Identification of functional cis-regulatory elements by sequential enrichment from a randomized synthetic DNA library

BMC Plant Biology

Research Article

Mario Roccaro¹  Imre E Somssich¹  Nahal Ahmadinejad²  Thomas Colby³ 

[1] Department of Plant Microbe Interaction, Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, 50829, Cologne, Germany;Department of Plant Microbe Interaction, Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, 50829, Cologne, Germany;INRES - Crop Bioinformatics, Universität Bonn, Katzenburgweg 2, 53115, Bonn, Germany;Mass Spectrometry Group, Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, 50829, Cologne, Germany;

关键词: Chromatin immunoprecipitation (ChIP);    Motif discovery;    Plant protoplasts;    Pol II CTD phosphorylation;    Synthetic DNA elements;   

DOI  :  10.1186/1471-2229-13-164

 received in 2013-07-08, accepted in 2013-10-08,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe identification of endogenous cis-regulatory DNA elements (CREs) responsive to endogenous and environmental cues is important for studying gene regulation and for biotechnological applications but is labor and time intensive. Alternatively, by taking a synthetic biology approach small specific DNA binding sites tailored to the needs of the scientist can be generated and rapidly identified.ResultsHere we report a novel approach to identify stimulus-responsive synthetic CREs (SynCREs) from an unbiased random synthetic element (SynE) library. Functional SynCREs were isolated by screening the SynE libray for elements mediating transcriptional activity in plant protoplasts. Responsive elements were chromatin immunoprecipitated by targeting the active Ser-5 phosphorylated RNA polymerase II CTD (Pol II ChIP). Using sequential enrichment, deep sequencing and a bioinformatics pipeline, candidate responsive SynCREs were identified within a pool of constitutively active DNA elements and further validated. These included bonafide biotic/abiotic stress-responsive motifs along with novel SynCREs. We tested several SynCREs in Arabidopsis and confirmed their response to biotic stimuli.ConclusionsSuccessful isolation of synthetic stress-responsive elements from our screen illustrates the power of the described methodology. This approach can be applied to any transfectable eukaryotic system since it exploits a universal feature of the eukaryotic Pol II.

【 授权许可】

CC BY   
© Roccaro et al.; licensee BioMed Central Ltd. 2013