Project Summary/Abstract Retrotransposons are ancient components of our genetic code that have self-replicated over millions of years and now make up 45% of our genome. Retrotransposon activation has serious implications for cell viability – retrotransposition is mutagenic by its nature and even expression of “dead” retrotransposons can trigger innate immune responses that cause inflammation. Therefore, the cell has evolved intricate epigenetic mechanisms to silence expression of retrotransposons by sequestering them in constitutive heterochromatin, which is marked the epigenetic modifications of H3K9 methylation and DNA CpG methylation. Dysregulation of H3K9 methylation and CpG methylation causes widespread re-activation of retrotransposons and occurs in multiple human diseases. However, very little is known about the precise molecular mechanisms that initiate retrotransposon silencing or maintain retrotransposons in a silent state. The broad goal of this research program is to define the precise molecular mechanisms the cell uses to suppress retrotransposon expression. A key component of the cells retrotransposon silencing machinery is the histone lysine methyltransferase Setdb-1, which deposits H3K9 methyl marks at retrotransposons. Because of its central role, Setdb-1 activity is tightly regulated by a complex cast of molecular players that control its catalytic activation, recruitment to retrotransposon sequences and its ability to spread histone methylation across the length of retrotransposons. Appropriate control of Setdb-1 activity is critical for retrotransposon silencing, but many aspects of Setdb-1 regulation remain enigmatic. We will use in vitro biochemical reconstitutions, Cryo-EM and genomics to decipher the precise mechanisms that underly regulation of Setbd-1. The work outlined in this proposal will explain how Setdb-1 is catalytically activated for retrotransposon silencing, how Setdb-1 reads the epigenetic code on retrotransposons and how Setdb-1 is recruited to retrotransposon sequences to establish H3K9 methylation patterns.