Unknown,Transcriptomics,Genomics,Proteomics

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RNA polymerase is poised for activation across the genome

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: NIEHS Microarray Core

PROVIDER: E-GEOD-6714 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA polymerase is poised for activation across the genome.

Muse Ginger W GW Gilchrist Daniel A DA Nechaev Sergei S Shah Ruchir R Parker Joel S JS Grissom Sherry F SF Zeitlinger Julia J Adelman Karen K

Nature genetics 20071111 12

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 genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-onc ...[more]

PMID: 17994021

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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.

Project description:The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate the differences between the two cell types, we performed strand-specific massively-parallel sequencing of RNA from C. albicans white and opaque cells. Combining the resulting data from both cell types, we first substantially re-annotated the C. albicans transcriptome, finding 1443 novel coding and non-coding transcriptionally active regions. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by the master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of the master regulator and the differential expression of adjacent genes, a discrepancy observed in many other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Indeed, many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also found that 5' and 3' untranslated regions (UTRs) of mRNAs in the circuit are unusually long and that 5' UTRs often differ in length between white and opaque cells. These observations suggest that the use of alternative promoters is widespread in the circuit and that important regulatory information is carried in the long UTRs. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes. RNA-Seq was applied to Candida albicans white and opaque cells to identify novel transcripts and UTRs that are differentially regulated between the two cell types. Two biological replicates each of white and opaque cell cultures. One of the white cell RNA samples was split just after isolation to allow a comparison of the poly(A)-selection and ribo-depletion sample preparation strategies.

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RNA polymerase is poised for activation across the genome

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RNA polymerase is poised for activation across the genome.

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