|Ms. Svenja Reinders (Max-Planck-Institute of Psychiatry)
|Establishing novel chromatin-associations by chromatin immunoprecipitation - The importance of Antibodies and Reagents
|It has been known for a long time that life experience can program gene activity later in life. More recently, epigenetics has attracted wide attention also in neuroscience as the potential underlying molecular mechanism. The stress hormone axis orchestrates the reaction of the body to stressful life situations, which also involves epigenetic programming. Dysregulation of the stress hormone axis, in turn, has been associated with major depression. Therefore, the question arises whether proteins regulated by the stress hormone axis are influencing epigenetic processes such as DNA or histone modifications? Many of the epigenetic regulators bind to DNA, and thus the first step to answering this question is to find out whether and where these proteins bind to DNA. The method of choice is Chromatin-Immunoprecipitation also called ChIP with subsequent next generation sequencing (i.e. ChIP-Seq). Here DNA and proteins are crosslinked together and then precipitated using an antibody directed against the protein of interest. As no binding sequences are known in the beginning, to establish a novel protein as DNA-associated requires a careful and lengthy optimization procedure leading to fine-tuning of the ChIP parameters. I will present different approaches and considerations to tackle such a research question covering the effects of buffers, antibodies and chrosslinking reagents at the example of FKBP51, a crucial protein of the stress hormone axis.