Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
As epigenetics assays go, chromatin immunoprecipitation is the metaphorical 800-lb gorilla. But there are other ways to interrogate histone modifications.
Some companies offer microarrays to assess the specificity of proteins that create and recognize histone modifications; others sell purified histones and/or nucleosomes for use as substrates in enzymatic reactions; and still others have developed methods for probing the genomic locations of specific modifications at the single-cell level. Some of these methods reveal a biochemical side of epigenetics that is otherwise overlooked by assays like Chromatin immunoprecipitation (ChIP), and others complement the technique.
In any event, histone analysis can be a complicated business. According to Davide Mantiero, research area content manager for epigenetics at antibody vendor Abcam, “The sheer variety of different modifications that can be found on a single histone or residue—and the range of enzymes that catalyze their addition—make analysis of histone modifications very complex. There are approximately 600-plus known, different histone modifications, and counting,” he says. And many are highly similar, meaning reagent specificity is a must.
The good news is there are so many offerings that researchers should have no problem finding the tool they need, whatever their application. Here we round up a few of the options.
Assessing substrate specificity
Epigenetic signals, including histone modifications, require a network of proteins to do their jobs.
Epigenetic signals, including histone modifications, require a network of proteins to do their jobs.There are “writers” to create the modifications, “readers” to interpret them and “erasers” to delete them. One tool researchers can use to probe the specificity of such proteins is the AbSurance™ Pro microarray from MilliporeSigma, the life science business of Merck KGaA, Darmstadt, Germany.
The AbSurance Pro microarray is a glass microarray containing multiple copies of some 275 modified histone peptides from all four core histone proteins (H2A, H2B, H3 and H4). Each peptide is present in 12 copies per array, and each slide contains two subarrays, meaning two samples can be tested simultaneously.
AbSurance Pro is a successor to the company’s AbSurance arrays, a series of membrane-based macro arrays that researchers could use to test the specificity of histone-modification-targeting antibodies, such as those used in ChIP. According to John Rosenfeld, external innovation manager at MilliporeSigma, researchers may use the Pro array for that purpose, as well. But unlike the original AbSurance array, he says, the Pro array contains multiply (as opposed to singly) modified peptides—“that’s an important difference”—and is intended mostly for protein-protein interaction studies.
For instance, Rosenfeld says, a researcher could incubate a polyhistidine-tagged BRD4 “reader” protein with the array, add a fluorescently labeled antibody to the His-tag® and finally read out the assay on an array slide reader to identify the protein’s targets. “It’ll give you quantitative values on which peptides are preferred across the panel.”
Measuring enzymatic activity
Histones are the targets of multiple regulatory enzymes. If researchers want to assess the enzymology of such proteins, for example to identify drug compounds that can alter enzyme activity, they require suitable substrates—in this case, recombinant histones.
MilliporeSigma offers a range of recombinant-histone monomers, dimers and tetramers, says Rosenfeld, including both “standard” histone proteins and some of the more common variants, such as H3.3 and H2AZ. And ActiveMotif offers intact nucleosomes, says technical product manager Kyle Hondorp.
“Sometimes the way a protein would react on a peptide vs. a recombinant protein vs. a histone octamer vs. a nucleosome – there could be very different activity levels and performance depending on the substrate choice,” Hondorp says. As a result, “nucleosomes are becoming interesting, because they are closest biologically to what happens in the sample.”
ActiveMotif’s recombinant nucleosomes currently are unmodified, but that could change in the future. “Customers would like to have specific modifications incorporated, and that is something we are exploring,” Hondorp says.
Measuring protein proximity at the single-cell level
ChIP enables researchers to measure histone-modification status across the entire genome, but only at the population level. “With ChIP you need from one million to 10 million cells,” says Rosenfeld. “Whatever you end up with is never from a single cell.”
There are assays, though, that enable researchers to validate their findings in single cells, including Promega’s NanoBRET™ assays. NanoBRET assays exploit the principle of bioluminescence resonance energy transfer (BRET), a transfer of light energy from one protein to another in close proximity. One protein is fused to luciferase, the second (via Promega’s HaloTag) to a fluorophore. If the two proteins are sufficiently close together, light emission from the luciferase will excite the fluorophore, producing a measurable fluorescent signal without external excitation.
According to Danette Daniels, senior research scientist at Promega, the company has developed more than 60 NanoBRET interaction-pair assays, including some that measure the proximity of bromodomain-containing proteins to different histone variants, “allowing for broad study of not only recruitment and displacement of readers from chromatin in real time, but also of important epigenetics enzymes or complexes.”
MilliporeSigma’s Duolink® assays offer an alternative strategy. Duolink is a proximity ligation assay in which antibodies to two antigens, each tagged with a short oligonucleotide, can—if within 40 nm of each other—prime a DNA-amplification reaction, leading to a spatially localized fluorescent signal that can be detected under a fluorescence microscope.
Duolink typically is used to measure the proximity of two proteins or post-translational modifications. Recently the company has extended the concept to investigate the proximity of a protein to a given DNA or RNA sequence—a method called Duolink In Situ hybridization, says Vikas Palhan, senior research and development scientist for molecular biotechnology at MilliporeSigma.
In one example, Palhan says, his team visualized the colocalization of 5-methylcytosine (an epigenetic modification of DNA) with the septin-9 promoter (targeted with biotinylated oligos) in colon cancer cells, as three fluorescent-red punctate dots corresponding to the three copies of chromosome 17 in those cells. In another case, the team measured EZH2 histone methyltransferase and the histone “mark” it makes, H3K27me3, in the presence and absence of putative EZH2 inhibitors. “We could see a dramatic suppression in the Duolink signal,” he says.
Creating histone marks at will
Researchers who wish to investigate the impact of specific modifications throughout the genome have a world of new possibilities, thanks to the CRISPR/Cas9 system.
Cas9 is a nuclease that, guided by a short guide RNA, can cut genomic DNA in live cells, kicking off the process of genome editing. But researchers have discovered clever ways to extend this system, by coupling new enzymatic activities to catalytically inactive (“dead”) Cas9 proteins.
For instance, MilliporeSigma offers a form of dead Cas9 fused to the p300 histone acetyltransferase. The resulting enzyme cannot cut DNA, Palhan says, but retains the ability to be guided to specific genomic loci using a guide RNA, where the p300 can unwind the local chromatin by acetylating histone H3 on residue lysine-27. As a result, researchers can assess the impact of artificially “opening” chromatin at a genomic site of their choosing.
Palhan describes how his colleague Qingzhou Ji used this system in mammalian cells to boost expression of endogenous Oct4, a notoriously difficult gene to modulate, achieving a three- to four-fold increase. “Three- to four-fold induction of endogenous Oct4 is a pretty big deal in the field,” he says.
Global detection of modifications
For those intent on detecting global changes in histone modification, ActiveMotif has for several years offered a series of ELISA kits specifically targeted to histone H3. These assays can indicate whether a population of cells has altered its histone-modification profile on a global level in response to some treatment—a finding that can be followed up with ChIP to gain a sequence-level perspective, says Hondorp. (Abcam also offers ELISA assays for histone modifications, Mantiero says, including kits to quantify “up to 21 histone H3 modifications and 10 histone H4 modifications simultaneously.”)
Recently ActiveMotif has multiplexed its assays into a Luminex bead-based array. Researchers can screen samples for differences in up to 13 different histone modifications at one time, Hondorp says—though for better accuracy, she recommends users limit themselves to no more than five.
According to Hondorp, these multiplexed assays are particularly empowering for researchers handling precious patient samples. “They don’t necessarily have enough material to run an ELISA—which may require micrograms of material per assay—for each modification. But with Luminex, they can run nanogram quantities in a single well and test five different modifications,” she says.
ChIP services
Of course, the most popular assay for understanding DNA-histone interactions research these days continues to be ChIP. Countless ChIP kits are on the market. But for those lacking the expertise, the time and/or the human resources, some companies do offer ChIP services.
Zymo Research, for instance, offers a ChIP-seq service that promises to convert a sample into biological insight in just 11 to 13 weeks, says Yap Ching Chew, the company’s director of epigenetics technologies. “Our record is four weeks,” she says.
To use the service, customers grow and treat their own cells, then crosslink them, freeze them and send the material to Zymo. The company then extracts the chromatin, performs the immunoprecipitation, purifies ChIP-enriched DNA, sequences the samples and performs the necessary bioinformatics. Total cost: $3,000 per sample.
According to Chew, the company offers a range of epigenetic services, including analysis of DNA methylation or hydroxymethylation, and can integrate those datasets as desired.
“Integrating different ‘omics data is a trendy thing,” says Keith Booher, service project manager at Zymo Research. “We can integrate gene-expression data with methylation data and ChIP-seq data to see what is going on in the cell—how is a treatment affecting gene expression, and what are the mechanisms driving that effect?”