ETV3 ChIP-seq
Large scale discovery of ERK2 substrates identifies transcriptional regulation by ETV3
Chromatin immunoprecipitation and high-throughput sequencing of transcriptional repressor ETV3. Two conditions consists of 75 x 10e6 293T cells transfected with human HA-tagged ETV3, while the third condition is a transfection control. One condition was treated with phorbol ester (PMA 100nM for 20 minutes) to activate signaling through ERK1/2. We have shown that this condition results in stoichiometric phosphorylation of ETV3. The second condition was treated with MEK inhibitor U0126 (10uM for 20 minutes), which results is complete loss of phosphorylation on ETV3. Each set of cells was crosslinked with 1% formaldehyde, sonicated, and subject to ChIP followed by high throughput sequencing.
The mitogen-activated protein kinase ERK2 is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAP kinases are heavily studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using an analog sensitive kinase to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are previously reported, across a wide range of pathways including regulation of transcription and translation, Rho GTPase activity and mRNA processing. Among the novel substrates, the ETS repressor factor ETV3 is heavily phosphorylated on sites within canonical and non-canonical ERK motifs. Phosphorylation of ETV3 regulates transcription by preventing binding to DNA at promoters for several thousand genes, including targets involved in negative feedback regulation of itself and of upstream signals.