Tools for Dissecting Epigenetic Pathways

Detailed studies have identified many of the components and basic principles of epigenetic mechanisms of gene expression. So far, the evidence indicates that stable but reversible alteration of gene expression is mediated by patterns of cytosine methylation and histone modification, the binding of nuclear proteins to chromatin, and interactions between these networks. The positioning and occupancy of nucleosomes are likely to be important factors in gene expression because these structures may modulate the binding of transcription factors as well as the movements of transcribing RNA polymerases, and there may be other, yet unknown factors that contribute to epigenetic mechanisms of gene regulation. The availability of specific, sensitive, and quantitative analytical tools has played a crucial role in dissecting epigenetic patterns and networks. Some of the important methods that have advanced our understanding of their architecture and function are described in this section.

Analytical techniques for monitoring the methylation state of DNA may be divided into two groups: those designed to determine the level of global methylation of studied genomes, and those designed to determine regional patterns of methylation, mostly CpG islands, of studied genomes. Established methods for measuring the methylation status include routine chromatographic methods, elec-tromigration methods, and immunoassays,76 and modifications of these older techniques continue to evolve.76 Semiautomatic detection of methylation at CpG

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islands,77 oligonucleotide-based microarrays, and tissue microarrays78-80 are some of the newer methods that have been reported. In addition, immunodetection offers the possibility of obtaining spatially resolved information on the distribution of 5mC on metaphase chromosomes.81

The choice of a proper method for a particular investigation rests with the aims and the specific requirements of the investigation. The important features of several widely used methods that employ 5mC as a marker for DNA methylation are summarized below and in Table 6.1. For details of protocols, the reader is referred to the recent review of Havlis and Trbusek76 as well as additional references cited in Table 6.1.

Table 6.1 Tools for Monitoring DNA Methylation in Epigenetic Disease

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