Mol Cell. 2008 Mar 28;29(6):742-54.
Identification of SUMO-dependent chromatin-associated transcriptional repression components by a genome-wide RNAi screen.
Stielow B, Sapetschnig A, Krüger I, Kunert N, Brehm A, Boutros M, Suske G.Institute of Molecular Biology and Tumor Research, Philipps-University of Marburg, Emil-Mannkopff-Strasse 2, D-35032 Marburg, Germany.
SUMO modification of many transcription factors is linked to transcriptional repression. The molecular mechanisms by which SUMO attachment represses transcription are largely unknown. Here we report a genome-wide RNA interference screen in Drosophila melanogaster cells for components regulating and mediating SUMO-dependent transcriptional repression. Analysis of >21,000 double-stranded RNAs (dsRNAs) identified 120 genes whose dsRNA-mediated knockdowns impaired SUMO-dependent transcriptional repression. Several of these genes encode chromatin-associated proteins, including the ATP-dependent chromatin remodeler Mi-2, the D. melanogaster ortholog of the C. elegans protein MEP-1, and the polycomb protein Sfmbt. Knockdown of these proteins did not impair SUMO conjugation, demonstrating that they act downstream of SUMO attachment. Biochemical analyses revealed that MEP-1, Mi-2, and Sfmbt interact with each other, bind to SUMO, and are recruited to promoters in a SUMOylation-dependent manner. Our results suggest that MEP-1, Mi-2, and Sfmbt are part of a common repression complex established by DNA-bound SUMO-modified transcription factors.
It is an interesting paper. The reporter assay was used as a screening tool. The output looks good.
Genome-wide High-Throughput RNA Interference Screen
A library of >21,000 dsRNAs on polystyrene 384-well plates representing the D. melanogaster genome was used as starting material (Boutros et al., 2004; Muller et al., 2005). As internal negative and positive controls, each 384-well plate contained wells with dsRNA targeting SUMO, GFP, and FLuc. Kc167 cells were dispensed at a density of 13,500 cells per well in 15 ul serum-free medium using an automated liquid dispenser (MultiDrop, Thermo Electron Corporation) and incubated for 60 min at 25 C. Subsequently, 20 ul serum-containing medium was added, and cells were cultured for 24 hr at 25 C. Transfection was performed with an automated liquid dispenser (MultiDrop) using the Effectene transfection reagent (QIAGEN). Each well obtained a total of 44 ng DNA (2 ng pPacUSp3, 40 ng [GC]2-FLuc, and 2 ng pPac-RLuc) in a volume of 20 ul. Five days posttransfection, firefly and Renilla luciferase activities were determined as described (Muller et al., 2005).
Firefly and Renilla luciferase values of individual wells were normalized to the median of the FLuc or RLuc values, respectively, of each plate. Then the ratio of each normalized FLuc and RLuc value, as well as the average of the duplicates, was calculated. Normalized FLuc/RLuc values higher than the lowest value obtained with dsRNA targeting D. melanogaster SUMO were considered as candidate dsRNAs leading to the initial selection of 585 candidate dsRNAs. Additional stringent selection criteria were applied to reduce the number of genes for further analyses. dsRNAs with the following features were not considered in secondary screens: (1) dsRNAs exhibiting an extreme viability phenotype leading to normalized FLuc values lower than 0.5, (2) dsRNAs that enhanced actin promoter-driven Renilla luciferase by more than 1.25-fold, and (3) dsRNAs with a specificity less than 70% or more than six potential 21 nt off-targets.
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