Control of gene silencing by long noncoding RNAs in trophoblast stem cells

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ABSTRACT Long noncoding RNAs (lncRNAs) make up a large portion of mammalian transcriptomes and have essential roles in diverse biological processes, including silencing of protein-coding genes during early development. In the trophoblast, lncRNAs have been shown to have augmented silencing potency through their relationship with highly conserved chromatin-modifying enzymes called Polycomb Repressive Complexes (PRCs). Specifically, the lncRNAs Xist, Airn, and Kcnq1ot1 direct PRCs to separate genomic domains that each span millions of base pairs, in which specific genes are silenced. Indeed, defective silencing by lncRNAs in the trophoblast leads to inappropriate expression of genes that can be a major cause of infertility, whereby the preimplantation embryo depends on the trophoblast for proper development. Despite the importance of the trophoblast in early embryogenesis, the molecular mechanisms that sustain it, particularly through regulation of gene networks by lncRNA silencing, are unknown. Recent work from our lab has shown that, in trophoblast stem cells (TSCs), the genomic domains silenced by Xist, Airn, and Kcnq1ot1 harbor PRCs that are distributed non-uniformly, with certain regions subject to greater levels of silencing than others. While the underlying features that establish this non-uniformity are not fully known, we and others have proposed that chromatin-associated factors are at least partly responsible. In our recent study, three-dimensional (3D) genome architecture and specific CpG island (CGI) DNA elements appeared to be important contributors. Thus, using TSCs as an ex vivo model for the trophoblast, I propose to investigate how chromatin conformation, contacts between chromatin and lncRNAs, and sequence-specific transcription factors bound to CGIs cooperate to dictate both regional and long-distance silencing in lncRNA-silenced domains. My studies will highlight specific factors that regulate epigenetic networks that sustain the trophoblast. Furthermore, my work will provide new paradigms for gene regulation by lncRNAs, whereby specific DNA-binding proteins control the intensity of silencing induced by lncRNAs on a region-by- region basis. In turn, these data may provide new insights on how to treat birth defects or prevent the infertility that results from altered dosage of lncRNA-silenced genes. My long-term goal is to pursue a career in chromatin and epigenetics research as a principle investigator at a major research institution. The training activities I propose are designed to prepare me for this goal, with the next immediate step to obtain a postdoctoral fellowship in chromatin and epigenetics. To these ends, my research plan will provide me with invaluable training in quantitative genomics and the skills needed to develop and rigorously test hypotheses via computational and molecular cell biology. I have enlisted the help of a collaborator and a co-sponsor whose computational expertise is complementary to that of my primary sponsor. My technical training will be accompanied by professional development in oral communication, scientific writing and evaluation, and networking, which will be instrumental to attaining my goal of becoming an independent investigator.