The identification of Dwg downstream targets using ChIP-seq

To study the autophagy related genes, we performed RNAi screens to identify new autophagic regulators. Dwg1 is one of new regulators of autophagy. We hypothesized that as an insulator, the Dwg protein might regulate autophagy through binding to insulator elements on chromatin and blocking enhancer functions. We therefore performed chromatin-immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify Dwg downstream targets. The results suggest that Dwg binds to the insulator elements present in/near the ATG genes, presumably suppressing their transcription. Overall design: S2R+ cells expressing Flag-Dwg were subjected to ChIP assays using SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer's protocol. DNA co-immunoprecipitation with either anti-Flag antibody or IgG control antibody was analyzed by deep DNA-sequencing or quantified by ChIP-qPCR.