PROTOCOL EXCHANGE | COMMUNITY CONTRIBUTED Subject terms: Genetic analysis Genetic modification Genomics Model organisms Nucleic acid based molecular biology Plant biology Proteomics Tissue culture Keywords: transcription factor rice protoplast chromatin immunoprecipitation luciferase assay Identification of genes directly regulated by a transcription factor in rice Supratim Basu, Arjun Krishnan, Madana Ambavaram, Lutfor Rahman, Venkategowda Ramegowda & Andy Pereira Pereira lab Abstract A protocol is described for identification of gene (promoter) targets regulated by an AP2/ERF transcription factor (TF) in rice. A tandem affinity purification TAP-tagged construct of the AP2 TF transformed into rice is used in chromatin immunoprecipitation (ChIP) assays to verify the putative AP2-regulated genes that are first identified from gene expression data and analysis of cis-regulatory elements in their promoters. Transcriptional activation/repression of the AP2 TF bound promoters is experimentally validated by a Dual Glo luciferase assay system (firefly luciferase luminescence normalized to renilla luciferase) using putative regulated promoters cloned upstream of the luciferase and co-transformed with a construct expressing the AP2 TF in rice protoplasts. Finally, the protocol describes the identification of the direct targets identified from ChIP and luciferase assay using a steroid receptor based inducible system. Introduction Environmental stresses like drought and high temperature primarily affect the grain yield of cereals by reducing photosynthesis and related carbon metabolism 1 . A comprehensive analysis of the gene network regulating these complex processes will help in development of genetic tools for maintaining yield under stress. The expression of stress related genes is Identification of genes directly regulated by a transcription facto... http://www.nature.com/protocolexchange/protocols/3423 1 of 13 6/19/15, 1:23 PM