Caitlin Smith
The intriguing field of epigenetics studies heritable changes in phenotype that are not a result of a change in genotype, or DNA sequence. Instead, other factors (“epi” means “in addition to”) such as environmental influences may change the expression of particular genes, altering processes as diverse as development, behavior, and disease mechanisms. “Genomic methylation studies of pluripotent and differentiated cells are shedding light on the epigenetics of embryonic development and also advancing stem cell and induced stem cell research,” says Jason Gioia, scientist at Zymo Research. “In cancer research, advances are being made in identifying epigenetic markers for early diagnosis, and in the therapeutic and preventative use of DNA methylation inhibitors.”
In epigenetics, two methods used to study the molecular bases of these phenomena are chromatin immunoprecipitation
(ChIP) and DNA methylation
analysis. Because chromatin can be fixed with DNA-binding proteins (such as transcription factors) cross-linked to it at sites of interaction, ChIP is often used to locate areas of epigenetic interest. In addition, areas of the genome enriched in adjacent cytosines and guanines (CpG islands) are important in epigenetics because these cytosines can be methylated, which halts expression of the gene. This article will spotlight recent advances in ChIP and DNA methylation analysis methods, leaving further coverage of their microarray applications for an upcoming Technology Spotlight.
ChIPing away at epigenetic mechanisms
Epigenetics tools have evolved quickly, including the newly accessible ChIP kits. Only recently, ChIP methodology was not so user-friendly, notes Suzan Oberle, senior product manager for epigenetics at Millipore. “The protocols were complex, labor intensive, and time-consuming, and there was a lack of specific, ‘ChIPable’ antibodies,” she says. “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.” But the introduction of products like Millipore’s EZ-Magna ChIP™ kits and others reduced assay time and improved sensitivity and reproducibility. Active Motif also offers their ChIP-IT™, ChIP-IT™ Express, and ChIP-IT™ Express HT kits based on magnetic beads. In addition, Active Motif offers the Re-ChIP-IT™ kit, to assay whether two different proteins or histone modifications localize to the same position on the genome.
Crucial tools for ChIP include high quality antibodies, because the binding of antibodies can be hindered by the immunoprecipitated chromatin and by the fixation that holds interacting partners in position for the assay. “‘ChIP-grade’ antibodies must be highly specific and recognize the epitope in the chromatin context and under fixed conditions,” says Oberle. “[Millipore’s] ChipAb+ line antibodies are validated on a lot-to-lot basis in an antibody-specific, quantitative ChIP assay. Furthermore, whenever possible, we challenge our antibodies with biological validation, which we believe is critically important.”
For genome-wide ChIP analysis, several companies offer ChIP on microarrays, known as ChIP-chip or ChIP-on-chip. For example, Roche NimbleGen offers an extensive line of epigenetic microarray solutions including their ChIP-Chip and DNA Methylation arrays. Their ChIP-Chip (and DNA Methylation) microarrays combine “ultra-high probe density with 2.1 million (2.1M) long, isothermal oligonucleotide probes (50 - 75mers) and superior design flexibility,” says Robert Brazas, global product manager for epigenetics at Roche NimbleGen. “These core features of Roche NimbleGen’s arrays provide greater information content per array, increased specificity, and higher detection sensitivity.” One of the challenges that Brazas sees for epigenetics researchers is reducing costs while increasing sample throughput. “The key is to develop arrays that enable high sample throughput without significantly decreasing the information content of the arrays,” he says. “Our current 2.1M feature arrays and future higher density arrays will enable the production of multiplex, high-content epigenetic arrays.” Other major microarray manufacturers include Affymetrix, Illumina, and Agilent, whose newest offerings will be detailed in an upcoming Technology Spotlight due to space constraints.
DNA methylation tools
Another important epigenetics tool, DNA methylation analysis, is also rapidly evolving. Zymo Research’s new EZ DNA Methylation-Direct Kit™ is unique in that it “performs complete bisulfite conversion of DNA directly from blood, tissues, or cells – no prior DNA purification step is needed,” says Gioia. “Frozen cells, cells from laser capture microdissection, and formalin fixed, paraffin-embedded cells can also be used.” Other epigenetics tools include Zymo’s new EZ Bisulfite DNA Clean-up Kit™, “for researchers who perform ‘home brew’ bisulfite conversion,” says Gioia, “and the EZ Methlyation Startup Kit™, which is designed for the first-time user and consolidates bisulfite conversion reagents, DNA methylation standards, and ZymoTaqTM polymerase into one product.” ZymoTaqTM polymerase is a new “hot start” polymerase designed for bisulfite-converted DNA.
To help study patterns of DNA methylation, which bears on developmental and disease processes, Invitrogen’s new MethylMiner™ Methylated DNA Enrichment Kit enriches methylated DNA for further types of analysis, such as “PCR⁄qPCR-based assays, bisulfite conversion followed by amplification, cloning and sequencing, direct sequencing, library preparation for high-throughput sequencing, and sample-prep for DNA microarray analysis,” says Jurgen Vanhauwe, senior manager for advanced sequencing applications at Invitrogen. “This kit enables superior enrichment and differential fractionation of double-stranded DNA based on CpG methylation density, with increased sensitivity over antibody-based methods. Fractionation permits important comparisons between samples and enables researchers to focus analysis on only the methylation densities of interest.” The protocol takes less than four hours and uses Dynabeads magnetic beads with an easy salt elution.
RNAs join in
DNA-binding proteins are joined by a newly discovered epigenetic factor – RNA. “Emerging technologies in the area of post-transcriptional regulation by noncoding RNAs (ncRNAs) and RNA binding proteins are uncovering previously underappreciated levels of regulation of genes and proteins,” says Oberle. “Getting an entire picture of DNA methylation, chromatin occupancy, and the impact of ncRNAs on gene expression is sure to keep researchers engaged for years to come.” Vanhauwe also notes that recent research “describing the ability to manipulate DNA methylation of promoters using small RNAs is a very exciting discovery.”
Invitrogen just released a tool for studying ncRNAs, the NCode™ Human and Mouse Non-coding RNA Microarrays. Vanhauwe says that they “provide first-generation high-density arrays to profile longer ncRNA (>200 bases), and to analyze mRNA simultaneously on the same array. In addition to the ncRNA content, probes targeting mRNA allow discovery of coordinated expression with associated protein-coding genes.”
Genes to behavior
It is sometimes easier to see the epigenetic effects wrought by these molecular mechanisms in less complex systems. An elegant example of epigenesis in honeybees was recently published by the research group headed by Ryszard Maleszka, professor in molecular genetics and evolution at the Australian National University (ref. 1). Genetically identical adult female honeybees are either fertile queens or sterile workers, depending on whether they were fed royal jelly as larvae. Using RNA interference to silence a DNA methyltransferase, Maleszka’s group found that RNAi-treated larvae became queens without feeding on royal jelly – in other words, interfering with DNA methylation had developmental effects similar to feeding larvae royal jelly.
“Because changes in epigenetic signatures are subtle and difficult to visualize, our model offers some obvious advantages,” says Maleszka. “One focus in my lab is to gain insights into the epigenomic mechanisms that have been selected by nature to control ‘behavioral’ networks, such as the social organization in insects. Central to this approach is the study of epigenetic processes that provide a link between the genome and environment. The biggest challenge for biology is to shift the current focus on a descriptive field producing comparative molecular portraits, to epigenesis and systems biology. As some people say, genes are only part of the story.”
References
(1) Kucharski R, Maleszka J, Foret S, Maleszka R. (2008) Nutritional control of reproductive status in honeybees via DNA methylation. Science 319: 1827 - 1830.