RNA polymerase II pausing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition.

Promoter-proximal RNA polymerase II (Pol II) pausing is implicated in the regulation of gene transcription. However, the mechanisms of pausing including its dynamics during transcriptional responses remain to be fully understood. We performed global analysis of short capped RNAs and Pol II Chromatin Immunoprecipitation sequencing in MCF-7 breast cancer cells to map Pol II pausing across the genome, and used permanganate footprinting to specifically follow pausing during transcriptional activation of several genes involved in the epithelial to mesenchymal transition (EMT). We find that the gene for EMT master regulator Snail (SNAI1), but not Slug (SNAI2), shows evidence of Pol II pausing before activation. Transcriptional activation of the paused SNAI1 gene is accompanied by a further increase in Pol II pausing signal, whereas activation of non-paused SNAI2 gene results in the acquisition of a typical pausing signature. The increase in pausing signal reflects increased transcription initiation without changes in Pol II pausing. Activation of the heat shock HSP70 gene involves pausing release that speeds up Pol II turnover, but does not change pausing location. We suggest that Pol II pausing is retained during transcriptional activation and can further undergo regulated release in a signal-specific manner.

Analysis of paired end Pol II ChIP-seq and short capped RNA-seq in MCF-7 cells.

While a role of promoter-proximal RNA Polymerase II (Pol II) pausing in regulation of eukaryotic gene expression is implied, the mechanisms and dynamics of this process are poorly understood. We performed genome-wide analysis of short capped RNAs (scRNAs) and Pol II chromatin immunoprecipitation sequencing (ChIP-seq) in human breast cancer MCF-7 cells to better understand Pol II pausing (Samarakkody, A., Abbas, A., Scheidegger, A., Warns, J., Nnoli, O., Jokinen, B., Zarns, K., Kubat, B., Dhasarathy, A. and Nechaev, S. (2015) RNA polymerase II pausing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition. Nucleic Acids Res43, 3938-3949). The data are available at the NCBI Gene Expression Omnibus under accession number GSE67041. For both ChIP and scRNA samples, we used paired end sequencing on the Illumina MiSeq instrument. For ChIP-seq, the use of paired end sequencing allowed us to avoid ambiguities in center-read definition. For scRNA seq, this allowed us to identify both the 5'-end and the 3'-end in the same run that represent, respectively, the transcription start sites and the locations of Pol II pausing. The sharpening of Pol II ChIP-seqmetagene profiles when aligned against 5'-ends of scRNAs indicates that these RNAs can be used to define the start sites for the majority of mRNA transcription events.