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. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Animal transcriptomes are dynamic, each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. We identified new genes, transcripts, and proteins using poly(A)+ RNA sequence from Drosophila melanogaster cultured cell lines, dissected organ systems, and environmental perturbations. We found 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. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long noncoding RNAs (lncRNAs) some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized arising from combinatorial usage of promoters, splice sites, and polyadenylation sites.

SUMMARY Determining the composition of protein complexes is an essential step towards understanding the cell as an integrated system. Using co-affinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly five thousand individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a novel statistical framework to define individual protein-protein interactions, led to the generation of a Drosophila Protein interaction Map (DPiM) encompassing 556 protein complexes. The high quality of DPiM and its usefulness as a paradigm for metazoan proteomes is apparent from the recovery of many known complexes, significant enrichment for shared functional attributes and validation in human cells. DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution.

Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5Ј expressed sequence tags (5Ј expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ∼40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5Ј ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. BF485518-BF503517, BF503521-BF506780, BG631888-BG631996, BG633696-BG637540, BG640063-BG641469, BI141709-BI142246, BI161485-BI173971, BI212109-BI216987, BI227448-BI233322, BI234009-BI243989, BI351612-BI354228, BI354231-BI355901, BI355935-BI358751, BI361285-BI376197, BI481532-BI487261, BI563331-BI593695, BI604243-BI620155, BI620158-BI635012, BI635064-BI638027, and BI638030-BI642053. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: J. Pringle and M. Fuller.]The identification of all expressed genes and the structure(s) of their transcripts are prerequisites for many structural and functional genomic studies. Gene-finding programs are valuable tools for identifying gene structure, but they are errorprone and suffer from the inability to predict untranslated regions (UTRs) (Ashburner 2000;Reese et al. 2000). Direct analysis of gene transcripts is the only proven way to establish gene structures with confidence. Generating a collection of expressed sequence tags (ESTs) from high quality cDNA libraries is a widely used approach for acquiring this information (Adams et al. 1991). The sequences of ESTs and full-length nonredundant cDNA collections provide ideal tools for genome annotation and for the further training of gene prediction algorithms. Our first D.melanogaster EST project yielded putative full-length clones corresponding to >5000 different genes (Rubin et al. 2000). This was acco...