Abstract
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.
Highlights
The process of epithelial to mesenchymal transition (EMT) involves the conversion of cells from an attached, epithelial morphology to a migratory, stem cell-like phenotype
Recent high-resolution re-analysis of existing polymerase II (Pol II) chromatin immunoprecipitation (ChIP)-sequencing data stipulated the existence of two polymerase peaks centered at the transcription start site (TSS) and 110 nt downstream of the TSS [45], in an apparent contradiction to Pol II positioning suggested by our short capped RNA (scRNA) sequencing in MCF-7 cells as well as previous work in other systems [15,26,35,46]
Because single-end ChIP-sequencing carries an uncertainty in centering of the read positions and hampers resolution of closely located peaks, we performed Pol II ChIPsequencing in paired end format to determine read center positions directly
Summary
The process of epithelial to mesenchymal transition (EMT) involves the conversion of cells from an attached, epithelial morphology to a migratory, stem cell-like phenotype. EMT is essential in normal development, but when activated in cancer cells it can lead to metastasis [1,2]. EMT is a complex process involving several genes, many of which are potential targets for anti-cancer therapy, including SNAI1, SNAI2, ZEB1 and HSP70 [3,4,5]. The products of SNAI1 (Snail) and SNAI2 (Slug) genes are master regulators of EMT and their elevated levels confer poor prognosis in breast cancer [6,7]. The Snail and Slug proteins promote EMT through repressing genes such as E-cadherin (CDH1), (which encodes a cell–cell adhesion protein) by binding to their promoter regions and recruiting repressor complexes including histone deacetylases (HDACs) [8,9,10]. One of the challenges in the prevention and treatment of metastatic cancers is that commonly used drugs, especially broad-spectrum agents including HDAC inhibitors, can act through multiple mechanisms [reviewed in [11]] and have variable effects on different cell types [12,13]
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