title: Fluorescence microscopy-based high-throughput screening for factors involved in gene silencing.
abstract: Gene silencing in eukaryotes is a highly controlled process. It involves the concerted action of histone and DNA-modifying enzymes as well as transcription factors and chromatin-associated proteins. To understand how epigenetic gene silencing is regulated, it is important to identify the factors involved in this process. Here we describe an assay that allows high-throughput screening for factors involved in gene silencing. This assay exploits the susceptibility of the viral cytomegalovirus (CMV) promoter to epigenetic silencing in embryonic stem cells (ESCs) and uses reporter constructs with an optical readout to determine the gene silencing potential of candidate factors. This approach allows to study mechanisms and kinetics of gene silencing in living cells and to evaluate the role of DNA methyltransferases, histone-modifying enzymes, and other chromatin-associated factors during gene silencing.
authors: Department of Biology, Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Munich, Germany.
authors: Sebastian
authors: S
authors: Bultmann
authors: Heinrich
authors: H
authors: Leonhardt
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D000090205:DNA Methyltransferase 3A; D053595:Embryonic Stem Cells / Q000166:cytology; D044127:Epigenesis, Genetic / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006649:Histocompatibility Antigens / Q000235:genetics; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering; D019076:Transgenes / Q000235:genetics; D000097584:DNA Methyltransferase 3B
chemical_list: D000199:Actins; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D006649:Histocompatibility Antigens; D011495:Histone-Lysine N-Methyltransferase; D034741:RNA, Small Interfering; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C445405:EHMT2 protein, human