Genome-wide identification of protein binding sites in mammalian cells

We present GWPBS-Cap, a method to capture genome-wide protein binding sites (PBSs) without using antibodies. Using this technique, we identified many protein binding sites with different binding strengths between proteins and DNA. The PBSs can be useful to predict transcription binding sites and the co-localization of multiple transcription factors in the genome. The results also revealed that active promoters contained more protein binding sites with lower NaCl tolerances. Taken together, GWPBS-Cap can be used to efficiently identify protein binding sites and reveal genome-wide landscape of DNA-protein interactions.

Biolayer Interferometry for DNA-Protein Interactions v1

Biolayer interferometry (BLI) is a widely utilized technique for determining the interaction dynamics between macromolecules. Most BLI instruments, such as the Octet RED96e used throughout this protocol, are completely automated and detect changes in the interference pattern of white light reflected off a biosensor tip. Biosensors are initially loaded with a stationary macromolecule, then introduced into a solution containing macromolecules of interest. Binding to the stationary molecules creates a change in optical wavelength that is recorded by the instrument in real-time. The majority of published BLI experiments assess protein-protein (such as antibody-substrate kinetics) or protein-small molecule (such as drug discovery) interactions. However, a less-appreciated assay for BLI analysis is DNA-protein interactions. In our laboratory, we have shown the practicality of using biotinylated-DNA probes to determine the binding kinetics of transcription factors to specific DNA sequences. The following protocol describes these steps, including the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis through GraphPad Prism.

Single-molecule simultaneous profiling of DNA methylation and DNA-protein interactions with Nanopore-DamID

DNA-protein interactions and cytosine methylation control eukaryotic gene expression. Here, we present an approach to simultaneously detect cytosine methylation and DNA-protein interactions from single molecules, through selective sequencing of adenine-labelled DNA. Applying this approach to LaminB1-associated heterochromatin domains, we identify strict CpG methylation maintenance at transcriptional start sites amid a generalised relaxation of methylation, potentially to prevent ectopic aberrant heterochromatic gene expression.

DNA–protein interaction studies: a historical and comparative analysis

DNA–protein interactions are essential for several molecular and cellular mechanisms, such as transcription, transcriptional regulation, DNA modifications, among others. For many decades scientists tried to unravel how DNA links to proteins, forming complex and vital interactions. However, the high number of techniques developed for the study of these interactions made the choice of the appropriate technique a difficult task. This review intends to provide a historical context and compile the methods that describe DNA–protein interactions according to the purpose of each approach, summarise the respective advantages and disadvantages and give some examples of recent uses for each technique. The final aim of this work is to help in deciding which technique to perform according to the objectives and capacities of each research team. Considering the DNA–binding proteins characterisation, filter binding assay and EMSA are easy in vitro methods that rapidly identify nucleic acid-protein binding interactions. To find DNA-binding sites, DNA-footprinting is indeed an easier, faster and reliable approach, however, techniques involving base analogues and base-site selection are more precise. Concerning binding kinetics and affinities, filter binding assay and EMSA are useful and easy methods, although SPR and spectroscopy techniques are more sensitive. Finally, relatively to genome-wide studies, ChIP–seq is the desired method, given the coverage and resolution of the technique. In conclusion, although some experiments are easier and faster than others, when designing a DNA–protein interaction study several concerns should be taken and different techniques may need to be considered, since different methods confer different precisions and accuracies.