MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate the expression of target genes by binding to the target mRNAs. Although a large number of animal miRNAs has been defined, only a few targets are known. In contrast to plant miRNAs, which usually bind nearly perfectly to their targets, animal miRNAs bind less tightly, with a few nucleotides being unbound, thus producing more complex secondary structures of miRNA/target duplexes. Here, we present a program, RNA-hybrid, that predicts multiple potential binding sites of miRNAs in large target RNAs. In general, the program finds the energetically most favorable hybridization sites of a small RNA in a large RNA. Intramolecular hybridizations, that is, base pairings between target nucleotides or between miRNA nucleotides are not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies, a Poisson approximation of multiple binding sites, and the calculation of effective numbers of orthologous targets in comparative studies of multiple organisms. We applied our method to the prediction of Drosophila miRNA targets in 3'UTRs and coding sequence. RNAhybrid, with its accompanying programs RNAcalibrate and RNAeffective, is available for download and as a Web tool on the Bielefeld Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/).

Micro RNAs are a large family of noncoding RNAs of 21-22 nucleotides whose functions are generally unknown. Here a large subset of Drosophila micro RNAs is shown to be perfectly complementary to several classes of sequence motif previously demonstrated to mediate negative post-transcriptional regulation. These findings suggest a more general role for micro RNAs in gene regulation through the formation of RNA duplexes.

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential, as it allows reduction of the sequence search space. Improvements in genome annotation and increased availability of cDNA sequences from other genomes will allow more sensitive screens. An increase in the number of confirmed targets is expected to reveal general structural features that can be used to improve their detection. While the screen is likely to miss some targets, our study shows that valid targets can be identified from sequence alone.

We have used the interaction trap, a yeast two-hybrid system, to identify proteins interacting with hairy, a basic-helix-loop-helix (bHLH) protein that represses transcription during Drosophila embryonic segmentation. We find that the groucho (gro) protein binds specifically to hairy and also to hairy-related bHLH proteins encoded by deadpan and the Enhancer of split complex. The C-terminal WRPW motif present in all these bHLH proteins is essential for this interaction. We demonstrate that these associations reflect in vivo maternal requirements for gro during neurogenesis, segmentation, and sex determination, three processes regulated by the above bHLH proteins, and we propose that gro is a transcriptional corepressor recruited to specific target promoters by hairy-related bHLH proteins.

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. We found that these transcriptional patterns can be dissected into distinct groups. We demonstrate that, in addition to signaling through the Toll and Imd pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, our results may imply a close link between the control of tissue repair and antimicrobial processes.

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.

The basic Helix-Loop-Helix (bHLH) proteins are transcription factors that play important roles during the development of various metazoans including fly, nematode, and vertebrates. They are also involved in human diseases, particularly in cancerogenesis. We made an extensive search for bHLH sequences in the completely sequenced genomes of Caenorhabditis elegans and of Drosophila melanogaster. We found 35 and 56 different genes, respectively, which may represent the complete set of bHLH of these organisms. A phylogenetic analysis of these genes, together with a large number (>350) of bHLH from other sources, led us to define 44 orthologous families among which 36 include bHLH from animals only, and two have representatives in both yeasts and animals. In addition, we identified two bHLH motifs present only in yeast, and four that are present only in plants; however, the latter number is certainly an underestimate. Most animal families (35/38) comprise fly, nematode, and vertebrate genes, suggesting that their common ancestor, which lived in pre-Cambrian times (600 million years ago) already owned as many as 35 different bHLH genes.

Dissecting components of key transcriptional networks is essential for understanding complex developmental processes and phenotypes. Genetic studies have highlighted the role of members of the Mef2 family of transcription factors as essential regulators in myogenesis from flies to man. To understand how these transcription factors control diverse processes in muscle development, we have combined chromatin immunoprecipitation analysis with gene expression profiling to obtain a temporal map of Mef2 activity during Drosophila embryonic development. This global approach revealed three temporal patterns of Mef2 enhancer binding, providing a glimpse of dynamic enhancer use within the context of a developing embryo. Our results provide mechanistic insight into the regulation of Mef2's activity at the level of DNA binding and suggest cooperativity with the bHLH protein Twist. The number and diversity of new direct target genes indicates a much broader role for Mef2, at all stages of myogenesis, than previously anticipated.

The cells along the dorsoventral boundary of the Drosophila wing imaginal disc have distinctive properties and their specification requires Notch activity. Later in development, these cells will form the wing margin, where sensory organs and specialised trichomes appear in a characteristic pattern. We find that Notch is locally activated in these cells, as demonstrated by the restricted expression of the Enhancer of split proteins in dorsal and ventral cells abutting the D/V boundary throughout the third larval instar. Furthermore other genes identified by their involvement in Notch signaling during neurogenesis, such as Delta and Suppressor of Hairless, also participate in Notch function at the dorsoventral boundary. In addition, Serrate, a similar transmembrane protein to Delta, behaves as a ligand required in dorsal cells to activate Notch at the boundary. Notch gain-of-function alleles in which Notch activity is not restricted to the dorsoventral boundary cause miss-expression of cut and wingless and overgrowth of the disc, illustrating the importance of localised Notch activation for wing development.

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

The CSL [CBF1/Su(H)/Lag2] proteins [Su(H) in Drosophila] are implicated in repression and activation of Notch target loci. Prevailing models imply a static association of these DNA-binding transcription factors with their target enhancers. Our analysis of Su(H) binding and chromatin-associated features at 11 E(spl) Notch target genes before and after Notch revealed large differences in Su(H) occupancy at target loci that correlated with the presence of polymerase II and other marks of transcriptional activity. Unexpectedly, Su(H) occupancy was significantly and transiently increased following Notch activation, suggesting a more dynamic interaction with targets than hitherto proposed.

The availability of 12 complete genomes of various species of genus Drosophila provides a unique opportunity to analyze genome-scale chromosomal rearrangements among a group of closely related species. This article reports on the comparison of gene order between these 12 species and on the fixed rearrangement events that disrupt gene order. Three major themes are addressed: the conservation of syntenic blocks across species, the disruption of syntenic blocks (via chromosomal inversion events) and its relationship to the phylogenetic distribution of these species, and the rate of rearrangement events over evolutionary time. Comparison of syntenic blocks across this large genomic data set confirms that genetic elements are largely (95%) localized to the same Muller element across genus Drosophila species and paracentric inversions serve as the dominant mechanism for shuffling the order of genes along a chromosome. Gene-order scrambling between species is in accordance with the estimated evolutionary distances between them and we find it to approximate a linear process over time (linear to exponential with alternate divergence time estimates). We find the distribution of synteny segment sizes to be biased by a large number of small segments with comparatively fewer large segments. Our results provide estimated chromosomal evolution rates across this set of species on the basis of whole-genome synteny analysis, which are found to be higher than those previously reported. Identification of conserved syntenic blocks across these genomes suggests a large number of conserved blocks with varying levels of embryonic expression correlation in Drosophila melanogaster. On the other hand, an analysis of the disruption of syntenic blocks between species allowed the identification of fixed inversion breakpoints and estimates of breakpoint reuse and lineage-specific breakpoint event segregation.

Like the neuroblasts of the central nervous system, sensory organ precursors of the peripheral nervous system of the Drosophila thorax arise as single spaced cells. However, groups of cells initially have neural potential as visualized by the expression of the proneural genes achaete and scute. A class of genes, known as the 'neurogenic genes', function to restrict the proportion of cells that differentiate as sensory organ precursors. They mediate cell communication between the competent cells by means of an inhibitory signal, Delta, that is transduced through the Notch receptor and results in a cessation of achaete-scute activity. Here we show that mutation of either the bHLH-encoding genes of the Enhancer of split complex (E(spl)-C) or groucho, like Notch or Delta mutants, cause an overproduction of sensory organ precursors at the expense of epidermis. The mutant cells behave antonomously suggesting that the corresponding gene products are required for reception of the inhibitory signal. Epistasis experiments place both E(spl)-C bHLH-encoding genes and groucho downstream of Notch and upstream of achaete and scute, consistent with the idea that they are part of the Notch signalling cascade. Since all competent cells produce both the receptor and its ligand, it was postulated that Notch and Delta are linked within each cell by a feedback loop. We show, that, like mutant Notch cells, cells mutant for E(spl)-C bHLH-encoding genes or groucho inhibit neighbouring wild-type cells causing them to adopt the epidermal fate. This inhibition requires the genes of the achaete-scute complex (AS-C) which must therefore regulate the signal Delta. Thus there is a regulatory loop between Notch and Delta that is under the transcriptional control of the E(spl)-C and AS-C genes.

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, PER and TIM, repress CLK-CYC-mediated transcription. To recognize additional direct CLK target genes, we designed a genome-wide approach and identified clockwork orange (cwo) as a new core clock component. cwo encodes a transcriptional repressor that synergizes with PER and inhibits CLK-mediated activation. Consistent with this function, the mRNA profiles of CLK direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that CWO acts preferentially in the late night to help terminate CLK-CYC-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks.

In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell-cell signaling pathway, which is utilized in imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH) transcriptional repressors, while 4 others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Here we present strong evidence from promoter-reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes were known previously to be direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins achaete and scute. Our extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. We next show that the very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mgamma are a property of small (200-300 bp), evolutionarily conserved transcriptional enhancer elements, which can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. We also demonstrate that the characteristic inactivity of the E(spl)mgamma enhancer in the notum and margin territories of the wing disc can be overcome by elevated activity of the N receptor. We conclude that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in which these modes of regulation are operative.

The Groucho (Gro)/TLE/Grg family of corepressors operates in many signaling pathways (including Notch and Wnt). Gro/TLE proteins recognize a wide range of transcriptional repressors by binding to divergent short peptide sequences, including a C-terminal WRPW/Y motif (Hairy/Hes/Runx) and internal eh1 motifs (FxIxxIL; Engrailed/Goosecoid/Pax/Nkx). Here, we identify several missense mutations in Drosophila Gro, which demonstrate peptide binding to the central pore of the WD (WD40) beta propeller domain in vitro and in vivo. We define these interactions at the molecular level with crystal structures of the WD domain of human TLE1 bound to either WRPW or eh1 peptides. The two distinct peptide motifs adopt markedly different bound conformations but occupy overlapping sites across the central pore of the beta propeller. Our structural and functional analysis explains the rigid conservation of the WRPW motif, the sequence flexibility of eh1 motifs, and other aspects of repressor recognition by Gro in vivo.

To elucidate mechanisms underlying the complex relationships between a host and its microbiota, we used the genetically tractable model Drosophila melanogaster. Consistent with previous studies, the microbiota was simple in composition and diversity. However, analysis of single flies revealed high interfly variability that correlated with differences in feeding. To understand the effects of this simple and variable consortium, we compared the transcriptome of guts from conventionally reared flies to that for their axenically reared counterparts. Our analysis of two wild-type fly lines identified 121 up- and 31 downregulated genes. The majority of these genes were associated with immune responses, tissue homeostasis, gut physiology, and metabolism. By comparing the transcriptomes of young and old flies, we identified temporally responsive genes and showed that the overall impact of microbiota was greater in older flies. In addition, comparison of wild-type gene expression with that of an immune-deficient line revealed that 53% of upregulated genes exerted their effects through the immune deficiency (Imd) pathway. The genes included not only classic immune response genes but also those involved in signaling, gene expression, and metabolism, unveiling new and unexpected connections between immunity and other systems. Given these findings, we further characterized the effects of gut-associated microbes on gut morphology and epithelial architecture. The results showed that the microbiota affected gut morphology through their impacts on epithelial renewal rate, cellular spacing, and the composition of different cell types in the epithelium. Thus, while bacteria in the gut are highly variable, the influence of the microbiota at large has far-reaching effects on host physiology.

OBJECTIVE

The guts of animals are in constant association with microbes, and these interactions are understood to have important roles in animal development and physiology. Yet we know little about the mechanisms underlying the establishment and function of these associations. Here, we used the fruit fly to understand how the microbiota affects host function. Importantly, we found that the microbiota has far-reaching effects on host physiology, ranging from immunity to gut structure. Our results validate the notion that important insights on complex host-microbe relationships can be obtained from the use of a well-established and genetically tractable invertebrate model.

A large fraction of the information content of metazoan genomes resides in the transcriptional and posttranscriptional cis-regulatory elements that collectively provide the blueprint for using the protein-coding capacity of the DNA, thus guiding the development and physiology of the entire organism. As successive whole-genome sequencing projects--including those of mice and humans--are completed, we have full access to the regulatory genome of yet another species. But our ability to decipher the cis-regulatory code, and hence to link genes into regulatory networks on a global scale, is currently very limited. Here we describe SCORE (Site Clustering Over Random Expectation), a computational method for identifying transcriptional cis-regulatory modules based on the fact that they often contain, in statistically improbable concentrations, multiple binding sites for the same transcription factor. We have carried out a Drosophila genomewide inventory of predicted binding sites for the Notch-regulated transcription factor Suppressor of Hairless [Su(H)] and found that the fly genome contains highly nonrandom clusterings of Su(H) sites over a broad range of sequence intervals. We found that the most statistically significant clusters are very heavily enriched in both known and logical targets of Su(H) binding and regulation. The utility of the SCORE approach was validated by in vivo experiments showing that proper expression of the novel gene Him in adult muscle precursor cells depends both on Su(H) gene activity and sequences that include a previously unstudied cluster of four Su(H) sites, indicating that Him is a likely direct target of Su(H).

Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells.

Cell-cell interactions mediated by the Notch receptor play an essential role in the development of the Drosophila adult peripheral nervous system (PNS). Transcriptional activation of multiple genes of the Enhancer of split Complex [E(spl)-C] is a key intracellular response to Notch receptor activity. Here we report that most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3' untranslated regions (3' UTRs). We present three lines of evidence that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes. First, K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species. Second, the wild-type m8 3' UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences. Finally, m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, we find that K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, we present sequence analyses that implicate genes of the iroquois Complex (Iro-C) and engrailed as additional targets of K box-mediated regulation.

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.

The proteins encoded by the myc proto-oncogene family are involved in cell proliferation, apoptosis, differentiation, and neoplasia. Myc acts through dimerization with Max to bind DNA and activate transcription. Homologs of the myc and max genes were cloned from the fruit fly Drosophila melanogaster and their protein products (dMyc and dMax) were shown to heterodimerize, recognize the same DNA sequence as their vertebrate homologs, and activate transcription. The dMyc protein is likely encoded by the Drosophila gene diminutive (dm), a mutation in which results in small body size and female sterility caused by degeneration of the ovaries. These findings indicate a potential role for Myc in germ cell development and set the stage for genetic analysis of Myc and Max.