Detecting transcriptionally engaged RNA polymerase in eukaryotic cells with permanganate genomic footprinting

Analysis of the distribution of RNA polymerase II on the genomes of Drosophila and human cells using in vivo protein-DNA crosslinking reveals that RNA polymerase II (Pol II) is concentrated at the 5′-ends of thousands of genes. This appears to be irrespective of transcription levels. Hence, a potential regulatory step in the transcription of many genes occurs after Pol II has associated with the promoter. The protein-DNA crosslinking technique widely used to monitor Pol II and other proteins on chromosomes in vivo, however, does not reveal if Pol II is transcriptionally engaged on DNA. Genomic footprinting with potassium permanganate provides one method for detecting transcriptionally engaged Pol II. Using this approach, we have determined that the Pol II associated with the promoters of many genes has initiated transcription but paused in the region 20–50 nucleotides from the start. Here we describe the application of this method in Drosophila and human cells. The method should prove useful in assessing if promoter bound Pol II has engaged in transcription and for investigating the establishment and regulation of transcriptionally engaged Pol II.