We report a technique, named targeted gene methylation (TAGM), for identifying in vivo protein-binding sites in chromatin. M.CviPI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing. Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34-fold occur proximal to native Pho4-binding sites. Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure. In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation. Notably, substantial amounts of methylation are targeted by Pho4-M.CviPI under repressive conditions when most of the transactivator is excluded from the nucleus. Thus, TAGM enables rapid detection of DNA-protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level. Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.T he interaction of proteins with chromosomal target sites, either directly or through recruitment by DNA-bound factors, is central to many processes, including transcriptional activation and repression, replication and repair of DNA, recombination, and chromosome segregation. Therefore, strategies are needed that can efficiently identify specific chromosomal sites at which factors act. Few techniques are capable of demonstrating these interactions in the context of native chromatin in living cells, and these methods have limitations (1). For example, with footprinting techniques, protection against chemical (e.g., dimethyl sulfate) or enzymatic probes expressed in cells, e.g., DNA methyltransferases (MTases) (2-6) or DNase I (7), requires close proximity of the interacting factor to DNA sites that are modified or cleaved by the footprinting agent. Footprinting methods also require that the factor resists displacement by the enzymatic or chemical probe. Moreover, because many proteins can exclude probe access, a footprint does not provide an unequivocal identity of the bound protein (8). To circumvent this latter problem, proteins have been fused to an endonuclease (9); however, the resulting DNA damage alters chromatin structure and activates checkpoint controls. Another method, chromatin immunoprecipitation (ChIP), uses in situ fixation with formaldehyde followed by immunoselection of DNA-bound complexes. The requirements for large numbers of cells and highly specific antibodies as well as low fixation efficiencies (Ϸ0.1-0.5%) (10, 11) present distinct disadvantages of ChIP analysis. The approach of tethering chromatin proteins to the Dam MTase, which methylates ...