Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster.

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.

RNA helicase Belle/DDX3 regulates transgene expression in Drosophila.

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 Dicer-1 (Dcr-1) also alleviates 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 show that genetic inhibition of Bel function leads to 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.

Interrogating the function of metazoan histones using engineered gene clusters.

Histones and their posttranslational 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 nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a 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 inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.

Prp22 and spliceosome components regulate chromatin dynamics in germ-line polyploid cells.

During Drosophila oogenesis, the endopolyploid nuclei of germ-line nurse cells undergo a dramatic shift in morphology as oogenesis progresses; the easily-visible chromosomes are initially polytenic during the early stages of oogenesis before they transiently condense into a distinct '5-blob' configuration, with subsequent dispersal into a diffuse state. Mutations in many genes, with diverse cellular functions, can affect the ability of nurse cells to fully decondense their chromatin, resulting in a '5-blob arrest' phenotype that is maintained throughout the later stages of oogenesis. However, the mechanisms and significance of nurse-cell (NC) chromatin dispersal remain poorly understood. Here, we report that a screen for modifiers of the 5-blob phenotype in the germ line isolated the spliceosomal gene peanuts, the Drosophila Prp22. We demonstrate that reduction of spliceosomal activity through loss of peanuts promotes decondensation defects in NC nuclei during mid-oogenesis. We also show that the Prp38 spliceosomal protein accumulates in the nucleoplasm of nurse cells with impaired peanuts function, suggesting that spliceosomal recycling is impaired. Finally, we reveal that loss of additional spliceosomal proteins impairs the full decondensation of NC chromatin during later stages of oogenesis, suggesting that individual spliceosomal subcomplexes modulate expression of the distinct subset of genes that are required for correct morphology in endopolyploid nurse cells.

At the crossroads of differentiation and proliferation: precise control of cell-cycle changes by multiple signaling pathways in Drosophila follicle cells.

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.

poly is required for nurse-cell chromosome dispersal and oocyte polarity in Drosophila.

During Drosophila oogenesis, nurse cells undergo changes in chromosomal morphology, first from the polytenic form to a transient condensed phase known as the five-blob configuration, then into a diffuse polytenic-polyploid state for the remainder of oogenesis. The mechanism by which nurse-cell chromosome dispersal is regulated remains elusive. Mutations in several genes, including the heterogeneous ribonucleoprotein genes squid (sqd) and hrb27C, the alternative splicing factor gene poly U binding factor 68 kDa (pUf68, also known as half-pint), and the germ-line-specific gene ovarian tumor (otu), that produce defects in nurse-cell chromosome dispersal also produce defects in oocyte polarity, suggesting a link between these two processes. Here, we characterize a novel gene named poly, which, when mutated in the germ line, disrupts nurse-cell chromosome dispersal, as well as localization of anteroposterior and dorsoventral determinants in the oocyte. We also show that poly interacts genetically with hrb27C and otu. We conclude that poly is required for nurse-cell chromosome dispersal and oocyte polarization in the Drosophila germ-line. In addition, our interaction data suggest that poly is probably a member of the characterized mRNP complex that mediates both processes.

Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded.

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 as another upstream component of the Hippo signaling pathway. We show that Kibra 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 Kibra as a potential tumor suppressor with relevance to neurofibromatosis.