Fluorescence microscopy-based high-throughput screening for factors involved in gene silencing

Methods Mol Biol. 2013;1042:237-44. doi: 10.1007/978-1-62703-526-2_17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics*
  • Animals
  • Cells, Cultured
  • Cytomegalovirus / genetics*
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • Embryonic Stem Cells / cytology
  • Epigenesis, Genetic / genetics
  • High-Throughput Screening Assays / methods*
  • Histocompatibility Antigens / genetics
  • Histone-Lysine N-Methyltransferase / genetics
  • Humans
  • Mice
  • Microscopy, Fluorescence / methods
  • Promoter Regions, Genetic / genetics*
  • RNA Interference*
  • RNA, Small Interfering
  • Transgenes / genetics

Substances

  • Actins
  • Histocompatibility Antigens
  • RNA, Small Interfering
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA methyltransferase 3A
  • DNA methyltransferase 3B
  • EHMT2 protein, human
  • Histone-Lysine N-Methyltransferase