Dissecting mechanisms of gene silencing by the lncRNA Kcnq1ot1 in mouse trophoblast stem cells

ABSTRACT A conserved long non-coding RNA (lncRNA) called Kcnq1ot1 is essential for proper mammalian development. Defects in proper gene silencing by Kcnq1ot1 result in Beckwith-Wiedemann syndrome, a birth defect with a high mortality rate whose prevalence is increased in children conceived by in vitro fertilization. Despite its critical role in development, the mechanisms that Kcnq1ot1 uses to silence genes remain poorly understood. While it is known that Kcnq1ot1 silences genes by recruiting histone-modifying enzymes called the Polycomb Repressive Complexes and G9a, it is not clear how sequence elements in Kcnq1ot1 facilitate these interactions. As one possible clue, another repressive lncRNA that is essential for development, Xist, has been shown to interact with RNA-binding proteins (RBPs), that in turn, recruit the Polycomb silencing enzymes. In my own studies, I have found that many of these same RBPs associate with Kcnq1ot1 at levels significantly higher than with other RNAs in the transcriptome, implying that they too are important for the function of Kcnq1ot1. The objective of my study is to further develop the mechanism of Kcnq1ot1 by identifying functional sequence domains in the transcript, identify the RBPs that bind these regions, and determine their involvement in recruiting the Polycomb and G9a silencing enzymes. If successful, I hope to not only elucidate the mechanism of Kcnq1ot1, which is conserved in mice and humans, but further our general understanding of lncRNA function in early development. Long term, this information could eventually contribute to optimization of in vitro fertilization and discovery of genes implicated in infertility. The activities I propose will provide me with critical training in RNA biology and genomics, molecular biology, developmental biology, teaching and mentorship, oral communication skills and networking, and scientific writing, each of which will be critical for my future career as a principal investigator at a primarily undergraduate institution.