Correction to: DNA copy number evolution in Drosophila cell lines.

Following publication of the original article [1], the authors reported the following errors.

Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines.

Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone .

Tools for Targeted Genome Engineering of Established Drosophila Cell Lines.

We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites.

Injury response checkpoint and developmental timing in insects.

In insects, localized tissue injury often leads to global (organism-wide) delays in development and retarded metamorphosis. In Drosophila, for example, injuries to the larval imaginal discs can retard pupariation and prolong metamorphosis. Injuries induced by treatments such as radiation, mechanical damage and induction of localized cell death can trigger similar delays.

DNA copy number evolution in Drosophila cell lines.

Background: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.

Results: Our work revealed dramatic duplications and deletions in all cell lines.

Comparative analysis of the transcriptome across distant species.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly.

Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines.

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes.

Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data.

Diversity and dynamics of the Drosophila transcriptome.

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing.

Tissue damage disrupts developmental progression and ecdysteroid biosynthesis in Drosophila.

In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g.

Cryptocephal, the Drosophila melanogaster ATF4, is a specific coactivator for ecdysone receptor isoform B2.

The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown.

Global patterns of tissue-specific alternative polyadenylation in Drosophila.

We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3' untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS.

The developmental transcriptome of Drosophila melanogaster.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages.

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction.

The transcriptional diversity of 25 Drosophila cell lines.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal).

Treatment of surfaces with poly-L-lysine for Drosophila cell cultivation.

Most commonly used Drosophila cell lines adhere only loosely to the substrate. For some purposes (e.g.

Transformation of Drosophila cell lines: an alternative approach to exogenous protein expression.

Techniques and experimental applications are described for exogenous protein expression in Drosophila cell lines. Ways in which the Drosophila cell lines and the baculovirus expression vector system differ in their applications are emphasized.

Drosophila cell culture and transformation.

INTRODUCTIONPermanent Drosophila cell lines derived from mixed embryonic tissues including the most commonly used lines, S2 and Kc, have been available for ~30 yr. More recently, lines derived from specific tissues, imaginal discs, and the larval central nervous system have come into use. Although cultured cells were originally used by Drosophilists mainly as convenient sources of DNA or carrier RNA, that situation has changed, as an armamentarium of techniques for using the cells has slowly but steadily evolved.

Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila.

During metamorphosis, the reorganization of the nervous system of Drosophila melanogaster proceeds in part through remodeling of larval neurons. In this study, we used in-vitro imaging techniques and immunocytochemistry to track the remodeling of the thoracic ventral neurosecretory cells. Axons of these neurons prune their larval arbors early in metamorphosis and a larger, more extensive adult arbor is established via branch outgrowth.

The Drosophila nucleosome remodeling factor NURF is required for Ecdysteroid signaling and metamorphosis.

Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, we performed whole genome expression analysis on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes.

Development of high-sensitivity ion trap ion mobility spectrometry time-of-flight techniques: a high-throughput nano-LC-IMS-TOF separation of peptides arising from a Drosophila protein extract.

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.

Methylation at lysine 4 of histone H3 in ecdysone-dependent development of Drosophila.

Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle.

Transcription activation by the ecdysone receptor (EcR/USP): identification of activation functions.

The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact receptor.

EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue.

The three Drosophila EcR isoforms differ only at their N termini; thus, they share the conserved ligand-binding domain transcriptional activation function (AF2) and only differ in the unconserved A/B region, which contains a second, isoform-specific, activation function (AF1). We have developed a dominant-negative mutant EcR (EcR-DN), expressed it in flies with the GAL4/UAS system, and used it to block ecdysone signaling in eight tissues or groups of tissues. Localized EcR-DN arrests ecdysone-dependent development in the target cells and often--because of a molting checkpoint--arrests development globally.