Evidence of abundant stop codon readthrough in Drosophila and other Metazoa

While translational stop codon readthrough is often used by viral genomes, it has been observed for only a handful of eukaryotic genes. We previously used comparative genomics evidence to recognize protein-coding regions in 12 species of Drosophila and showed that for 149 genes, the open reading frame following the stop codon has a protein-coding conservation signature, hinting that stop codon readthrough might be common in Drosophila. We return to this observation armed with deep RNA sequence data from the modENCODE project, an improved higher-resolution comparative genomics metric for detecting protein-coding regions, comparative sequence information from additional species, and directed experimental evidence. We report an expanded set of 283 readthrough candidates, including 16 double-readthrough candidates; these were manually curated to rule out alternatives such as A-to-I editing, alternative splicing, dicistronic translation, and selenocysteine incorporation. We report experimental evidence of translation using GFP tagging and mass spectrometry for several readthrough regions. We find that the set of readthrough candidates differs from other genes in length, composition, conservation, stop codon context, and in some cases, conserved stem–loops, providing clues about readthrough regulation and potential mechanisms. Lastly, we expand our studies beyond Drosophila and find evidence of abundant readthrough in several other insect species and one crustacean, and several readthrough candidates in nematode and human, suggesting that functionally important translational stop codon readthrough is significantly more prevalent in Metazoa than previously recognized.National Institutes of Health (U.S.) (U54 HG00455-01)National Science Foundation (U.S.) (CAREER 0644282)Alfred P. Sloan Foundatio

Myc-Dependent Genome Instability and Lifespan in Drosophila

The Myc family of transcription factors are key regulators of cell growth and proliferation that are dysregulated in a large number of human cancers. When overexpressed, Myc family proteins also cause genomic instability, a hallmark of both transformed and aging cells. Using an in vivo lacZ mutation reporter, we show that overexpression of Myc in Drosophila increases the frequency of large genome rearrangements associated with erroneous repair of DNA double-strand breaks (DSBs). In addition, we find that overexpression of Myc shortens adult lifespan and, conversely, that Myc haploinsufficiency reduces mutation load and extends lifespan. Our data provide the first evidence that Myc may act as a pro-aging factor, possibly through its ability to greatly increase genome instability

A Cis-Regulatory Map of the Drosophila Genome

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships

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. 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

Versatile P(acman) BAC Libraries for Transgenesis Studies in Drosophila melanogaster

We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95percent of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using Phi C31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns

Broad Complex Evolution, Function and Expression: Insights From Tissue Reorganization During Metamorphosis

Broad Complex (BRC) is an ecdysone-pathway gene essential for entry into and progression through metamorphosis in D. melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. Alternative splicing, of a protein-binding BTB-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC), produces four BRC isoforms. Highly conserved orthologs of BTBBRC and all four ZBRC were found in silico from Diptera, Lepidoptera, Hymenoptera and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometalolous orders. Five Tramtrack subfamily members were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, revealing that Z3BRC is the basal member. All four ZBRC domains, including Z4BRC which has no known essential function, are evolving in a manner consistent with selective constraint. Transgenic rescue and immunohistochemistry were used to explore how different BRC isoforms contribute to their shared tissue-morphogenesis functions at the onset of metamorphosis, when BRC is required for CNS reorganization. As predicted, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. Each isoform had a unique expression pattern in the CNS, with no substantial three-way overlap among them. Z4 is strongly expressed in a novel subset of CNS neurons. The most prominent localizations of BRC-Z1, -Z2, -Z3 corresponded with glia, neuroblasts and neurons, respectively. There appears to be a switch from BRC-Z2 in proliferating cells to BRC-Z1 and BRC-Z3 in differentiating cells. The temporal-requirement and spatial-distribution data suggest that BRC-dependent CNS morphogenesis is the result of multicellular interactions among different cell types at different times. BRC-Z1-expressing glia in prepupae may mediate the final steps of CNS morphogenesis. Lastly, BRC is required for migration and programmed cell death of the ring gland, the site of ecdysone and juvenile hormone production. Therefore, BRC may function in ecdysone auto-regulation

Myc-Dependent Genome Instability and Lifespan in <i>Drosophila</i>

The Myc family of transcription factors are key regulators of cell growth and proliferation that are dysregulated in a large number of human cancers. When overexpressed, Myc family proteins also cause genomic instability, a hallmark of both transformed and aging cells. Using an in vivo lacZ mutation reporter, we show that overexpression of Myc in Drosophila increases the frequency of large genome rearrangements associated with erroneous repair of DNA double-strand breaks (DSBs). In addition, we find that overexpression of Myc shortens adult lifespan and, conversely, that Myc haploinsufficiency reduces mutation load and extends lifespan. Our data provide the first evidence that Myc may act as a pro-aging factor, possibly through its ability to greatly increase genome instability.</p

Evolution of H3K27me3-marked chromatin is linked to gene expression evolution and to patterns of gene duplication and diversification

Histone modifications are critical for the regulation of gene expression, cell type specification, and differentiation. However, evolutionary patterns of key modifications that regulate gene expression in differentiating organisms have not been examined. Here we mapped the genomic locations of the repressive mark histone 3 lysine 27 trimethylation (H3K27me3) in four species of Drosophila, and compared these patterns to those in C. elegans. We found that patterns of H3K27me3 are highly conserved across species, but conservation is substantially weaker among duplicated genes. We further discovered that retropositions are associated with greater evolutionary changes in H3K27me3 and gene expression than tandem duplications, indicating that local chromatin constraints influence duplicated gene evolution. These changes are also associated with concomitant evolution of gene expression. Our findings reveal the strong conservation of genomic architecture governed by an epigenetic mark across distantly related species and the importance of gene duplication in generating novel H3K27me3 profiles

Myc overexpression increases mutation frequency.

<p>(A) Schematic representation of the <i>lacZ</i> reporter we used to quantitate mutation frequency, and potential mutation events that can occur in this transgene. Adapted from Garcia et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074641#pone.0074641-Garcia3" target="_blank">[29]</a> (B) Western analyses showing 9-fold overexpression of Myc compared to the loading control of γ-tubulin. Samples are wing imaginal discs of the genotypes hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc larvae (shown as “Myc”). Larvae were heat shocked (37°C) for 45 min during the 3^rd larval instar and allowed to develop for 12 hours. Eight discs were loaded per lane. (C) <i>lacZ</i> mutation frequency of hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc larvae (shown as “Myc”). Larvae are collected 3 days after a 45 minute heat shock at 37°C during the 1^st instar larval stage and separated by sex. Experiments shown are from at least four biological repeats (D) <i>lacZ</i> mutation frequency of hs-FLP; <i>lacZ</i> #9/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #9/+; act>CD2>Gal4, UAS-GFP/UAS-Myc (shown as “Myc”) larvae. (E) A selection (at least 48) plasmids were chosen to digest with AvaI to determine plasmid size per experiment for hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc (shown as “Myc”) larvae. Male and female data are shown separately for these analyses and do not show any differences in mutation spectra. White filled area of bar represents frequency of size change mutation (genome rearrangement), where as black solid area shows frequency of mutant plasmids that do not show a size change (point mutations). *indicates statistical significance of p<0.001 (student’s t-test).</p