N6-methyladenine DNA modification in Drosophila.

DNA N(6)-methyladenine (6mA) modification is commonly found in microbial genomes and plays important functions in regulating numerous biological processes in bacteria. However, whether 6mA occurs and what its potential roles are in higher-eukaryote cells remain unknown. Here, we show that 6mA is present in Drosophila genome and that the 6mA modification is dynamic and is regulated by the Drosophila Tet homolog, DNA 6mA demethylase (DMAD), during embryogenesis. Importantly, our biochemical assays demonstrate that DMAD directly catalyzes 6mA demethylation in vitro. Further genetic and sequencing analyses reveal that DMAD is essential for development and that DMAD removes 6mA primarily from transposon regions, which correlates with transposon suppression in Drosophila ovary. Collectively, we uncover a DNA modification in Drosophila and describe a potential role of the DMAD-6mA regulatory axis in controlling development in higher eukaryotes.

Muscle-derived Dpp regulates feeding initiation via endocrine modulation of brain dopamine biosynthesis.

In animals, the brain regulates feeding behavior in response to local energy demands of peripheral tissues, which secrete orexigenic and anorexigenic hormones. Although skeletal muscle is a key peripheral tissue, it remains unknown whether muscle-secreted hormones regulate feeding. In Drosophila, we found that decapentaplegic (dpp), the homolog of human bone morphogenetic proteins BMP2 and BMP4, is a muscle-secreted factor (a myokine) that is induced by nutrient sensing and that circulates and signals to the brain. Muscle-restricted dpp RNAi promotes foraging and feeding initiation, whereas dpp overexpression reduces it. This regulation of feeding by muscle-derived Dpp stems from modulation of brain tyrosine hydroxylase (TH) expression and dopamine biosynthesis. Consistently, Dpp receptor signaling in dopaminergic neurons regulates TH expression and feeding initiation via the downstream transcriptional repressor Schnurri. Moreover, pharmacologic modulation of TH activity rescues the changes in feeding initiation due to modulation of dpp expression in muscle. These findings indicate that muscle-to-brain endocrine signaling mediated by the myokine Dpp regulates feeding behavior.

Dendrite regeneration of adult Drosophila sensory neurons diminishes with aging and is inhibited by epidermal-derived matrix metalloproteinase 2.

Dendrites possess distinct structural and functional properties that enable neurons to receive information from the environment as well as other neurons. Despite their key role in neuronal function, current understanding of the ability of neurons to regenerate dendrites is lacking. This study characterizes the structural and functional capacity for dendrite regeneration in vivo in adult animals and examines the effect of neuronal maturation on dendrite regeneration. We focused on the class IV dendritic arborization (c4da) neuron of the Drosophila sensory system, which has a dendritic arbor that undergoes dramatic remodeling during the first 3 d of adult life and then maintains a relatively stable morphology thereafter. Using a laser severing paradigm, we monitored regeneration after acute and spatially restricted injury. We found that the capacity for regeneration was present in adult neurons but diminished as the animal aged. Regenerated dendrites recovered receptive function. Furthermore, we found that the regenerated dendrites show preferential alignment with the extracellular matrix (ECM). Finally, inhibition of ECM degradation by inhibition of matrix metalloproteinase 2 (Mmp2) to preserve the extracellular environment characteristics of young adults led to increased dendrite regeneration. These results demonstrate that dendrites retain regenerative potential throughout adulthood and that regenerative capacity decreases with aging.

A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor.

The C-terminal binding protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and feature a prominent intrinsically disordered region in the C terminus. In the mammalian system, CtBP proteins lacking the C-terminal domain (CTD) are able to function as transcriptional regulators and oligomerize, putting into question the significance of this unstructured domain for gene regulation. Yet, the presence of an unstructured CTD of ∼100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of maintaining this domain over evolutionary time. To uncover the significance of the CtBP CTD, we functionally tested naturally occurring Drosophila isoforms of CtBP that possess or lack the CTD, namely CtBP(L) and CtBP(S). We used the CRISPRi system to recruit dCas9-CtBP(L) and dCas9-CtBP(S) to endogenous promoters to directly compare their transcriptional impacts in vivo. Interestingly, CtBP(S) was able to significantly repress transcription of the Mpp6 promoter, while CtBP(L) was much weaker, suggesting that the long CTD may modulate CtBP's repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfected Mpp6 reporter gene. The context-specific differences in activity of these two developmentally regulated isoforms suggests that the CTD may help provide a spectrum of repression activity suitable for developmental programs.

The ATAC acetyltransferase complex coordinates MAP kinases to regulate JNK target genes.

In response to extracellular cues, signal transduction activates downstream transcription factors like c-Jun to induce expression of target genes. We demonstrate that the ATAC (Ada two A containing) histone acetyltransferase (HAT) complex serves as a transcriptional cofactor for c-Jun at the Jun N-terminal kinase (JNK) target genes Jra and chickadee. ATAC subunits are required for c-Jun occupancy of these genes and for H4K16 acetylation at the Jra enhancer, promoter, and transcribed sequences. Under conditions of osmotic stress, ATAC colocalizes with c-Jun, recruits the upstream kinases Misshapen, MKK4, and JNK, and suppresses further activation of JNK. Relocalization of these MAPKs and suppression of JNK activation by ATAC are dependent on the CG10238 subunit of ATAC. Thus, ATAC governs the transcriptional response to MAP kinase signaling by serving as both a coactivator of transcription and as a suppressor of upstream signaling.

Protein tyrosine phosphatase 69D is a substrate of protein O-mannosyltransferases 1-2 that is required for the wiring of sensory axons in Drosophila.

Mutations in protein O-mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, here we reveal that Dg alone cannot account for the phenotypes of POMT mutants, and identify Protein tyrosine phosphatase 69D (PTP69D) as a gene interacting with POMTs in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, we reveal that PTP69D is required for the wiring of larval sensory axons. We also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, we further characterized the glycosylation of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. We found that the PTP69D construct carries many O-linked mannose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs. These modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of receptor-type protein tyrosine phosphatase functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.

DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2.

Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5-/- tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.

Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.

Dietary nutrients provide macromolecules necessary for organism growth and development. In response to animal feeding, evolutionarily conserved growth signaling pathways are activated, leading to increased rates of cell proliferation and tissue growth. It remains unclear how different cell types within developing tissues coordinate growth in response to dietary nutrients and whether coordinated growth of different cell types is necessary for proper tissue function. Using the early Drosophila larval brain, we asked whether nutrient-dependent growth of neural stem cells (neuroblasts), glia, and trachea is coordinated and whether coordinated growth among these major brain cell types is required for neural development. It is known that in response to dietary nutrients and PI3-kinase activation, brain and ventral nerve cord neuroblasts reactivate from quiescence and ventral nerve cord glia expand their membranes. Here, we assay growth in a cell-type specific manner at short time intervals in the brain and determine that growth is coordinated among different cell types and that coordinated growth is mediated in part through activation of PI3-kinase signaling. Of the 7 Drosophila insulin-like peptides (Dilps), we find that Dilp-2 is required for PI3-kinase activation and growth coordination between neuroblasts and glia in the brain. Dilp-2 induces brain cortex glia to initiate membrane growth and make first contact with quiescent neuroblasts. Once reactivated, neuroblasts promote cortex glia growth to ultimately form a selective membrane barrier. Our results highlight the importance of bidirectional growth signaling between neural stem cells and surrounding cell types in the brain in response to nutrition and demonstrate how coordinated growth among different cell types drives tissue morphogenesis and function.

Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex.

Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3' supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3' supplementary region upon target recognition.

Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila.

Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome.

Roles of matrix metalloproteinases in development, immunology, and ovulation in fruit Fly (Drosophila).

Matrix metalloproteinase (MMP), a protease enzyme, participates in proteolytic cleavage of extracellular matrix proteins from Drosophila and mammals. But, recent studies have revealed other physiologically important roles of MMP in Drosophila. MMP contributes to cardioblast movement and distribution of collagen proteins during cardiogenesis in developing Drosophila. Tissue remodeling, especially tracheal development is also maintained by MMP. MMP regulates certain immunological functions in Drosophila such as wound repairing, plasmatocyte assemblage at the injured site of the basement membrane and glial response to axon degeneration in Drosophila nervous system. But, the contribution of MMP to tumor formation and metastasis in Drosophila has made it an interesting topic among researchers. Ovulation and egg laying are also found to be affected positively by MMP in Drosophila.

A major role for noncoding regulatory mutations in the evolution of enzyme activity.

The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating.

Intramembrane proteases catalyse the signal-generating step of various cell signalling pathways, and continue to be implicated in diseases ranging from malaria infection to Parkinsonian neurodegeneration. Despite playing such decisive roles, it remains unclear whether or how these membrane-immersed enzymes might be regulated directly. To address this limitation, here we focus on intramembrane proteases containing domains known to exert regulatory functions in other contexts, and characterize a rhomboid protease that harbours calcium-binding EF-hands. We find calcium potently stimulates proteolysis by endogenous rhomboid-4 in Drosophila cells, and, remarkably, when rhomboid-4 is purified and reconstituted in liposomes. Interestingly, deleting the amino-terminal EF-hands activates proteolysis prematurely, while residues in cytoplasmic loops connecting distal transmembrane segments mediate calcium stimulation. Rhomboid regulation is not orchestrated by either dimerization or substrate interactions. Instead, calcium increases catalytic rate by promoting substrate gating. Substrates with cleavage sites outside the membrane can be cleaved but lose the capacity to be regulated. These observations indicate substrate gating is not an essential step in catalysis, but instead evolved as a mechanism for regulating proteolysis inside the membrane. Moreover, these insights provide new approaches for studying rhomboid functions by investigating upstream inputs that trigger proteolysis.

Identification and functional analysis of dsRNases in spotted-wing drosophila, Drosophila suzukii.

RNAi efficiency in insects is different from species to species; some species in Coleoptera are relatively more amenable to RNA interference (RNAi) than other species. One of the major factors is the presence of dsRNA-degrading enzymes, called dsRNases, in saliva, gut, or hemolymph in insects, which degrade the double-stranded RNA (dsRNA) introduced, resulting in the low efficacy of RNAi. In this study, we report a dsRNA-degrading activity in the gut homogenates from the spotted-wing drosophila, Drosophila suzukii, by ex vivo assay. Then, we identified two Drosophila suzukii dsRNase genes, named DrosudsRNase1 and DrosudsRNase2. In silico analysis shows that the gene structures are similar to dsRNases found in other insects. When dsRNases expressed in Sf9 cells were compared for their dsRNA degrading activities, dsRNase1 was more vital than dsRNase2. Both dsRNases were expressed highly and exclusively in the gut compared to the rest of body. Also, they were highly expressed during larval and adult stages but not in embryonic and pupal stages, suggesting the dsRNases protect foreign RNA molecules received during the feeding periods. DsRNase1 was expressed at a higher level in adults, whereas dsRNase2 showed more expression in early larvae. Our study on the tissue and development-specific patterns of dsRNases provides an improved understanding of the RNAi application for the management of D. suzukii.

Structural insights into a unique preference for 3' terminal guanine of mirtron in Drosophila TUTase tailor.

Terminal uridylyl transferase (TUTase) is one type of enzyme that modifies RNA molecules by facilitating the post-transcriptional addition of uridyl ribonucleotides to their 3' ends. Recent researches have reported that Drosophila TUTase, Tailor, exhibits an intrinsic preference for RNA substrates ending in 3'G, distinguishing it from any other known TUTases. Through this unique feature, Tailor plays a crucial role as the repressor in the biogenesis pathway of splicing-derived mirtron pre-miRNAs. Here we describe crystal structures of core catalytic domain of Tailor and its complexes with RNA stretches 5'-AGU-3' and 5'-AGUU-3'. We demonstrate that R327 and N347 are two key residues contributing cooperatively to Tailor's preference for 3'G, and R327 may play an extra role in facilitating the extension of polyuridylation chain. We also demonstrate that conformational stability of the exit of RNA-binding groove also contributes significantly to Tailor's activity. Overall, our work reveals useful insights to explain why Drosophila Tailor can preferentially select RNA substrates ending in 3'G and provides important values for further understanding the biological significances of biogenesis pathway of mirtron in flies.

Krüppel homolog 1 represses insect ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes.

In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in both Drosophila and Bombyx Application of a JH mimic on ex vivo cultured PGs from Drosophila and Bombyx larvae induces Kr-h1 expression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown of Drosophila Kr-h1 promotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication in Drosophila PG cells and that the reduction of PG size mediated by Kr-h1 overexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.

Capicua controls Toll/IL-1 signaling targets independently of RTK regulation.

The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. Here, we characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. We show that Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-κB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, our results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.

Metallophosphoesterase regulates light-induced rhodopsin endocytosis by promoting an association between arrestin and the adaptor protein AP2.

Light-induced endocytosis of rhodopsin in the retina is critical for preventing photoreceptor hyperactivity and for the survival of photoreceptor cells. In Drosophila, this process is mediated by arrestin1 (Arr1). Because Arr1 lacks a clathrin-binding domain required for receptor internalization and the C-terminal sequence that interacts with the ß-subunit of the clathrin adaptor protein AP2, the mechanism of how Arr1 mediates endocytosis of the major rhodopsin Rh1 is unclear. Here, using several approaches, including Arr binding and pulldown assays, immunofluorescence techniques, and EM imaging, we found that Drosophila metallophosphoesterase (dMPPE) is involved in light-induced rhodopsin endocytosis. We observed that the photoreceptor cells of a dmppe mutant exhibit impaired light-induced rhodopsin endocytosis and that this impairment is independent of dMPPE phosphoesterase activity. Furthermore, dMPPE directly interacted with Arr1 and promoted the association of Arr1 with AP2. Of note, genetic dmppe deletion largely prevented retinal degeneration in norpA (encoding phospholipase C) mutants, which were reported previously to contribute to retinal degeneration, by suppressing Rh1 endocytosis. Our findings demonstrate that Arr1 interacts with AP2 and that dMPPE functions as a critical regulator in Rh1 endocytosis and retinal degeneration.

A calcium-mediated actin redistribution at egg activation in Drosophila.

Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.

Genetic analysis of the RNA polymerase II CTD in Drosophila.

The Carboxy-terminal Domain (CTD) of RNA polymerase II (Pol II) plays essential roles in regulating gene expression in eukaryotes. Here, we describe multiple genetic approaches for studying the CTD in Drosophila that complement pre-existing molecular analyses of the Pol II CTD in other experimental models. These approaches will allow one to assess the effects of any CTD mutations in a developmentally complex organism. The approaches discussed in this work can in principle, be applied to analyze other transcription components in eukaryotes.