by Jeffrey M. Perkel
Check the pages of most biology textbooks or research publications and you're sure to find at least one illustration of gene expression represented in geometric shapes. With lines for DNA, rectangles for coding and regulatory elements, and circles or squares for proteins, these images, like simplified biological circuits, are both easy to make and to digest.
There's just one problem: They're almost always incomplete.
Genetic material doesn’t sit naked in the nucleus waiting to be activated or repressed; it is organized in a complex, hierarchical, and sophisticated structure called chromatin. In chromatin, DNA winds around octameric protein spindles called nucleosomes, which are themselves organized in ever higher order structures. Seemingly homogeneous, chromatin is in fact highly variable, and site-to-site differences in everything from how tightly DNA wraps around nucleosomes, to the spacing of those nucleosomes, can alter gene expression patterns. In such an environment, the influence of any given transcription factor binding is just one more variable.
The term to describe this layer of regulation, mediated by chemical rather than sequence variation, is epigenetics. According to Gerald Schock, associate global business director for epigenetics technologies at Qiagen, epigenetics involves "heritable changes in gene regulation and expression that occur without a change in the nuclear DNA sequence."
"Without a change in the nuclear DNA sequence" is the key element in that definition. Epigenetic mechanisms involve chemical changes to chromatin, but not of the sequence itself. Rather, if the genome is a text, then epigenetics is its typesetting – the bold and italics that adds nuance and emphasis to the otherwise indistinguishable string of words.
Increasingly, researchers are recognizing the importance of this chromatin typography – chemical modifications such as DNA methylation and histone acetylation that govern gene expression patterns in biological processes from normal development to the onset of cancer to stem cell differentiation. And armed with an ever-evolving toolbox of epigenetics kits and reagents, these researchers are probing the epigenome with unprecedented precision and scale.
DNA methylation
Epigenetic modifications, or "marks," basically come in two flavors: DNA methylation and histone modification. (Another epigenetic process, involving regulation by non-coding RNAs, will not be covered in this article.)
Histone modifications change the DNA's protein scaffold. Nucleosomes are octamers of histone proteins, each of which contains an N-terminal tail that can be extensively modified with methyl groups, acetyl groups, phosphates, and so on. Histone H3 acetylated on lysine-9 (H3K9ac) tends to mark transcriptionally active regions, whereas H3K27me3 (histone H3 trimethylated on lysine-27) tends to flag transcriptionally silent ones.
DNA methylation is a chemical alteration of the DNA itself, generally a conversion of cytosine to 5-methylcytosine in the context of CpG dinucleotides (that is: 5'-…NNmCGNN…-3'). One approach to detecting methylation uses sodium bisulfite treatment to chemically convert non-methylated cytosine residues to uracil while leaving 5-mC unchanged. A number of companies offer bisulfite-conversion kits, including Active Motif (MethylDetector™ Bisulfite Modification Kit), EMD Millipore (CpGenome™ Turbo Bisulfite Modification Kit), Qiagen (EpiTect Plus Bisulfite Kits), and Sigma-Aldrich (Imprint® Bisulfite DNA Modification Kit), among others.
Bisulfite conversion offers nucleotide-level methylation resolution, which can be detected using PCR-based methods or sequencing analysis. But sodium bisulfite is challenging. For one thing, for the technique to be reliable, researchers require complete chemical conversion – that is, they need to be sure that any C remaining in treated DNA really was methylated in the original sample. Additionally, says Sriharsa Pradhan, division head of RNA biology at New England Biolabs, the reagent is "extremely harsh," fragmenting DNA in the process of modifying it. (This problem can be overcome with reagents for optimizing buffer conditions, such as the EpiTect DNA Protect technology from Qiagen.)
There are other problems, too. DNA polymerases don't expect to find uracils in the DNA they are copying, making amplification of converted DNA difficult. At the recent American Association for Cancer Research (AACR) meeting, New England Biolabs launched a variant of Taq DNA polymerase specifically designed to circumvent this problem, says Pradhan. Another problem is that, by converting a four-base alphabet into a three-base alphabet, the treatment complicates primer design. "Bisulfite-converted DNA is less complex than genomic DNA," says Schock, "we have only three bases available to design primers." To circumvent this problem, Qiagen offers a set of control DNAs for validating primer design as well as some 60,000 predesigned PyroMark CpG Assays for sequencing human and mouse loci using its Pyrosequencing technology, says Schock.
Some researchers eschew bisulfite treatment in favor of more gentle, enzymatic approaches. These researchers selectively cleave methylated (or unmethylated) DNA with methylation-sensitive and –insensitive restriction enzymes and then PCR amplify across the cleavage site. HpaII and MspI, for instance, both recognize 5-CCGG, but the former cannot cleave methylated DNA, whereas the latter can. The OneStep qMethyl™ Kit from Zymo Research is based on this principle.
A variety of kits are available to specifically isolate methylated DNA. The CpG MethylQuest Kit from EMD Millipore, for instance, uses a methyl-binding domain protein (MBD2b) as a capture reagent to essentially immunoprecipitate methylated DNA. The MethylCollector™ Ultra Kit from Active Motif uses two proteins, MBD2b and MBD3L1, to do the same thing, collecting the DNA on magnetic beads. (A related technique, called MeDIP (methyl-DNA immunoprecipitation), uses antibodies to methylated DNA rather than MBDs.)
To measure global DNA methylation shifts, Sigma-Aldrich has introduced the Imprint Global DNA Methylation Quantification Kit (MDQ1), which not only captures methylated DNA in a sandwich ELISA, it also quantifies it. The kit is faster and simpler to use than conventional methods for analyzing global hypo- and hyper-methylation, such as HPLC and MALDI-TOF MS, says Savita Bagga, global epigenetics product manager. "This requires no technical training, anyone who has done an ELISA can use it." The technique, says Bagga, "enables the researcher to identify which gene or area of the genome needs to be investigated further, ... but this is a quick quantification and in 3.5 hours you can know which samples to pursue."
5-hydromethylcytosine
One new epigenetic mark that’s generating considerable buzz, says Pradhan, is 5-hydroxymethylcytosine (5-hmC), produced from 5-methylcytosine by the TET family of enzymes. Until recently, researchers could not distinguish the two methyl forms, but a growing number of tools is now available to help.
Active Motif, for instance, offers a hMeDIP kit that uses an antibody to 5-hmC to specifically pull down hydroxymethylated DNA sequences. "It's basically a methylated DNA immunoprecipitation technique," says Kyle Hondorp, product manager at Active Motif. "By using the antibody for the 5-hydroxymethylcytosine modification, we're able to enrich for samples that contain that mark."
In March 2011, Epigentek released the EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit which offers a high throughput format for immunoprecipitating hydroxymethylated DNA.
Both New England Biolabs and Zymo Research have launched kits for detecting 5-hmC that rely on selective modification of the modified base with a glucosyltransferase. Zymo's Quest 5-hmC Detection Kit uses hydroxymethylcytosine glucosyltransferase and uridine diphosphoglucose to selectively glucosylate 5-hmC residues, thereby rendering them immune to cleavage with the nuclease MspI. NEB's EpiMark 5-hmC and 5-mC Analysis Kit uses T4 beta-glucosyltransferase to accomplish the same thing. In either case, the assay can be read out using PCR, enabling researchers to interrogate the methylation status of specific loci.
To get a wider view, researchers can use restriction enzymes like MspJ1, FspE1 and LpnP1. Each of these enzymes will excise a 32-bp fragment with a methylated or hydroxymethyled cytosine in the center -- unless that residue is glucosylated to block digestion of glucosylated 5-hydroxymethylcytosine, that is. According to Pradhan, the resulting fragment pool can then be sequenced to get a bisulfite-free, nucleotide-resolution view of the entire methylome. "That's more of a genome-wide analysis," Pradhan says.
Histone modification
Going whole-genome is a growing trend in the methylation field. "Probably one of the biggest changes I've seen is moving from looking at a handful of loci to looking at the whole genome," says Michael Sturges, Senior Product Manager, Epigenetics, at EMD Millipore.
One of the best methods for getting a genome-wide view of the epigenome is chromatin immunoprecipitation, or ChIP. In ChIP, protein-DNA complexes in genomic DNA are crosslinked. The genomic DNA is then sheared and immunoprecipitated with antibodies to the proteins or modifications of interest, to enrich for sequences that were associated with that protein or mark. Finally, the cross-link is reversed, at which point the protein or modification's distribution can be probed on a genomic scale via sequencing or microarrays. Alternatively, ChIP-enriched sequences can be probed using PCR to interrogate specific sites.
ChIP kits are widely available from such companies as Sigma-Aldrich (the newly released Imprint Ultra Chromatin Immunoprecipitation Kit, for low abundance targets), Epigentek (ChromaFlash™ One-Step ChIP Kit, chromatin to ready-to-use DNA in less than 60 minutes), EMD Millipore (Magna ChIP2 and the new Magna ChIP-Seq kits, for microarray and next-gen sequencing readout, respectively), and Active Motif (ChIP-IT™ Express Chromatin Immunoprecipitation Kits), among others. Also available are kits for RNA-binding proteins, such as Millipore's Magna RIP (RNA immunoprecipitation) kit.
When it comes to ChIP not just any antibody will do, Sturges says; not all are capable of recognizing their antigens in the context of chromatin. So, look for ChIP-validated antibodies, says Sturges. EMD Millipore, for instance, has compiled a panel of some 55 "rigorously screened" ChIP-validated antibodies in its ChIP Ab+ product line, Sturges says. The company's portfolio of RIP-validated antibodies is also growing, he says, and currently includes some 30 antibodies.
Sturges recommends probing RNA polymerase II, H3K9ac, and H3K4me3 as marks of transcriptional activation, and H3K27me3 and H3K9me, as well as DNA methylation, as marks of transcriptional silencing.
Another confounding issue is the impact of nearby modifications on antibody binding. An antibody may bind its target modification well, even in the context of chromatin, but not if the next residue in the chain is also methylated, for instance. To address this level of validation, researchers can try Active Motif's MODified™ Histone Peptide Array. Containing some 384 modified histone peptides in duplicate, this array, says marketing manager Chris Wasden, lets researchers probe the enzymatic activities of different epigenetic enzymes as well as the specificity of ChIP antibodies. "You can look at each spot and say, okay, this is the binding we got at the single spot. But, if three amino acids down there's also a methylation, now my binding has been cut in half," says Wasden. "If I'm going to do ChIP, I need to keep that in mind."
Active Motif's diverse portfolio of epigenetics tools also includes recombinant enzymes for adding or removing epigenetic marks, recombinant histones for building custom nucleosomes in vitro, and assays for enzyme activity. PerkinElmer Life & Analytical Sciences also offers assays for enzyme activity. Sara Howland, the company's Product Portfolio Director, Drug Discovery Reagents of Bio-discovery, says PerkinElmer's 's AlphaLISA® and LANCE® Ultra-based assays are especially useful for drug discovery efforts. "You are measuring the enzyme's ability to modify the protein or peptide [substrate]," in the presence of compound libraries, Howland explains. "When you see your enzyme has been inhibited, those are potential lead compounds."
Chromatin accessibility
Fundamentally, says Bio-Rad Laboratories marketing manager Viresh Patel, epigenetic marks are not important to the cell in and of themselves; rather, it is how they influence the chromatin state that matters.
"DNA methylation, histone modifications, and non-coding RNA expression – those events all tend to contribute to the chromatin state within a living cell. And that chromatin state is defined as being open and accessible to transcriptional machinery, or closed or inaccessible. And that state determines how likely a gene is to be expressed or not," Patel says.
Bio-Rad's EpiQ™ Chromatin Analysis Kit allows researchers to probe that "openness." The kit measures a region's accessibility to nuclease digestion in just six hours and with minimal manipulation, Patel says. "While epigenetic marks contribute to the chromatin state, the EpiQ kit measures the functional state of chromatin inside of cells," he says.
Siddarth Dey, a graduate student at the University of California, Berkeley, has used EpiQ to probe the impact of mutations in the HIV-1 Tat protein on the chromatin state of viral DNA. Though his lab has experience with ChIP, Dey says he chose EpiQ for its speed. "EpiQ gives a quick view of the promoter, and if it looks interesting, you can go in and study it in more detail," he says.
Services
Not every researcher has the time, inclination, or wherewithal to learn the ins and outs of epigenetics. For these researchers, several companies offer epigenetic services. Zymo Research, for instance, recently launched a bisulfite sequencing service called EpiQuest. Active Motif now effers a 5-hmC-MeDIP sequencing service through the recently acquired service firm, GenPathway.
"For people that don't have the expertise, or … all they really want is the sequencing of this one event, the services are a great opportunity for them to get the data they need without investing the time to learn how to do all this stuff," Wasden says.
The image at the top of this article is from Zymo Research's QUEST 5-hmC Detection Kit™.