Mechanistic Insights into Mammalian DNA Methylation ABSTRACT DNA methylation is a major epigenetic mechanism that is essential for transcriptional silencing of retrotransposons, genomic imprinting and X-chromosome inactivation. Aberrant DNA methylation patterns lead to genomic and chromosomal instability and silencing of tumor suppressor genes, which contribute to the development of cancer and many other human diseases. DNA methylation patterns in mammalian genomes are dynamically established and maintained by two groups of DNA methyltransferases (DNMTs): DNMT3A and DNMT3B, which together establish DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which propagates DNA methylation patterns in differentiated cells. The molecular mechanisms of both groups of DNMTs remain a long-standing and fundamental question. The long-term goals of the PI's research are: (a) to provide a comprehensive understanding of how the DNA methylation machinery is regulated, and (b) to determine the relationship between the regulation of DNA methylation and human diseases. The biochemical and cellular functions of DNMTs are subject to both intramolecular and intermolecular regulations. How these regulations cooperate in controlling mammalian DNA methylation has not been well characterized. Our research program will focus on addressing these important challenges of mammalian DNA methylation through an approach that integrates structural biology with biochemistry, molecular biology and cell biology. The objective of this application is to provide a deep mechanistic understanding of DNMT1-mediated maintenance DNA methylation and DNMT3A-mediated de novo DNA methylation. We will provide structural basis for the conformational dynamics of DNMT1 and its regulatory protein UHRF1, the interaction between DNMT1 and UHRF1, and the substrate recognition of DNMT3A. Guided by these structural studies, we will further investigate how the regulations of DNMTs influence genomic DNA methylation. Together, these studies will bring our mechanistic understanding of mammalian DNA methylation to the next level.