets, yet the composition of the underlying silencing complex is unknown. Here we demonstrate that the double CHHC zinc finger protein gametocyte-specific factor 1 (Gtsf1) is an essential factor for Piwi-mediated transcriptional repression in Drosophila. Cells lacking Gtsf1 contain nuclear Piwi loaded with piRNAs, yet Piwi's silencing capacity is ablated. Gtsf1 interacts directly with a small subpool of nuclear Piwi, and loss of Gtsf1 phenocopies loss of Piwi in terms of deregulation of transposons, loss of H3K9 trimethylation (H3K9me3) marks at euchromatic transposon insertions, and deregulation of genes in proximity to repressed transposons. We propose that only a small fraction of nuclear Piwi is actively engaged in target silencing and that Gtsf1 is an essential component of the underlying Piwi-centered silencing complex.[Keywords: transposon control; piRNA pathway; Piwi; Gtsf1; transcriptional silencing; heterochromatin formation] Supplemental material is available for this article. The nuclear Piwi-piRNA complex inhibits transcription of TEs by an unknown mechanism. Target silencing is accompanied by histone H3 Lys 9 trimethylation (H3K9me3) and appears to require the HP1 family member Su(var)205 (Shpiz et al. 2011;Wang and Elgin 2011;Sienski et al. 2012;Le Thomas et al. 2013;Rozhkov et al. 2013). The piRNA pathway factor Maelstrom (Mael) is required for Piwi-guided transcriptional silencing, yet loss of Mael (in contrast to loss of Piwi) does not result in significant loss of H3K9me3 marks at repressed TE insertions (Sienski et al. 2012). Whether H3K9me3, a mark for heterochromatin, is required for transcriptional silencing or accompanies it is therefore not fully understood.Conceptually, TE silencing by Piwi might resemble the much better understood process of RNAi-induced heterochromatin formation in Schizosaccharomyces pombe (Moazed 2009;Grewal 2010;Castel and Martienssen 2013). In both systems, a nuclear Argonaute protein guides the transcriptional silencing of targets, and this is accompanied by H3K9 methylation. Fission yeast AGO1 assembles in the trimeric RNA-induced transcriptional silencing (RITS) complex and recruits the histone methyltransferase complex CLRC that mediates H3K9 methylation. In analogy, vertebrate and invertebrate nuclear PIWI proteins are likely to assemble a silencing complex that interacts with nascent RNA at the target locus and induces transcriptional silencing by recruiting factors impacting RNA polymerase II (Pol II) recruitment and/or the chromatin status. However, besides Mael and possibly Su(var)205, no factor is known to be required for

p21(Waf1) (p21) was described as a cyclin-dependent kinase inhibitor, but other p21 activities have subsequently been described, including its ability to inhibit apoptosis in some models. Comparative work on the human colon cancer isogenic cell lines HCT116 and HCT116p21(-/-) led to the proposal that p21 protects colon cancer cells against apoptosis by genotoxic drugs. We asked whether p21 also protected from cell death induced by non-genotoxic drugs, such as tyrosine kinase inhibitors. We found that p21-deficient cells were dramatically more sensitive towards imatinib and gefitinib than parental cells. Interestingly, HCT116p21(-/-) also showed higher basal activity of protein kinases as c-Abl, c-Src, and Akt. We generated HCT116p21(-/-) sublines with inducible p21 expression and found that p21 did not rescue the hypersensitivity to imatinib. Moreover, down-regulation of p21 by enforced c-Myc expression or by p21 siRNA did not sensitize parental HCT116 cells. We found that, in HCT116p21(-/-) cells, p53 showed higher stability, higher transcriptional activity and phosphorylation in serines associated with p53 activity. Furthermore, silencing of p53 with siRNA and inactivation of p53 with a dominant negative mutant rescued the hypersensitive response to kinases inhibitors, 5-fluorouracil and adriamycin in HCT116p21(-/-) cells. Consistently, HCT116p53(-/-) cells are more resistant to imatinib than parental cells, suggesting that imatinib activity is partly dependent on p53 in colon cancer cells. We conclude that high p53 activity, rather than p21 deficiency, is the mechanism responsible for hypersensitivity to drugs of HCT116p21(-/-) cells. Therefore the role of p21 on apoptosis of HCT116 colon cancer cells should be re-evaluated.