Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Global Analysis of the Drosophila NELF complex

ABSTRACT: To determine the physiological targets of the NELF complex, and provide insight into the mechanism of NELF activity in vivo. Experiment Overall Design: Drosophila melanogaster S2 cells, obtained from the Drosophila Genomics Resource Center, were untreated, or treated with dsRNA for 72 hours, as described in Armknecht, S. et al. (2005) Methods in Enzymology, 392: pp. 55-73. Total RNA was then extracted using the RNeasy RNA extraction kit, with on column DNAse digestion (Qiagen), according to manufacturerâs protocol. Gene expression analysis was conducted using Drosophila Genome 2.0 GenechipÂ® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3â Amplification One-Cycle Target labeling kit according to manufacturerâs protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the GenechipÂ® Operating Software (Version 1.2.0.037) and imported into the Rosetta Resolver system (Version 6.0).

ORGANISM(S): Drosophila melanogaster

SUBMITTER: NIEHS Microarray Group

PROVIDER: E-GEOD-6141 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly.

Gilchrist Daniel A DA Nechaev Sergei S Lee Chanhyo C Ghosh Saikat Kumar B SK Collins Jennifer B JB Li Leping L Gilmour David S DS Adelman Karen K

Genes & development 20080701 14

The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes invol ...[more]

PMID: 18628398

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Project description:Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of gene. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region. To determine the importance of polymerase stalling for transcription regulation, we performed a genome-wide search for Drosophila genes with promoter-proximally stalled Pol II. Our data reveal that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals, indicating a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues. Keywords: gene expression, ChIP-chip, transcriptional regulation Drosophila Schneider cells (S2) were untreated or treated with dsRNA against LacZ or NELF for 96 hours. RNA isolation and immunoprecipitations (IPs) were performed for each of the test samples. Each treatment group (untreated, dsRNA-LacZ, dsRNA-NELF) was cultured in duplicate. Each RNA sample was amplified according to Affymetrix Eukaryotic One-cycle Protocol and hybridized to an Affymetrix Drosophila Genome 2.0 array, resulting in two biological replicates for each treatment group. Immunoprecipitations were performed either in the presence of Pol II Rpb3 or Ser2P Pol II CTD antibody or mock-immunoprecipitated with Protein-A agarose in the absence of antibody. Each IP was paired with a genomic DNA sample obtained at the time of the experiment. A separate labeling reaction was completed for each array type on which the IP/genomic DNA comparison was made (Agilent Dm3, Dm7 and Whole Drosophila Genome 2 chip set) and each of the two biological replicates were assayed.

Project description:Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter, or subsequently, through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a key difference between genes that use these distinct regulatory strategies lies in the chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor nucleosomal occlusion of the promoter. Pausing of polymerase maintains these genes in an active state by inhibiting the formation of repressive promoter chromatin. In contrast, promoters of housekeeping genes that lack paused Pol II are deprived of nucleosomes regardless of polymerase binding, but show higher nucleosome occupancy downstream. Our results suggest that the â??defaultâ?? chromatin state of a gene instructs its regulation, and that highly-regulated promoters have evolved to encourage competition between nucleosomes and paused Pol II for promoter occupancy. Drosophila melanogaster S2 cells (Drosophila Genomics Resource Center) were untreated, or treated with dsRNA for 96 hours, as described in Armknecht, S. et al. (2005) Methods in Enzymology, 392: pp. 55-73. Total RNA was then extracted using the RNeasy RNA extraction kit, with on column DNAse digestion (Qiagen), according to manufacturerâ??s protocol. Gene expression analysis was conducted using Drosophila Genome 2.0 GenechipÂ® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3â?? Amplification One-Cycle Target labeling kit according to manufacturerâ??s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using either GenechipÂ® Operating Software (Version 1.2.0.037) or Affymetrix GeneChip Command Console (V. 1.1) and imported into the Rosetta Resolver system (Version 6.0) as outlined in the scan and data processing protocols.

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Dataset Information

Global Analysis of the Drosophila NELF complex

Publications

NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly.

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