Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery.

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 PIWI-piRNA 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 Piwi-mediated 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 Piwi-piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.

Drosophila Gtsf1 is an essential component of the Piwi-mediated transcriptional silencing complex.

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.

Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.

Eukaryotic 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.

HCT116 cells deficient in p21(Waf1) are hypersensitive to tyrosine kinase inhibitors and adriamycin through a mechanism unrelated to p21 and dependent on p53.

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.