Chromatin immunoprecipitation sequencing (ChIP-seq) combines two powerful techniques to analyze protein interactions with DNA. The first of these, ChIP, uses an antibody specific to the DNA-binding protein under investigation to immunoprecipitate chromatin fragments. Next, the bound DNA within the complex is sequenced. This provides a detailed understanding of gene regulation yet, despite widespread use, ChIP-seq has been limited by various problems. This article discusses how ChIP-seq methods are evolving to overcome these.
To set the scene, it’s important to appreciate how traditional ChIP-seq works. Essentially, cells are fixed with formaldehyde before fragmentation and solubilization of the chromatin, usually by sonication or enzymatic digestion. Next, an antibody attaches to the DNA-binding protein of interest, typically a transcription factor. The antibody-bound chromatin fragments are immunoprecipitated, then the DNA is sequenced to identify protein binding sites. “ChIP-seq allows researchers to study gene expression profiles in both health and disease and it is widely recognized as an invaluable tool for biomarker discovery,” notes Julian Pampel of antibodies-online.com. “However, to increase its utility and reliability, several protocol modifications have been necessary.”
One significant modification has been to eliminate the need for formaldehyde fixation, which, due to epitope masking, is a major cause of complications within ChIP-seq experiments. Epitope masking can prevent antibody binding during immunoprecipitation. It can also generate misleading data by allowing the capture of unwanted chromatin fragments that have become cross-linked to the fragments of interest. The evolution of native ChIP-seq has addressed this issue by removing formaldehyde fixation from protocols, instead using micrococcal nucleases for chromatin digestion, yet native ChIP-seq can suffer from incomplete extraction of protein-DNA complexes, potentially leading to the loss of important information.
Another limitation of native ChIP-seq is that it results in sampling from the entire genome. Using sonication or enzymatic digestion to fragment the chromatin causes the cells and their nuclei to be disrupted. Consequently the whole genome is solubilized, meaning that deep sequencing is needed to resolve the targeted protein binding sites above background. This extends timelines and can also impact on data quality.
With the development of chromatin endogenous cleavage (ChEC) and chromatin immunocleavage (ChIC), the need to incorporate sonication/enzymatic digestion into protocols has been eliminated. Both methods involve tethering an inactive micrococcal nuclease (MN) to a chromatin protein of interest; upon activation, the MN cleaves the DNA, which can then be extracted and sequenced. Although these techniques represent important ChIP-seq advances, a drawback of ChEC is that the chromatin-MN fusion proteins are expressed in vivo, meaning that a different construct must be produced for each chromatin protein under investigation. ChIC avoids this requirement by using an antibody to bind the protein of interest and a Protein A-MN fusion for DNA cleavage, however some ChIC protocols incorporate formaldehyde fixation, reintroducing one of the original ChIP-seq problems.
Drawing on the advantages of both ChEC and ChIC, CUT&RUN (cleavage under targets and release using nuclease) relies on targeted digestion to fragment chromatin. The defining feature of this technique is that it is performed on intact cells or nuclei without
formaldehyde fixation or sonication/enzymatic digestion. Instead, an antibody binds the target protein in situ and tethers a Protein A-MN or Protein A and Protein G MN fusion protein at the Fc region. The nuclease then cleaves around the chromatin complex, which diffuses out of the nucleus, leaving the remainder of the undigested genome behind. The target complexes can then be harvested in supernatant ahead of sequencing. This approach avoids the issue of cross-linking and greatly reduces background in comparison to native ChIP-seq.
By removing the need for chromatin fragmentation, protein-DNA interactions are more likely to be maintained in their physiological state, offering potential for more accurate detection of protein binding sites. An additional benefit of the CUT&RUN method is that it speeds up protocols by reducing the number of sequencing reads required because only relevant chromatin complexes are included in downstream sequencing. Furthermore, the technique uses less sample than traditional ChIP-seq, and it is also an ideal method to study rare cell populations since it delivers extremely high sensitivity – down to just 100 cells for profiling a specific histone modification.
To learn more about CUT&RUN and how it can benefit your ChIP-seq research, click here.