RNA interference (RNAi) is generally associated with post-transcriptional gene silencing, but there is growing evidence that small RNAs and RNAi components also have a role in transcriptional gene silencing in the nucleus. This study shows that, in Drosophila, the RNAi components Dicer-2 (Dcr2) and Argonaute-2 (AGO2) are present in the nucleus, associating with transcriptionally active gene loci and interacting with RNA polymerase II. Dcr2 and AGO2 are associated with sense and antisense transcripts and seem to have a role in regulating transcription, particularly during heatshock. RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21–30 nucleotide) RNAs1,2,3,4,5,6. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation7,8,9,10. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

Lung cancer is the leading cause of cancer death in the world and there is no current treatment able to efficiently treat the disease as the tumor is often diagnosed at an advanced stage. Moreover, cancer cells are often resistant or acquire resistance to the treatment. Further knowledge of the mechanisms driving lung tumorigenesis, aggressiveness, metastasization, and resistance to treatments could provide new tools for detecting the disease at an earlier stage and for a better response to therapy. In this scenario, Yes Associated Protein (YAP) and Trascriptional Coactivator with PDZ-binding motif (TAZ), the final effectors of the Hippo signaling transduction pathway, are emerging as promising therapeutic targets. Here, we will discuss the most recent advances made in YAP and TAZ biology in lung cancer and, more importantly, on the newly discovered mechanisms of YAP and TAZ inhibition in lung cancer as well as their clinical implications.

Lung cancer is the leading cause of cancer-related deaths, worldwide. Non-small cell lung cancer (NSCLC) is the most prevalent lung cancer subtype. YAP and TAZ have been implicated in lung cancer by acting as transcriptional co-activators of oncogenes or as transcriptional co-repressors of tumor suppressor genes. Previously we reported that YAP and TAZ regulate microRNAs expression in NSCLC. Among the set of regulated miRNAs, the oncogenic miR-25, 93, and 106b, clustering within the MCM7 gene were selected for further studies. We firstly identified Transforming Growth Factor-β (TGF-β) Receptor 2 (TGFBR2), a member of the TGF-β signaling, as a target of the miRNA cluster, which exhibited prognostic value because of its tumor suppressor activity. We found that YAP/TAZ-mediated repression of TGFBR2 occurs both: post-transcriptionally through the miR-106b-25 cluster and transcriptionally by engaging the EZH2 epigenetic repressor that we reported here as a novel target gene of YAP/TAZ. Furthermore, we document that YAP/TAZ and EZH2 cooperate in lung tumorigenesis by transcriptionally repressing a specific subset of tumor suppressor genes, including TGFBR2. Our findings point to YAP/TAZ and EZH2 as potential therapeutic targets for NSCLC treatment.

The Hippo pathway plays a critical role for balancing proliferation and differentiation, thus regulating tissue homeostasis. The pathway acts through a kinase cascade whose final effectors are the Yes-associated protein (YAP) and its paralog transcriptional co‑activator with PDZ‑binding motif (TAZ). In response to a variety of upstream signals, YAP and TAZ activate a transcriptional program that modulates cellular proliferation, tissue repair after injury, stem cell fate decision, and cytoskeletal reorganization. Hippo pathway signaling is often dysregulated in gastric cancer and in Helicobacter pylori-induced infection, suggesting a putative role of its deregulation since the early stages of the disease. In this review, we summarize the architecture and regulation of the Hippo pathway and discuss how its dysregulation fuels the onset and progression of gastric cancer. In this setting, we also focus on the crosstalk between Hippo and other established oncogenic signaling pathways. Lastly, we provide insights into the therapeutic approaches targeting aberrant YAP/TAZ activation and discuss the related clinical perspectives and challenges.

Polycomb group (PcG) proteins are part of a conserved cell memory system that conveys epigenetic inheritance of silenced transcriptional states through cell division. Despite the considerable amount of information about PcG mechanisms controlling gene silencing, how PcG proteins maintain repressive chromatin during epigenome duplication is still unclear. Here we identified a specific time window, the early S phase, in which PcG proteins are recruited at BX-C PRE target sites in concomitance with H3K27me3 repressive mark deposition. Notably, these events precede and are uncoupled from PRE replication timing, which occurs in late S phase when most epigenetic signatures are reduced. These findings shed light on one of the key mechanisms for PcG–mediated epigenetic inheritance during S phase, suggesting a conserved model in which the PcG–dependent H3K27me3 mark is inherited by dilution and not by de novo methylation occurring at the time of replication. During embryonic development, pluripotent cells divide and use their potential to differentiate into a variety of cells with identical genomes but different phenotypes. The emerging concept suggests that the DNA sequence information is not the sole determinant of cell identity. Indeed, epigenetic mechanisms, acting via chromatin organization, control transcriptome complexity and contribute to maintain cell fate. Polycomb-group proteins (PcG) are epigenetic transcriptional regulators that maintaining gene silencing programs through cell division. During S phase, in addition to DNA, the entire epigenome needs to be duplicated. A key question that remains to be addressed is how epigenetic marks are transmitted to subsequent generations. In this study we propose a model for PcG epigenetic inheritance during replication. We found that, during S phase, PcG engagement and characteristic H3K27me3 histone mark deposition on target sites are restricted to a brief interval occurring before DNA replication of the same regions. By increasing the dose of PcG binding the system would prevent potential weakening of silencing control, which is challenged at the time of replication, allowing proper transmission of epigenetic marks to the next generation and preservation of cell identity.

Lung cancer is the first cause of cancer death worldwide and the Hippo pathway transcriptional coactivators YAP/TAZ have a pro-oncogenic role in this context. In order to understand the mechanisms through which YAP/TAZ elicit their oncogenic role in different systems, many studies are focused on YAP/TAZ target genes involved in the regulation of cell proliferation/survival and migration. However, there is scarce evidence on the role of YAP/TAZ in microRNA regulation while there is increasing evidence supporting the role of microRNAs in the main oncogenic processes. Here, we showed that YAP/TAZ were able to regulate several microRNAs in non-small cell lung cancer (NSCLC) cell lines. In detail, we focused on a cluster of three oncogenic microRNAs (miR-25, 93 and 106b) hosted in the MCM7 gene that were overexpressed in lung tumors compared to normal tissues. In addition, similar behavior was observed in breast cancer and head and neck tumor casuistries, where they showed a prognostic role. In NSCLC cells, YAP/TAZ induced the transcription of the MCM7 gene and its hosted miRs, thereby promoting cell proliferation through the post-transcriptional inhibition of the p21 cell cycle regulator. Accordingly, p21 was maintained at low levels in lung tumors compared to normal tissues. Conversely, its expression was restored in NSCLC cells upon YAP/TAZ interference or upon treatment with the statin cerivastatin. In summary, we provide evidence for a novel mechanism of modulation supporting the protumorigenic functions of the YAP/TAZ factors through the modulation of a bioncogenic locus consisting of one gene and three hosted microRNAs.