Oct 9, 2018

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Journal of Visualized Experiments : JoVE
Anna M ChiarellaNathaniel A Hathaway

Abstract

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitativ...Continue Reading

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Mentioned in this Paper

Real-Time Polymerase Chain Reaction
Flow Cytometry
Sma-2 protein, C elegans
Histone antigen
Cellular Process
Genome
Genes
Regulation of Biological Process
Transcription, Genetic
Environment

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