Mechanistic Insights into Mammalian DNA Methylation ABSTRACT DNA methylation in mammals is a major epigenetic mechanism that is essential for transcriptional silencing of retrotransposons, genomic imprinting, and X-chromosome inactivation. Aberrant DNA methylation leads to genomic and chromosomal instabilities and silencing of tumor suppressor genes, which contribute to the development of cancers and many other human diseases. Mammalian DNA methylation is established and maintained by two groups of DNA methyltransferases (DNMTs): de novo DNMTs (DNMT3A and DNMT3B), which are responsible for establishing the DNA methylation patterns during gametogenesis and early embryogenesis, and maintenance DNMT (DNMT1), which propagates DNA methylation during mitotic division. We have a long-standing interest in mechanistic understanding of mammalian DNA methylation, which has led us to unravel the molecular basis of DNMT1-mediated maintenance DNA methylation and DNMT3A/3B- mediated de novo DNA methylation. The activities of these DNMTs are governed not only by their intrinsic enzymatic specificities, but also by an intricate network of cellular factors, such as histone modifications and chromatin modifiers. However, the mechanism underlying the functional regulation of the DNA methylation machinery remains poorly understood, partly due to the lack of structural and dynamic information on DNMTs under the chromatin environment. Our research program focuses on addressing this important challenge through an approach that integrates structural biology with biochemistry, molecular biology, and cell biology. We have two long-terms goals: to provide a comprehensive understanding of the structure and mechanism of DNA methylation machinery, and to identify the relationship between DNA methylation, gene regulation, and human diseases. Our work in the past has led to structure-function understanding of the protein-protein and protein-DNA interactions underpinning discrete steps of mammalian DNA methylation. We have identified multilayered mechanisms, involving intricate interplay between intramolecular and intermolecular interactions, for the substrate specificity and chromatin association of both maintenance and de novo DNA methylation machinery. Our goal in the next five years is to gain a mechanistic understanding of mammalian DNA methylation at the chromatin level, with emphasis on four new directions: (i) structural understanding of mammalian DNA methylation in the chromatin context, (ii) dynamic characterization of mammalian DNA methylation, (iii) protein engineering targeting specific DNA methylation pathways, and (iv) deciphering the regulatory network of mammalian DNA methylation. Together, these combined studies promise to lead us toward a comprehensive mechanistic understanding of mammalian DNA methylation.