Identification of genes directly regulated by a transcription factor in rice

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.