Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies.

Piwi-interacting RNAs (piRNAs) silence transposons in animal germ cells. piRNAs are thought to derive from long transcripts spanning transposon-rich genomic loci and to direct an autoamplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. Here, we describe strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. We find that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. We also detect a second, Ago3-independent piRNA pathway centered on Piwi. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germline.

Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster.

N-Acetylglucosamine ß-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.

Meiotic recombination is suppressed near the histone-defined border of euchromatin and heterochromatin on chromosome 2L of Drosophila melanogaster.

In Drosophila melanogaster, the borders between pericentric heterochromatin and euchromatin on the major chromosome arms have been defined in various ways, including chromatin-specific histone modifications, the binding patterns of heterochromatin-enriched chromosomal proteins, and various cytogenetic techniques. Elucidation of the genetic properties that independently define the different chromatin states associated with heterochromatin and euchromatin should help refine the boundary. Since meiotic recombination is present in euchromatin, but absent in heterochromatin, it constitutes a key genetic property that can be observed transitioning between chromatin states. Using P element insertion lines marked with a su(Hw) insulated mini-white gene, meiotic recombination was found to transition in a region consistent with the H3K9me2 transition observed in ovaries.

Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc).

O-linked N-acetylglucosamine transferase (OGT) reversibly modifies serine and threonine residues of many intracellular proteins with a single beta-O-linked N-acetylglucosamine residue (O-GlcNAc), and has been implicated in insulin signaling, neurodegenerative disease, cellular stress response, and other important processes in mammals. OGT also glycosylates RNA polymerase II and various transcription factors, which suggests that it might be directly involved in transcriptional regulation. We report here that the Drosophila OGT is encoded by the Polycomb group (PcG) gene, super sex combs (sxc). Furthermore, major sites of O-GlcNAc modification on polytene chromosomes correspond to PcG protein binding sites. Our results thus suggest a direct role for O-linked glycosylation by OGT in PcG-mediated epigenetic gene silencing, which is important in developmental regulation, stem cell maintenance, genomic imprinting, and cancer. In addition, we observe rescue of sxc lethality by a human Ogt cDNA transgene; thus Drosophila may provide an ideal model to study important functional roles of OGT in mammals.

Characterization of Gfat1 (zeppelin) and Gfat2, Essential Paralogous Genes Which Encode the Enzymes That Catalyze the Rate-Limiting Step in the Hexosamine Biosynthetic Pathway in Drosophila melanogaster.

The zeppelin (zep) locus is known for its essential role in the development of the embryonic cuticle of Drosophila melanogaster. We show here that zep encodes Gfat1 (Glutamine: Fructose-6-Phosphate Aminotransferase 1; CG12449), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism. We also describe the isolation and characterization of lethal mutants in the euchromatic paralog, Gfat2 (CG1345), and demonstrate that ubiquitous expression of Gfat1+ or Gfat2+ transgenes can rescue lethal mutations in either gene. Gfat1 and Gfat2 show differences in mRNA and protein expression during embryogenesis and in essential tissue-specific requirements for Gfat1 and Gfat2, suggesting a degree of functional evolutionary divergence. An evolutionary, cytogenetic analysis of the two genes in six Drosophila species revealed Gfat2 to be located within euchromatin in all six species. Gfat1 localizes to heterochromatin in three melanogaster-group species, and to euchromatin in the more distantly related species. We have also found that the pattern of flanking-gene microsynteny is highly conserved for Gfat1 and somewhat less conserved for Gfat2.

The Drosophila cohesin subunit Rad21 is a trithorax group (trxG) protein.

The cohesin complex is a key player in regulating cell division. Cohesin proteins SMC1, SMC3, Rad21, and stromalin (SA), along with associated proteins Nipped-B, Pds5, and EcoI, maintain sister chromatid cohesion before segregation to daughter cells during anaphase. Recent chromatin immunoprecipitation (ChIP) data reveal extensive overlap of Nipped-B and cohesin components with RNA polymerase II binding at active genes in Drosophila. These and other data strongly suggest a role for cohesion in transcription; however, there is no clear evidence for any specific mechanisms by which cohesin and associated proteins regulate transcription. We report here a link between cohesin components and trithorax group (trxG) function, thus implicating these proteins in transcription activation and/or elongation. We show that the Drosophila Rad21 protein is encoded by verthandi (vtd), a member of the trxG gene family that is also involved in regulating the hedgehog (hh) gene. In addition, mutations in the associated protein Nipped-B show similar trxG activity i.e., like vtd, they act as dominant suppressors of Pc and hh(Mrt) without impairing cell division. Our results provide a framework to further investigate how cohesin and associated components might regulate transcription.

Genetic and Molecular Analysis of Essential Genes in Centromeric Heterochromatin of the Left Arm of Chromosome 3 in Drosophila melanogaster.

A large portion of the Drosophila melanogaster genome is contained within heterochromatic regions of chromosomes, predominantly at centromeres and telomeres. The remaining euchromatic portions of the genome have been extensively characterized with respect to gene organization, function and regulation. However, it has been difficult to derive similar data for sequences within centromeric (centric) heterochromatin because these regions have not been as amenable to analysis by standard genetic and molecular tools. Here we present an updated genetic and molecular analysis of chromosome 3L centric heterochromatin (3L Het). We have generated and characterized a number of new, overlapping deficiencies (Dfs) which remove regions of 3L Het. These Dfs were critically important reagents in our subsequent genetic analysis for the isolation and characterization of lethal point mutations in the region. The assignment of these mutations to genetically-defined essential loci was followed by matching them to gene models derived from genome sequence data: this was done by using molecular mapping plus sequence analysis of mutant alleles, thereby aligning genetic and physical maps of the region. We also identified putative essential gene sequences in 3L Het by using RNA interference to target candidate gene sequences. We report that at least 25, or just under 2/3 of loci in 3L Het, are essential for viability and/or fertility. This work contributes to the functional annotation of centric heterochromatin in Drosophila, and the genetic and molecular tools generated should help to provide important insights into the organization and functions of gene sequences in 3L Het.

Cooperative Control of Ecdysone Biosynthesis in Drosophila by Transcription Factors Séance, Ouija Board, and Molting Defective.

Ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. In this study, we report that three zinc finger-associated domain (ZAD)-C2H2 zinc finger transcription factors-Séance (Séan), Ouija board (Ouib), and Molting defective (Mld)-cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster Séan and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier, respectively, two Halloween genes. Remarkably, loss-of-function mutations in séan, ouib, or mld can be rescued by the expression of neverland, spookier, or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila Given that neverland and spookier are located in constitutive heterochromatin, Séan, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin.

Heterochromatic genes in Drosophila: a comparative analysis of two genes.

Centromeric heterochromatin comprises approximately 30% of the Drosophila melanogaster genome, forming a transcriptionally repressive environment that silences euchromatic genes juxtaposed nearby. Surprisingly, there are genes naturally resident in heterochromatin, which appear to require this environment for optimal activity. Here we report an evolutionary analysis of two genes, Dbp80 and RpL15, which are adjacent in proximal 3L heterochromatin of D. melanogaster. DmDbp80 is typical of previously described heterochromatic genes: large, with repetitive sequences in its many introns. In contrast, DmRpL15 is uncharacteristically small. The orthologs of these genes were examined in D. pseudoobscura and D. virilis. In situ hybridization and whole-genome assembly analysis show that these genes are adjacent, but not centromeric in the genome of D. pseudoobscura, while they are located on different chromosomal elements in D. virilis. Dbp80 gene organization differs dramatically among these species, while RpL15 structure is conserved. A bioinformatic analysis in five additional Drosophila species demonstrates active repositioning of these genes both within and between chromosomal elements. This study shows that Dbp80 and RpL15 can function in contrasting chromatin contexts on an evolutionary timescale. The complex history of these genes also provides unique insight into the dynamic nature of genome evolution.

A genetic and molecular characterization of two proximal heterochromatic genes on chromosome 3 of Drosophila melanogaster.

Heterochromatin comprises a transcriptionally repressive chromosome compartment in the eukaryotic nucleus; this is exemplified by the silencing effect it has on euchromatic genes that have been relocated nearby, a phenomenon known as position-effect variegation (PEV), first demonstrated in Drosophila melanogaster. However, the expression of essential heterochromatic genes within these apparently repressive regions of the genome presents a paradox, an understanding of which could provide key insights into the effects of chromatin structure on gene expression. To date, very few of these resident heterochromatic genes have been characterized to any extent, and their expression and regulation remain poorly understood. Here we report the cloning and characterization of two proximal heterochromatic genes in D. melanogaster, located deep within the centric heterochromatin of the left arm of chromosome 3. One of these genes, RpL15, is uncharacteristically small, is highly expressed, and encodes an essential ribosomal protein. Its expression appears to be compromised in a genetic background deficient for heterochromatin protein 1 (HP1), a protein associated with gene silencing in these regions. The second gene in this study, Dbp80, is very large and also appears to show a transcriptional dependence upon HP1; however, it does not correspond to any known lethal complementation group and is likely to be a nonessential gene.

Neonectria and Cylindrocarpon: the Nectria mammoidea group and species lacking microconidia.

Neonectria (Hypocreales: Nectriaceae) species having Cylindrocarpon anamorphs that lack microconidia and chlamydospores include: Neo. discophora var. discophora, Neo. discophora var. rubi, stat nov. et comb. nov., Neo. lucida, comb. nov., Neo. viridispora, sp. nov. and Neo. westlandica, comb. nov. Perithecia of these species are red and perithecial anatomyis of the N. mammoidea type, with a palisade of hypha-like cells in the outer perithecial wall. These species occur on recently dead or dying trees. Perithecia of Neo. betulae, sp. nov and Neo. dumontii, sp. nov. are anatomically and biologically similar to those of Neo. discophora. The only known culture of Neo. betulae remained sterile, while Neo. dumontii has not been cultured; their anamorphs are presumed to be Cylindrocarpon. Analyses of mit ssu rDNA sequences indicate that Neonectria/Cylindrocarpon is monophyletic. Within the genus, species having N. mammoidea type perithecia are paraphyletic. Most species cluster with Neo. discophora, but Neo. westlandica and Neo. trachosa are basal to a clade that includes species that do not have a N. mammoidea-type perithecium. Nectria fuckeliana clusters independently of Neonectria and Nectria. Although reported to have a Cylindrocarpon anamorph, fresh ascospore isolates of N. fuckeliana did not produce Cylindrocarpon macroconidia but produced acremonium- or verticillium-like anamorphs. A key to nectriaceous species of Neonectria that have Cylindrocarpon anamorphs that lack microconidia and chlamydospores and/or that have a N. mammoidea type perithecial wall anatomy is presented. New combinations are proposed for other species formerly included in Nectria that have non-microconidial Cylindrocarpon anamorphs: Neonectria cinnamomea, Neo. jungneri, Neo. platycephala, Neo. phaeodisca and Neo. verrucospora.

dSet1 is the main H3K4 di- and tri-methyltransferase throughout Drosophila development.

In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. We report here that an essential locus within chromosome 3L centric heterochromatin encodes the previously uncharacterized Drosophila melanogaster ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). Our results suggest that dSet1 acts as a "global" or general H3K4 di- and trimethyl HMT in Drosophila. Levels of H3K4 di- and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). We present biochemical evidence that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence that these members are functionally required for H3K4 methylation. Taken together, our results suggest that dSet1 is responsible for the bulk of H3K4 di- and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans.

Essential loci in centromeric heterochromatin of Drosophila melanogaster. I: the right arm of chromosome 2.

With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. We undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster. We also utilized available RNA interference lines to knock down the expression of genes in 2Rh as another approach to identifying essential genes. In total, we verified the existence of eight novel essential loci in 2Rh: CG17665, CG17683, CG17684, CG17883, CG40127, CG41265, CG42595, and Atf6. Two of these essential loci, CG41265 and CG42595, are synonymous with the previously characterized loci l(2)41Ab and unextended, respectively. The genetic and molecular analysis of the previously reported locus, l(2)41Ae, revealed that this is not a single locus, but rather it is a large region of 2Rh that extends from unextended (CG42595) to CG17665 and includes four of the novel loci uncovered here.

The pink gene encodes the Drosophila orthologue of the human Hermansky-Pudlak syndrome 5 (HPS5) gene.

Hermansky-Pudlak syndrome (HPS) consists of a set of human autosomal recessive disorders, with symptoms resulting from defects in genes required for protein trafficking in lysosome-related organelles such as melanosomes and platelet dense granules. A number of human HPS genes and rodent orthologues have been identified whose protein products are key components of 1 of 4 different protein complexes (AP-3 or BLOC-1, -2, and -3) that are key participants in the process. Drosophila melanogaster has been a key model organism in demonstrating the in vivo significance of many genes involved in protein trafficking pathways; for example, mutations in the "granule group" genes lead to changes in eye colour arising from improper protein trafficking to pigment granules in the developing eye. An examination of the chromosomal positioning of Drosophila HPS gene orthologues suggested that CG9770, the Drosophila HPS5 orthologue, might correspond to the pink locus. Here we confirm this gene assignment, making pink the first eye colour gene in flies to be identified as a BLOC complex gene.