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), 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™.
