Group Prof. Dr. M. Thomm
Group member: Robert Reichelt, Thomas Fouqueau
Studying genome wide Protein-DNA interactions through Chromatin immunoprecipitation (ChIP) in Pyrococcus furiosus
Chromatin immunoprecipitation (ChIP) is a powerful tool to study protein-DNA interactions in vivo. Treatment of living Pyrococcus furiosus cells with formaldehyde leads to the covalent fixation of protein-DNA interactions. After harvesting the cells, sonication disrupts the cells and genomic DNA can be sheared to a fragment size smaller than 1000 base pairs. Immunoprecipitation of the protein of interest with a specific antibody enriches the genomic DNA fragments bound to this protein. Enrichment of specific genomic loci (e.g. promoter regions) can be analysed by realtime PCR and shows binding of the protein of interest to these regions in vivo. Moreover combining ChIP with high-throughput sequencing (ChIP-Seq) allows the study of protein-DNA interactions in a genome wide scale in vivo.
Schematic overview Chromatin Immunoprecipitation (ChIP) workflow:
Currently we are establishing this method to look for genome wide binding sites of various transcriptional regulators (e.g. TrmBL1) and to reveal their global function in archaeal transcription.