by Caitlin Smith
To witness what occurs in a meeting of DNA and protein would be a fantastic voyage for anyone studying gene regulation – or any number of central processes such as transcription, replication, repair, and epigenetic silencing. A central (but not sole) technique to convey this important information is the chromatin immunoprecipitation (ChIP) assay, which identifies regions of the genome with which particular proteins interact.
These interacting proteins may be histones, transcription factors, or other DNA-binding proteins. For non-histone proteins, which bind DNA less tightly than do histones, the DNA and proteins are crosslinked, followed by shearing of the chromatin into 200-1000 nucleotide fragments. Immunoprecipitation with antibodies specific for the proteins of interest co-precipitate any DNA regions to which a protein was interacting at the time of crosslinking. Agarose or magnetic beads are used to isolate the complex, after which reverse crosslinking releases the DNA for analysis.
The analytical power of ChIP and its descendents is already being sought across many applications. “The field of epigenetic analysis is expanding from studies of localized DNA-binding sites toward whole genome analysis of histone modifications and methylation profiling,” says Suzan Oberle, senior product manager in epigenetics at Millipore. “ChIP combined with high-density oligonucleotide array analysis (ChIP-chip) or coupled with next generation DNA sequencing (ChIP-Seq) allows precise, genome-wide mapping of binding sites.” A range of options abound, so tailor the assay to your needs.
ChIP assays and reagents
Even the basic ChIP assays, and their faster cousins, such as those using magnetic beads, have improved dramatically. Earlier ChIP assays could mean 3 days of complex protocol. “Optimizing ChIP assays can be challenging, especially for new users, and in fact ChIP didn’t really start to catch on until the availability of user-friendly kits to standardize and simplify the procedure,” says Oberle. Today, most standard ChIP assays take about 1 day, as for Millipore’s EZ MagnaChIP kits. Other ChIP assays include (but are not limited to) Abcam’s ChIP Kits, Active Motif’s ChIP-IT Express Kits, Aviva’s ChIP kits,Imgenex’s QuikChIP Kits, Miltenyi Biotec’s µMACS Protein A or G MicroBeads for ChIP, and Sigma-Aldrich’s Imprint ChIP Kit.
Oberle believes that ChIP assays are facing a challenge to validate reagents for greater reliability. “‘ChIP-grade’ antibodies must be highly specific and recognize the epitope in the chromatin context and under fixed conditions,” expains Oberle. “Even if an antibody
works in immunoprecipitation and Western blotting, it still may not be suitable for ChIP. Moreover, lot-to-lot differences, especially among polyclonal antibodies, highlight the need for reagents that are specifically verified as suitable for ChIP.” To address this, Millipore offers their ChipAb+ line of antibodies validated by lot in quantitative ChIP assays. “ChIPAb+ antibodies include a negative control antibody for the ChIP assay and aliquots of qPCR primer sets to validate the performance of each antibody lot,” says Oberle. “Unless we’ve tested the antibody in ChIP ourselves, every single lot, we don’t promote it as a ChIPAb+ product. Furthermore, whenever possible, we challenge our antibodies with biological validation, which we believe is critically important.”
Jeff Falk, director of technology and business applications at Aviva Systems Biology, agrees that the emergence of ChIP-grade antibodies is an important new development. “The increasing availability of ChIP-grade transcription factor [(TF)] antibodies is rapidly opening up the ChIP field and enabling the study of regulatory pathways that were previously blocked from ChIP analysis by the lack of good antibodies,” he says. “We have found that only about 1 in 4 antibodies that work on a Western blot will actually be suitable for ChIP-based profiling,” he says. “Consequently, it is imperative that you have a large collection of TF antibodies available that recognize different epitopes on the same TF protein. Aviva has addressed this need by aggressively producing multi-epitope antibodies to the entire TF protein family.” Other companies offering ChIP-grade antibodies include Abcam and Santa Cruz Biotechnology.
Another important permutation of the ChIP assay is the development of locus-specific ChIP-qPCR arrays, according to Falk. These give a detailed “cataloging of precisely what factor interactions are occurring and the site of the interaction in the promoter/enhancer region,” says Falk. “These assays are particularly useful in monitoring changes in factor binding interactions as a result of compound treatments or effects of expression in different tissue types, and Aviva has incorporated them into 96-well screening assays for high profile loci such as IL17A &F involved in T-cell activation or APOA1 which is a key player in cardiovascular disease.”
Speeding up with ChIP-on-chip and ChIP-Seq
ChIP-on-chip is the combination of ChIP with microarray
technology, such as offered by Affymetrix or Agilent, and Illumina. This technology is particularly powerful for applications such as epigenetics, as well as studies of specific genetic regulatory proteins, because it affords a genome-wide analysis of any location in the genome that a protein of interest might bind.
In contrast to ChIP-on-chip, which may require sets of tiling arrays to achieve the needed level of resolution, ChIP-sequencing, or ChIP-Seq, gives better positional resolution for mapping binding sites, and greater signal-to-noise levels, with a single sequencing run of the ChIP-DNA pairings. “We provide a ChIP-Seq technology that allows researchers to precisely map the in vivo DNA-protein binding location on a truly whole genome scale at a fraction of the cost of traditional ChIP-Chip experiments,” says Chris Streck, product manager for gene expression and regulation at Illumina. “This system combines the powerful Illumina sequencing technology with custom built analysis software tools enabling researchers to use DNA input amounts as low as 10 ng while providing high resolution mapping to identify the specific DNA-binding sites on the genome.” Streck says that this new technology allows scientists to integrate ChIP-Seq data with analyses for epigenetics, gene expression, and microRNA work, for example. “Many of our customers can now directly correlate the occupancy of a specific transcription factor or other DNA-binding factor on the promoter regions of microRNAs or messenger RNAs and get a truly global view of the regulatory circuitry in a single experiment,” says Streck.
Falk also believes that sequencing has expanded the ChIP field. “We now have the sensitivity to get very precise binding interaction information for a given factor at any spot throughout the genome,” he says. “Furthermore, the increased sensitivity of ChIP-Seq now provides us with a tool to look not only at direct factor binding interactions, but to also start characterizing indirect co-factor interactions that are thought to convey much of the transcriptional regulatory specificity.”
Non-ChIP options
While the impressive advancement of ChIP technology deserves attention, it is not the only method available to detect DNA-protein interactions. For example, in Thermo Fisher Scientific’s LightShift EMSA Kits, a mixture of biotin-labeled DNA duplexes and nuclear extract is electrophoresed, then blotted to a membrane. After UV cross-linking, the labeled DNA is probed with streptavidin-HRP and detected by chemiluminescence. Another option is Promega’s HaloCHIP System, in which proteins of interest are expressed as HaloTag fusion proteins. After formaldehyde crosslinking, the DNA-HaloTag fusion protein complexes are captured with a HaloLink resin. The purified DNA is released after reversing the crosslinking. As ChIP technologies evolve, we may also see non-ChIP methods advance for researchers who could benefit from other systems.