Identification of synthetic cis -regulatory DNA elements (SynCREs) via Pol II ChIP and validation. (A) Synthetic DNA element (SynEs) library was cloned into a binary vector upstream of the CaMV 35S-derived TATA-box to drive expression of the luciferase reporter gene (LUC), and transfected into parsley protoplasts. Pol II phosphorylation at Ser-5 is indicated. DNA-Pol II complexes were formaldehyde cross-linked and the SynEs mediating LUC expression were immunoprecipitated with an antibody (H14; indicated in red) specific to Ser-5 phosphorylated Pol II. The first chromatin immunoprecipitated SynEs (ChIP) were specifically PCR amplified and re-cloned to construct a subsequent library, which was used for a second transfection round and ChIP. By repeating these steps, an additional enrichment for SynEs mediating LUC expression was obtained. (B) Scheme for SynEs library screening, ChIP, and sample treatments. Three Pol II ChIP rounds were performed on transfected parsley protoplasts untreated or induced by Pep25, to enrich for responsive SynCREs. Eight samples were sequenced (boxed) comprising the main library, two sub-libraries and six samples. (C) Comparison of LUC activities in parsley protoplasts between SynEs contained in a first round ChIP (I ChIP) sample and in a subsequent ChIP-derived sample (II ChIP), either untreated (control) or Pep25 induced. The II ChIP sample showed higher LUC activity than the I ChIP sample. Both samples showed increased luciferase activities under inducing conditions compared to control. LUC activity was measured as counts per second (cps) of photon emission using the TopCount®. (D) Comparison of LUC activities between SynEs contained in a II ChIP sample and following a third ChIP (III ChIP) round, either under untreated or Pep25-induced conditions. The III ChIP sample showed higher LUC activity than the II ChIP sample. Increase of LUC activity observed in samples after each subsequent round of ChIP indicates enrichment for responsive SynCREs.