DNA-protein interactions are central to the regulation of living systems. Thus, effective methods for mapping and characterization of these interactions are crucial to a better understanding of how both chromatin structure and function contribute to genome function. We have found cytidine-5 DNA methyltransferases (C5 DMTases) to be highly effective probes of chromatin structure in living cells (1-3). When expressed in vivo, DMTases bind to and methylate accessible DNA target sequences but are obstructed by DNA-bound and other DNA-associated proteins to a degree dictated by the spans and strengths of their respective interactions. The DNA remains otherwise unaltered, resulting in minimal impact to the physiology of the cell during the short induction times commonly applied. In addition, no cell permeabilization or nuclei isolation is required

From: Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina © Humana Press Inc., Totowa, NJ


PCR off single (a) strand with a1/a2 primers m m

(a') 5' TuTuGCATuTATTATuGATuTuTAGCTGAuuTATAATuATTAAGuTATuT 3' □ deam. (a) strand cytidine deamination


original DNA

cytidine deamination

(b') 3' AGAGCGTAGATAATAGuTAGAGATCGAuTGGATATTAGTAATTuGATAGA 5' □ deam. (b) strand b1 PCR off single (b) strand with b1/b2 primers


Fig. 1. Detection of methylated cytidine by bisulfite genomic sequencing. M.CviPI sites (GC) are shown in bold. An obstructing DNA-bound protein is shown as a dashed partial ellipse. Unmodified cytidine residues are deaminated to uridine. Cytidine residues that have been methylated (m) resist deamination. Note that the a' and b' DNA strands are no longer complementary following deamination and thus can and must be amplified separately with dedicated primer pairs (a1/a2 or b1/b2). PCR products are then sequenced using ddGTP (a1 or b1 primer) or ddCTP (a2 or b2 primer) terminators.

since the only necessary cofactor, the methyl donor S-adenosyl methionine, is naturally present in the cell. This approach allows for accurate analysis of the true in vivo chromatin state by allowing detection not only of nucleosomes but also of other more easily disrupted nonhistone protein-DNA interactions (1,2).

5-Methyl-cytidine (m5C) yields a positive signal when isolated DNA containing the modification is subjected to the polymerase chain reaction (PCR)-based process of bisulfite genomic sequencing; the strength of the sequencing signal is directly proportional to the fraction of templates methylated at the site of interest across the population (4,5). Deamination by bisulfite ion chemically converts unmethylated cytidine residues in the DNA to uridine (6). Subsequent PCR amplification of a region of interest yields product in which uridine has been replaced by thymidine, resulting in a net unmethylated C to T transition. Because methylated cytidines resist deamination, corresponding residues are propagated as cytidine in subsequent steps ofthe process. The location and relative amount of cytidine, and hence DNA methylation, are determined by sequencing using 2',3'-dideoxyguanosine terminator with dATP, dCTP, and dTTP. A diagram of the deamination/amplification/sequencing process is shown in Fig. 1. By comparing the relative strength of signals within a region in experimental vs control populations (e.g., intact chromatin vs naked DNA, inducing vs repressive conditions, mutant vs wild type, and so on), conclusions can be drawn about the respective chromatin states. DMTases with short and/or degenerate recognition sequences will yield higher resolution owing to the higher frequency of naturally occurring sites.

Many common yeast plasmid vectors can be used for cloning the appropriate coding and regulatory sequences for expression of DMTases in vivo in yeast. Such vectors should include an antibiotic resistance gene (such as the P-lac-tamase gene, bla, conferring ampr) for E. coli cloning purposes and an appropriate yeast-selectable marker (such as LYS2) for selection of yeast transformants. We have constructed a series of yeast integrating plasmids (YIps) for use in DMTase expression, comprising estrogen-, doxycycline-, and galactose-induci-ble systems, with a variety of C5-DMTases including M.CviPI (Gm5C) (7), M.SssI (m5CG) (8), and M.Hhal (Gm5CGC) (9). In addition, fusions of M.CviPI and M.HhaI have been constructed that confer additional functional and regulatory characteristics, such as a Pho4-M.CviPI fusion expressed from the endogenous PHO4 promoter (10). Ongoing work in our and other laboratories suggests that these approaches can probably be applied across a variety of experimental host organisms as well as to in vitro analysis of DNA-protein complexes (2). The Materials and Methods sections address the preparation and execution of an experiment using pSH1052, a LYS2--marked HO-targeted YIp that codes for all necessary exogenous components of an estrogen-inducible free M.CviPI expression system.

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