Cytosine-5 DNA methyltransferases (C5 DMTases) are effective reagents for analyzing chromatin and footprinting DNA-bound factors in vivo. Cytosine methylation in accessible regions is assayed positively by the PCR-based technique of bisulfite sequencing. In this article, we outline two complementary uses for the DNA methyltransferase CviPI (M.CviPI, GC specificity) in probing chromatin organization. First, we describe the use of the naturally occurring, free enzyme as a diffusible probe to map changes in nucleosome structure and to footprint factor interactions at cis-regulatory sequences. In a second application, termed targeted gene methylation (TAGM), the DMTase is targeted via in-frame fusion to a DNA-binding factor. The rapid accumulation of DNA methylation enables highly sensitive detection of factor binding. Both strategies can be applied with any C5 DMTase, such as M.SssI, which also possesses a short-recognition specificity (CG). A description of methods for constructing C5 DMTase-expressing strains of Saccharomyces cerevisiae and analyzing chromatin regions is provided. We also include comprehensive protocols for the isolation and bisulfite treatment of genomic DNA as well as the subsequent bisulfite sequencing steps. Data demonstrating the efficacy of both DMTase probing techniques, theoretical considerations, and experimental analyses are presented at GAL1 and PHO5.
- Saccharomyces cerevisiae
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)