Transcription profiling of Drosophila E2f/Rb genetic pathway manipulation
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ABSTRACT: We demonstrate that the cell cycle regulators, E2f /Dp and Rb, control the transcription of ribosomal proteins (RP) in Drosophila embryos. Mutation of E2f1 or Dp and over expression of Rbf1 increase and reduce RP transcription, respectively. Although E2f/Dp/Rb might exert this effect through a repressor complex, the regulatory regions of RP genes do not show an enrichment of canonical E2f binding sites. In addition, E2f1, Dp and Rbf1 also regulate the expression of RACK1, a ribosomal component and a negative regulator of cell cycle progression. These findings strengthen the coupling of cell cycle regulation to protein biosynthesis. Experiment Overall Design: Our study examines the genomic response of intact Drosophila embryos to the genetic manipulation of E2F/Rb pathway molecules. For over-expression experiments, Arm-Gal4 stocks were crossed to UAS-gene stocks for the following genes: E2f1/Dp, E2f2/Dp, Rbf1, and E2f1336-805 (a dominant negative form of E2f1 lacking the DNA binding domain) [16]. The E2f17172 and E2f191 null alleles were balanced by TM3[Kr-GFP]. Dpa2 and Dpa4 null alleles were balanced by CyO[Kr-GFP]. 14-16 hr E2f17172/E2f191 null mutant embryos were hand selected based on the absence of fluorescent Bolwigâs organs in Kr-GFP balanced stocks using a Zeiss stereomicroscope equipped with epifluorescence. E2f17172/E2f191 and Dpa2/Dpa4 null mutant embryos (8-10h) were selected using the COPASTM SELECT system for automated sorting of multi-cellular organisms. Eighteen RNA samples, two from each cross, were obtained from approximately 200 to 700 embryos per sample. For each cross, 2 h embryo collections were aged for 8 to 14 h at 25oC, quick frozen in an ethanol/dry ice mixture, and stored at -80 oC. The RNA was prepared for microarray analysis in accordance with Affymetrix instructions.
ORGANISM(S): Drosophila melanogaster
SUBMITTER: William Ward
PROVIDER: E-GEOD-7655 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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