Abstract
In eukaryotes, the circadian clock controls 24h rhythms in physiology, metabolism, and behavior via cell autonomous transcriptional feedback loops. These feedback loops keep circadian time and control rhythmic outputs by driving rhythms in transcription; thus, it is important to determine when clock transcription factors bind their target sequences in vivo to promote or repress transcription. Interactions between proteins and DNA can be measured in cells, tissue, or whole organisms using a technique called chromatin immunoprecipitation (ChIP). The principle underlying ChIP is that protein is cross-linked to associated chromatin to form a protein-DNA complex, the DNA is then sheared, and the protein of interest is immunoprecipitated. The cross-links are then removed from the antibody-protein-DNA complex, and the associated DNA fragments are purified. The DNA is then used to quantify specific targets by real-time quantitative PCR or to generate libraries for global analysis of protein target sites by high-throughput sequencing (ChIP-seq). ChIP has been widely used in circadian biology to assess rhythmic binding of clock components, RNA polymerase II, and rhythms in chromatin modifications such as histone acetylation and methylation. Here, we present a detailed method for ChIP analysis in Drosophila that can be used to assess protein-DNA-binding rhythms at specific genomic target sites. With minor modifications, this technique can be used to assess protein-DNA-binding rhythms at all target sites via ChIP-seq. ChIP analysis has revealed the relationship between clock factor binding, transcription, and chromatin modifications and promises to reveal circadian transcription networks that control phase and tissue specificity.
