RNA Fluorescence In Situ Hybridization Tagging and Recovery of Associated Proteins to Analyze In Vivo Chromatin Interactions

Abstract

This chapter presents a method designed to detect tertiary chromatin interactions between specific DNA sequences in vivo. This method is used to show that the distal β-globin locus control region is intimately associated with an actively transcribed β-globin gene in erythroid cells. The technique, called RNA FISH TRAP (fluorescence in situ hybridization tagging and recovery of associated proteins), utilizes the targeting power of in situ hybridization to tag proteins near a specific, transcriptionally active gene locus. A hapten-labeled, antisense DNA probe is hybridized to the intron sequences of a nascent primary transcript associated with an actively transcribed gene in formaldehyde-fixed cells. A hapten-specific antibody conjugated with horseradish peroxidase (HRP) is then directed to the DNA probe, thereby localizing HRP activity to the specific gene locus. The HRP is then used to catalyze the covalent attachment of a tag (in this case, a biotinylated tyramide) to proteins in the immediate vicinity of the gene. This tag can then be used in affinity purification procedures to recover proteins and chromatin complexes near the site of transcription. This technique helps to bridge the gap between light microscopy and electron microscopy in that it allows the recovery and analysis of sequences that are engaged in functional interactions or specifically juxtaposed to a defined gene locus in vivo. Permutations of this technique will undoubtedly aid in the understanding of higher order chromatin structure and the role of chromatin interactions in various nuclear processes.