ChIP-ing away at DNA–protein interactions

ChIP captures interactions between DNA and proteins. IMAGE COURTESY OF RICHARD WHEELER, A WIKIMEDIA COMMONS USER

What is it?

The chromatin immunoprecipitation assay, popularly known as ChIP, is a technique used to capture and examine interactions between DNA and proteins. The basic principle behind ChIP is the achievement of a selective enrichment of genomic material when antibodies bind to and pull out protein–DNA complexes in vivo.

How does it work?

The assay begins with capturing a snapshot of protein-DNA interactions by fixing live cells with a crosslinking agent, such as formaldehyde. Once the interactions are preserved or frozen within the cell, chromatin is extracted and broken down either by physical agitation or by enzymatic fragmentation. These chromatin fragments are subjected to immunoprecipitation where specific antibodies recognize and selectively precipitate target proteins. Any DNA sequences attached to the proteins of interest are co-immunoprecipitated as part of the chromatin–DNA crosslinked complex. Finally, the crosslinking process is reversed to allow for further analysis. The separated DNA can be identified and quantified further using standard PCR amplification, cloning and sequencing.

Some protocols call for the preservation of the native state of the chromatin, especially when mapping for DNA targets of proteins, such as histone modifiers. In such cases, researchers avoid crosslinking and instead use enzymes to cut up the native chromatin into intact DNA–histone complexes for further purification and analysis.

How did it come about?

In the early 1980s, when researchers avidly were pursuing DNA recombination and gene-mapping projects, a student working with John Lis of Cornell University set out to explore an overlooked aspect of genetics: protein–DNA interactions in vivo. The student, David Gilmour, along with members of the Lis laboratory, became the first to use ultraviolet light to crosslink covalently proteins and DNA in bacterial cells. However, one of the limitations of using ultraviolet light is that it covalently links DNA with only proteins that are in direct contact with nucleic acid segments. In 1988, while mapping the genomic locations of histones, Alexander Varshavsky and colleagues at the Massachusetts Institute of Technology successfully replaced ultraviolet light with formaldehyde. Formaldehyde is a global crosslinker that conjugates DNA to all associated peptides, including elements of protein complexes that that do not interact directly with genes. The reagent increased the versatility of ChIP assays and is still used as a crosslinker in many experiments.

What are its applications?

Data from ChIP analyses augment our understanding of the mechanisms behind transcriptional and epigenetic gene regulation and spatiotemporal expression of regulatory elements in cells, tissues and sometimes organisms. Researchers often combine ChIP assays with DNA microarray assays, called ChIP-on-chip, to investigate the DNA targets of specific proteins, such as transcription factors or regulatory elements, on a genomewide scale. A dynamic and cost-effective version of ChIP-on-chip is the ChIP-seq. It involves high-throughput sequencing of multiple DNA fragments to map precisely global genomic binding sites of proteins of interest. ChIP-seq, which boasts high signal-to-noise ratios and robust outputs, has widened the field of comparative genome analyses and is a valuable approach for studying disease processes and cellular function.

Aditi S. Iyengar Aditi S. Iyengar earned her Ph.D. in cancer biology from Louisiana State University Health Sciences Center at New Orleans and completed her postdoctoral research at Massachusetts General Hospital, Boston.