PROJECT SUMMARY Epigenetic gene repression by heterochromatin is necessary for multi-cellular organismal development and genome stability. Mis-regulation of heterochromatin is a cause of multiple diseases through perturbed gene expression patterns. The long-term goals of our research program are to determine at a molecular level how noncoding RNAs (ncRNAs) participate in heterochromatin formation and function. Many ncRNAs act in the nucleus to regulate gene expression through association with chromatin regulatory machinery. We and others have shown that certain RNA-mediated heterochromatin pathways require intermolecular RNA-RNA interactions between ncRNAs and nascent RNA that serve as the trigger to form heterochromatin. However, many systems where RNA is implicated have unexplored molecular mechanisms of action. We will address three high-level major outstanding questions in the field: 1) Which heterochromatin systems are controlled through RNA-RNA interactions? 2) How is heterochromatin built around nascent RNA? 3) How are RNA binding proteins re- purposed from mRNA processing functions to contribute to RNA-mediated heterochromatin formation? Our research program focuses on multiple heterochromatin systems that incorporate different species of noncoding RNAs. We study long noncoding RNAs (lncRNAs), such as HOTAIR and pericentromeric transcripts, which can inhibit heterochromatic histone modifiers to control their activity. In addition, we have developed the first biochemical system to study the human nuclear piRNA pathway, which uses base-pairing of the small piRNA to target nascent transcripts of repeats that then are suppressed via heterochromatin. Finally, we address how RBPs such as the N6-methyladenosine reader YTHDC1 can work with ncRNAs to promote gene repression. We use biochemical, structural, cell biological, and genomic approaches to study these models of RNA-regulated heterochromatin. Mechanistic insight into these pathways will provide a fuller understanding of how they work normally and in disease, which will prove useful in targeting for pharmacological intervention. Relevance to public health Noncoding RNAs are produced from regions of the human genome originally thought to be "junk" DNA. Many ncRNAs participate in epigenetic mechanisms of gene regulation and mis-regulation can lead to diseases such as cancer. ncRNAs are therefore clear candidates to provide a missing link to understanding the molecular mechanisms of many human diseases for which there is a "hidden heritability" factor that has not yet been identified.