Summary The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra (Kbr) as another upstream component of the Hippo signaling pathway. We show that Kbr functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function, and implicate Kbr as a potential tumor suppressor with relevance to neurofibromatosis.

SUMMARY Histones and their post-translational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have non-histone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a facile platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike conclusions drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity during development. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.

Here, we discuss the findings to date about genes and pathways required for regulation of somatic follicle-cell proliferation and differentiation during Drosophila oogenesis and demonstrate how loss of these genes contributes to the tumorigenic potential of mutant cells. Follicle cells undergo cell-fate determination through stepwise activation of multiple signaling pathways, including the Notch, Hedgehog, Wingless, janus kinase/STAT, and JNK pathways. In addition, changes in DNA replication and cellular growth depend on the spatial and temporal activation of the mitotic cycle-endocycle and endocycle-gene amplification cell-cycle switches and insulin-dependent monitoring of cellular health; systemic loss of these pathways contributes to loss of controlled cellular proliferation, loss of differentiation/growth, and aberrant cell polarity in follicle cells. We also highlight the effects of the neoplastic and Hippo pathways on the cell cycle and cellular proliferation in promoting normal development and conclude that lack of coordination of multiple signaling pathways promotes conditions favorable for tumorigenesis.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dcr-1 also rescued the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we showed that genetic inhibition of Bel function led to the de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited. Our findings have demonstrated a novel involvement of Bel in regulating transgene expression and its loss triggers a transgene silencing mechanism mediated by protein regulators implicated in RNA processing, piRNA biogenesis, chromatin remodeling and the microRNA pathway.