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RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.

ABSTRACT: It is widely assumed that the key rate-limiting step in gene activation is the recruitment of RNA polymerase II (Pol II) to the core promoter. Although there are well-documented examples in which Pol II is recruited to a gene but stalls, a general role for Pol II stalling in development has not been established. We have carried out comprehensive Pol II chromatin immunoprecipitation microarray (ChIP-chip) assays in Drosophila embryos and identified three distinct Pol II binding behaviors: active (uniform binding across the entire transcription unit), no binding, and stalled (binding at the transcription start site). The notable feature of the approximately 10% genes that are stalled is that they are highly enriched for developmental control genes, which are either repressed or poised for activation during later stages of embryogenesis. We propose that Pol II stalling facilitates rapid temporal and spatial changes in gene activity during development.

SUBMITTER: Zeitlinger J

PROVIDER: S-EPMC2824921 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Chromatin immunoprecipitation of Drosophila embryos to investigate RNA polymerase stalling at developmental control genes

Project description:Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. We determine the genome-wide occupancy of the dorsoventral determinants Dorsal, Twist and Snail in the Drosophila embryo using ChIP-chip analysis. There is a tight correlation between the limits of known enhancers and the in vivo binding of these proteins. The analysis predicts substantially more target genes than previously estimated, including Dpp signaling components and genes encoding anteroposterior segmentation determinants. Thus, the ChIP-chip data uncovers a much larger network, which integrates diverse patterning processes during development.

2007-11-15 | E-TABM-322 | biostudies-arrayexpress

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Comparative genomic analysis of Drosophila melanogaster and vector mosquito developmental genes.

Project description:Genome sequencing projects have presented the opportunity for analysis of developmental genes in three vector mosquito species: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A comparative genomic analysis of developmental genes in Drosophila melanogaster and these three important vectors of human disease was performed in this investigation. While the study was comprehensive, special emphasis centered on genes that 1) are components of developmental signaling pathways, 2) regulate fundamental developmental processes, 3) are critical for the development of tissues of vector importance, 4) function in developmental processes known to have diverged within insects, and 5) encode microRNAs (miRNAs) that regulate developmental transcripts in Drosophila. While most fruit fly developmental genes are conserved in the three vector mosquito species, several genes known to be critical for Drosophila development were not identified in one or more mosquito genomes. In other cases, mosquito lineage-specific gene gains with respect to D. melanogaster were noted. Sequence analyses also revealed that numerous repetitive sequences are a common structural feature of Drosophila and mosquito developmental genes. Finally, analysis of predicted miRNA binding sites in fruit fly and mosquito developmental genes suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. The results of this study provide insight into the evolution of developmental genes and processes in dipterans and other arthropods, serve as a resource for those pursuing analysis of mosquito development, and will promote the design and refinement of functional analysis experiments.

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Mechanism of RNA polymerase II stalling by DNA alkylation.

Project description:Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

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The developmental transcriptome of Drosophila melanogaster.

Project description: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. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

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Argonaute2 attenuates active transcription by limiting RNA Polymerase II elongation in Drosophila melanogaster.

Project description:Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.

| S-EPMC6200757 | biostudies-literature

UV damage-induced RNA polymerase II stalling stimulates H2B deubiquitylation.

Project description:Histone H2B monoubiquitylation plays an important role in RNA polymerase II (RNAPII) elongation. Whether this modification responds to RNAPII stalling is not yet known. We report that both yeast and human cells undergo a rapid and significant H2B deubiquitylation after exposure to UV irradiation. This deubiquitylation occurs concurrently with UV-induced transcription arrest and is significantly reduced in a DNA damage-bypassing RNAPII yeast mutant. Consistent with these results, yeast deubiquitylases Ubp8 and Ubp10 are associated with the RNAPII complex. Moreover, simultaneous deletion of Ubp8 and Ubp10 leads to a lack of H2B deubiquitylation after UV exposure. Consequently, nucleotide excision repair at an actively transcribed gene locus is decreased, whereas UV-induced RNAPII degradation is increased in ubp8?ubp10? mutant cells. These results indicate that eukaryotic cells respond to RNAPII arrest by deubiquitylating H2B to coordinate DNA repair and RNAPII degradation.

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