Chem-seq

Chem-seq is a technique that is used to map genome-wide interactions between small molecules and their protein targets in the chromatin of eukaryotic cell nuclei. The method employs chemical affinity capture coupled with massively parallel DNA sequencing to identify genomic sites where small molecules interact with their target proteins or DNA. It was first described by Lars Anders et al. in the January, 2014 issue of 'Nature Biotechnology'. Chem-seq is a technique that is used to map genome-wide interactions between small molecules and their protein targets in the chromatin of eukaryotic cell nuclei. The method employs chemical affinity capture coupled with massively parallel DNA sequencing to identify genomic sites where small molecules interact with their target proteins or DNA. It was first described by Lars Anders et al. in the January, 2014 issue of 'Nature Biotechnology'. A substantial number of small-molecule ligands, including therapeutic drugs, elicit their effects by binding specific proteins associated with the genome. Mapping the global interactions of these chemical entities with chromatin in a genome-wide manner could provide insights into the mechanisms by which a small molecule influences cellular functions. When combined with other chromatin analysis techniques such as ChIP-seq, Chem-seq can be utilized to investigate the genome-wide effects of therapeutic modalities and to understand the effects of drugs on nuclear architecture in various biological contexts. In a broader sense, these methods will be useful to enhance our understanding of the therapeutic mechanisms through which small molecules modulate the function and activity of genome-associated proteins. Through the identification of the cellular targets of a drug, it becomes possible to gain an increased understanding of the causes of side effects and toxicity in the early stages of drug development, which should help to reduce the attrition rate in development. Chem-seq relies on the ability to create a biotinylated version of a small molecule of interest to allow for downstream affinity capture. Chem-seq can be carried out either In vitro or In vivo, although the results from each have proven to be highly similar. In vivo Chem-seq During In vivo Chem-seq, cultured cells in medium are treated simultaneously with either a biotinylated version of the small molecule under study or DMSO (as a control) and 1% formaldehyde for the crosslinking of DNA, proteins and small molecules. DNA is then extracted from the cells, sonicated and enriched for regions containing the biotinylated molecule of interest by incubation with streptavidin magnetic beads, which have a very high affinity for biotin. The enriched DNA fraction is then purified, eluted from the beads and subjected to next generation sequencing. Genomic regions enriched in the Chem-seq library relative to the control are associated with the small molecule under study. In vitro Chem-seq In vitro Chem-seq begins with the crosslinking of cultured cells in medium with 0.5% formaldehyde. Cell nuclei are then harvested from the cells and their DNA is extracted. This extract is sonicated before being incubated with streptavidin magnetic beads that are bound to a biotinylated form of our compound of interest. This provides an opportunity for the small molecule of interest to interact with its target genomic regions. These genomic regions are then isolated using a magnet and subjected to next generation sequencing and analysis to determine regions enriched for our small molecule of interest. Chem-seq was tested on three classes of drugs using MM1.S multiple myeloma cells to: 1) Investigate the genome-wide binding of the bromodomain inhibitor JQ1 to the BET bromodomain family members BRD2, BRD3 and BRD4