Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Chromatin immunoprecipitation (ChIP)—a methodology that enables researchers to identify where on a cell’s DNA a given protein binds—has been a boon to understanding how that DNA is regulated, repaired and replicated. Certain post-translational modifications (PTMs) found on nucleosomal histone proteins may indicate that a gene is poised for transcription, for example, while a different set of PTMs effectively discourages recruitment of transcriptional machinery. Similarly, the binding of a known transcription factor (TF), co-activator or co-repressor may help elucidate the networks and pathways in which those sequences and factors are involved. Associating binding with developmental processes, cell cycle, disease state or treatment can yield powerful insights, as well.
Here we look at some ways to get the most out of ChIP—whether for checking the epigenetic status of your favorite gene of interest or globally mapping the binding sites of an in silico-discovered regulatory protein.
What is ChIP?
A traditional ChIP process begins by crosslinking cells’ DNA to whatever proteins are in close proximity. The cells are then lysed and the nuclear material is shorn. An antibody specific for the DNA-binding protein of interest is used to pull out the DNA-protein complexes, which can then be captured by Protein A or G, typically linked to agarose or magnetic beads. DNA is released from the complexes by reversing the crosslinks—typically using heat, or alternatively a proteinase—after which it is purified and analyzed.
Unlike the gel-shift assay that it has largely replaced, “ChIP is the perfect assay for looking at those protein-DNA interactions in real time,” says Chris Fry, associate director at Cell Signaling Technology. “It’s a much more relevant assay.”
And although every step and reagent can have an impact, Fry points to chromatin preparation and the precipitating antibody itself as the “two key factors in ensuring that you have a successful ChIP.”
Fix and shear
Formaldehyde is the typical crosslinking reagent of choice—“it basically just freeze-frames the cells at one point in time,” points out David Klinkebiel, director of the Nebraska Epigenomics Core Facility at the University of Nebraska Medical Center. But sometimes it’s not necessary, and sometimes it’s not enough.
Formalin fixation can alter the epitopes that the antibodies recognize, which can potentially be a problem.
Thus researchers looking at histones—specifically H3 and H4, which are tightly associated with the DNA, and who may or may not want to examine histones H2A and H2B—may forego crosslinking, using a process called native ChIP (nChIP).
On the other hand, sometimes the bait is co-factors or quaternary complexes that are not in direct contact with DNA (or the researcher does not know if they are). In this case, the researcher may wish to link the protein chains to each other by adding fixatives with longer crosslinkers, such as ethylene glycol bis(succinimidyl succinate) (EGS) or disuccinimidyl glutarate (DSG) [1].
The length of fixation time should be optimized to maximize integrity of the complex while minimizing structural damage.
Many protocols suggest isolating the nucleus from the cytosolic fraction to reduce background and enhance sensitivity.
The shearing process can also affect the integrity of the complex. There are two principal approaches to shearing DNA for ChIP, says Fry: sonication and enzymatic digestion, with sonication being the traditional and most commonly used method. “But we’ve found that depending on the buffer you’re using, the sonicator and the amount of sonication, you often lose signal for transcription factors (TFs) and co-factors when doing ChIP,” he adds. More care must be taken, and more optimization is required, than when using micrococcal nuclease (MNase), which gently digests DNA between nucleosomes.
Yet sonicators have “come a long way,” Klinkebiel says. A Covaris sonicator, for example, is “fairly gentle on the DNA-protein complex—it doesn’t knock a lot of proteins off—and is fairly easy to work with.” For labs that don’t have their own, these often can be found in core facilities.
Pull it down
Probably the biggest problems researchers face are finding an antibody specific to their protein and knowing that it’s recognizing the form of the protein they’re interested in, says Klinkebiel. A good first step is to look at what other people have used; that’s great if you can find it, but it should still be validated in the researcher’s own hands.
Commercial antibodies are increasingly being tested and validated specifically for ChIP.
The antibody should be robust and able to immunoprecipitate proteins out of a crosslinked protein-DNA mixture.
It should be specific, sensitive and reproducible. “We take a multi-application approach to validation—it increases confidence in specificity and performance,” shares Fry. “For example, we begin by testing by Western blot. For every target, we always try to test different cell lines that are known to be high- or low-expressing for a given target, based on RNA levels.” He adds, “We’ll test in other applications, like immunofluorescence, flow cytometry and immunohistochemistry. The beauty of these types of assays is that we can also look at changes in cellular localization, again to show us that the antibody is seeing the specific protein.”
Validation of post-translationally modified (PTM) proteins should be even more rigorous, to assure that the antibody can distinguish one PTM from another and to distinguish between proteins with and without neighboring modifications, Fry elaborates. Here peptide competition ELISAs, peptide dot blots and peptide arrays are often used. Of course, not every end user is expected to do every test on every antibody, but Fry advises researchers to “always look at the data” associated with the antibody, at the very least as a starting point for their own validation.
When a qualified antibody is not available, researchers can opt for a genetic fusion tag such FLAG, His or Myc (for which qualified antibodies are available). Another option is Promega’s HaloTag® system, in which the genetic tag covalently and irreversibly binds to its surface capture ligand. “At that point, you can do very, very stringent washes to remove nonspecific interactors and DNA,” explains Danette Daniels, group leader and senior scientist at Promega.
Whatcha got?
These days, the DNA is typically analyzed either by next-generation sequencing (ChIP-seq) or by qPCR (ChIP-qPCR). ChIP-on-chip—that is, microarray analysis—has largely fallen by the wayside: “There hasn’t been demand for several years now,” notes George Watts, the co-director of the University of Arizona Cancer Center Genomics Shared Resource, which still lists ChIP-on-chip as a service.
Requiring far less starting sample, qPCRanalysis is an option that is far less challenging than ChIP-seqand is ideal when looking at specific, known sequences. QIAGEN’s EpiTect ChIP PCR Arrays, for example, are 96- and 384-well primer arrays designed for a variety of biological pathways or disease states.
ChIP-seq is an effective tool to map interactions between DNA and DNA-associated proteins at the genome-wide level, notes Blanca Valle, scientific support specialist at EpiGentek Group. But it is “often technically challenging, requiring considerable protocol optimization and large sample input quantities.”
Input and throughput are key pain points that Fry thinks may soon be solved by multiplexing using DNA barcodes. Recent publications have demonstrated proof of concept, he notes, “definitely for histones, and some for TFs and co-factors. I don’t know of a kit yet.”
Klinkebiel confesses that kitsare changing so fast that he can hardly keep up with them. Numerous kits are currently available that are optimized for ChIP-qPCR or ChIP-seq, for low cell numbers or high throughput or fast protocols, for sonication or MNase, optimized for this buffer or that. Some come with all the necessary reagents—including negative and positive controls and post-ChIP DNA purification columns—and others don’t. There are even all-in-one kits for ChIP and next-generation sequencing (NGS) library preparation. Klinkebiel’s advice? “Give the company a call and tell them what you’re wanting to do, and ask what kit they would recommend. Then go their website, pull down the protocol, read it and figure out if it’s something you can do, if you have all the equipment.”
Reference
[1]Thermo Fisher Scientific application note: “A step-by step guide to successful chromatin immunoprecipitation(ChIP) assays,” 2016.
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