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June 01, 2011 (Vol. 31, No. 11)

PCR Assay for Chromatin Accessibility

Epigenetics Tool for the Rapid Determination of Chromatin State for Gene-Expression Studies

  • Click Image To Enlarge +
    Figure 1. Chromatin is found in two states: euchromatin (transcriptionally competent) and heterochromatin (transcriptionally silent). Epigenetic events such as DNA methylation and histone modification may alter chromatin between these states.

    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.

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