In order to be interpreted properly, the DNA in our cells must be accurately recognized by many different DNA-binding proteins such as transcription factors and chromatin remodelers. To understand which sequences DNA-binding proteins recognize, scientists often use a technique called chromatin immunoprecipitation (ChIP). In this technique, chromatin, the combination of DNA and protein, is isolated from cells and then processed into smaller fragments. Next, the targeted chromatin is precipitated using an antibody to a DNA-binding protein of interest and the DNA that directly contacts the protein of interest is isolated and analyzed. The procedure is meant to reveal associations that exist in cells but the associations must be retained through the precipitation procedure, which takes place outside of the true cellular context. Researchers in the Henikoff Laboratory (Basic Sciences Division) have developed techniques to isolate targeted DNA-protein complexes directly from the cell nucleus rather than following a precipitation step.
In one such method called ChEC (Chromatin Endogenous Cleavage), a DNA-binding protein of interest is genetically fused to micrococcal nuclease (MNase), an enzyme that chews away DNA when there is a high concentration of calcium. Cells that express this DNA-binding protein-MNase fusion are lightly permeabilized and calcium is added, triggering MNase to cleave unprotected DNA. The targeted chromatin can then be collected and sequenced. However, fusing MNase to the DNA binding protein of interest is not always feasible or straightforward. Thus, researchers in the Henikoff lab adapted a technique2 that uses purified MNase tagged with Protein A (ProtA). The ProtA-MNase is added to permeabilized cells along with an antibody specific to the DNA-binding protein of interest. MNase is targeted to the DNA-binding protein of interest through the ProtA-IgG (part of the antibody) interaction, and the DNA near the MNase is digested, liberating the targeted protein-DNA complexes. In a recent publication in eLife, they report results using this new technique, which demonstrate that the coverage and dynamic range of this technique outperforms traditional ChIP-seq and "achieves at least parity with modern ChIP-seq methods, including those that we developed for this and previous studies", said Dr. Henikoff.
Their new method, which they term CUT&RUN (Cleavage Under Targets and Release Using Nuclease), has fewer steps than ChIP-seq, improving recovery and ease-of-use. Also, using an antibody against the DNA-binding protein of interest avoids the need for engineering and expressing an MNase fusion protein. It also prevents potential artifacts or changes in DNA-binding affinity that may be caused by fusing the MNase enzyme to the DNA-binding protein. In effect, all that is needed is a specific antibody to the DNA-binding protein of interest and purified ProtA-MNase. This allows scientists to investigate DNA-protein interactions in cells that are more difficult to manipulate genetically as well as rare cell types. Finally, it has a high dynamic range, requiring few cells to achieve a high signal-to-noise ratio. Dr. Henikoff explains: "The advantage of CUT&RUN is that it provides better quality data that allows for lower sequencing costs (by ~10-fold). Currently and likely in the future, sequencing costs limit ChIP-seq and so a 10-fold reduction in cost is likely the biggest practical advantage of our method."
Scientists such as those in the Henikoff Lab studying transcription factor binding and chromatin remodeling complexes now have a simpler, practical protocol to generate a high-resolution map of DNA-protein binding sites found inside intact cell nuclei.
Skene PJ, Henikoff S. 2017. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife. 2017;6:e21856
2. Schmid M, Durussel T, Laemmli UK. 2004. ChIC and ChEC; genomic mapping of chromatin proteins. Molecular Cell. 16:147-157.
This research was funded by Howard Hughes Medical Institute.