June 1, 2011 (Vol. 31, No. 11)
Viresh Patel, Ph.D.
Epigenetics Tool for the Rapid Determination of Chromatin State for Gene-Expression Studies
Epigenetics is the study of inherited phenotypic changes caused by mechanisms other than mutations in the underlying DNA sequence. In mammalian cells, most of the chromatin—a complex of DNA, proteins, and histones—exists in a condensed, transcriptionally silent form called heterochromatin. The transcriptionally active form of chromatin is called euchromatin; it exists in a relaxed, less condensed state.
Histone subunits and DNA can be chemically modified as a result of environmental factors. These chemical modifications, called epigenetic markers (or marks), which include DNA methylation and histone tail modification (Figure 1), influence chromatin structure by altering its electrostatic nature or by modulating the affinity of chromatin-binding proteins. By altering chromatin structure, epigenetic changes have a profound effect on the expression of the genes present in the genomic regions affected.
Histones are strongly alkaline proteins that package and order DNA into structural units called nucleosomes. They are the major protein component of chromatin and can undergo several covalent chemical modifications, including methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation. A clear correlation has been established between histone acetylation and active transcription. Conversely, many histone methylation events are correlated with transcriptional silencing. Different histone modifications likely function in different ways; acetylation at one position will have a different effect than acetylation at another position.
Multiple modifications exist simultaneously and likely work together to influence chromatin state and gene expression. The concept of multiple dynamic modifications regulating gene expression in a systematic and reproducible fashion is known as the histone code.
In eukaryotes, DNA can be modified by methylation of cytosine bases. The enzymes that carry out this modification are called DNA methyltransferases. Aberrant or increased levels of methylation has been correlated with gene silencing and the development of several cancers. Recently, a second cytosine modification, 5-hydroxymethylcytosine (5-hmC), has been characterized in eukaryotes and research efforts are focused to understand its function.
Methods Used to Study Epigenetics
The most common technique for assessing DNA methylation involves the use of sodium bisulfite to convert unmethylated and methylated cytosine residues to uracil and cytosine, respectively. Methylated cytosines can then be identified through various downstream nucleic acid analysis methods, including PCR, qPCR, HRM, and sequencing. This commonly used technique can both identify individual methylation sites and quantify the level of methylation for a particular genomic region.
Other techniques for methylation analysis include the use of restriction enzymes, which are either resistant or sensitive to DNA methylation, and methylated DNA immunoprecipitation (MeDIP), which utilizes antibodies to isolate methylated DNA fragments for analysis.
The interaction between proteins (for example, histones) and DNA is most often studied using a technique known as chromatin immunoprecipitation (ChIP). ChIP is a preparatory method that uses highly specific antibodies against DNA-binding proteins to isolate DNA fragments that bind transcription factors or other DNA-binding proteins.
The isolated fragment can be characterized by using various nucleic acid analysis techniques, including PCR, qPCR, sequencing, and microarray hybridization. This can help determine whether specific proteins are associated with specific genomic regions. ChIP is also useful for identifying regions of the genome that are associated with specific histone modifications (for example, activating or repressive).
Researchers are interested in understanding the role of epigenetic changes, including DNA methylation and histone modifications, in disease (cancer) development and cell differentiation and reprogramming. The methods described thus far provide detailed molecular information about epigenetic markers that can be correlated to changes in gene-expression levels. However, they do not provide direct information regarding the chromatin state associated with these epigenetic marks.
Furthermore, several of these methods are time-consuming and may require up to five days to complete. New tools that deliver additional information about the functional state of chromatin in a shorter time are needed to accelerate the pace of epigenetics research.
The EpiQ™ chromatin analysis kit developed by Bio-Rad Laboratories is a real-time PCR assay for the quantitative assessment of chromatin states in cultured cells. Using this kit, chromatin structure data can be obtained within six hours directly from cultured cells (a few as 5 x 104) without the need for nuclei isolation.
In situ chromatin states can be identified based on how accessible the DNA is to nucleases. The DNA in heterochromatin is inaccessible to outside proteins, including exogenous nucleases, rendering it protected from nuclease digestion and available for subsequent quantitation by qPCR. Analysis of heterochromatin using the EpiQ kit reveals a minimal Cq shift between digested and undigested samples (Figure 2).
In contrast, the DNA in euchromatin is accessible to exogenous nucleases, making it susceptible to nuclease digestion and unavailable for qPCR. Analysis of euchromatin using the EpiQ kit shows a large Cq shift between digested and undigested samples (Figure 3).
By combining in situ chromatin digestion, genomic DNA purification, and real-time PCR, the chromatin state for several gene promoters can be studied simultaneously using the EpiQ chromatin analysis kit. The kit helps quantify the impact of epigenetic events, such as DNA methylation and histone modification, on gene-expression regulation through chromatin state changes.
The EpiQ chromatin analysis kit requires cultured (adherent or suspension) cells as starting material and includes the necessary supplies for performing chromatin assessment. Kit components include buffers for cell permeabilization and in situ chromatin digestion, optimized nuclease, materials for genomic DNA purification, control assays (qPCR primers) for chromatin assessment of a reference (epigenetically silenced) and control (constitutively expressed) gene, and the EpiQ chromatin SYBR® Green supermix, a real-time PCR reagent designed to amplify genomic DNA. Experimental results for user-specified gene targets are analyzed against the reference target to quantify the extent of chromatin accessibility.
Making Connections in Cancer Cell Proliferation
Together with a group of Japanese researchers, Ian Marc Bonapace, Ph.D., discovered the UHRF1 protein in the late 1990s. Since its discovery, UHRF1 has been shown to play an important role in gene regulation; specifically, it controls the epigenetic machinery that reinstalls correct modifications on DNA and chromatin during cell proliferation.
The main objective of Dr. Bonapace’s studies is to determine ways UHRF1 can be used to improve cancer treatment. “We believe that, at minimum, this protein can be very useful as a marker for cancer aggressiveness,” explains Bonapace. “But we hope to discover in the future that hampering the activity of UHRF1 could be a way to stop tumor growth and metastasis in the body.”
Early in 2010, Dr. Bonapace obtained the EpiQ kit. To test its efficacy and applicability to his research efforts, he conducted an experiment to compare the expression of three different oncosupressors in two different lines of prostate cancer.
Dr. Bonapace explains that prior to obtaining the EpiQ kit, determining if genes are expressed or not required days of work using the more resource-intensive chromatin immunoprecipitation method. “With this kit,” he explains, “you can determine immediately if genes are repressed or not. Then you know whether or not to go through the more time-consuming and money-consuming experiments to study why.”
Viresh Patel, Ph.D. (Viresh_Patel@bio-rad.com), is a marketing manager in the gene-expression division of Bio-Rad Laboratories.