TESTING MECHANISMS OF LONG NON-CODING RNA REGULATION AT THE XIST – TSIX LOCUS IN SINGLE CELLS

Abstract

Antisense long non-coding RNAs (antisense lncRNAs) are ubiquitous in the human genome and important for many diseases and biological functions. Despite their importance, the mechanisms by which they exert their function are largely unknown. To test mechanisms of antisense lncRNAs, I used the Xist/Tsix locus in mouse embryonic stem cells as a model system. Xist and Tsix are antisense lncRNAs critical for initiating and regulating X chromosome inactivation respectively. Many studies have proposed different mechanisms for these lncRNAs, but a consensus for which mechanisms are used has not been reached. One reason for this uncertainty is a lack of quantitative single-molecule studies, which are critical for validating these mechanisms. To bridge this gap, I developed an automated image processing pipeline called CellDissect to determine nuclear and cellular boundaries in microscopy images. Next, I developed software to design probes for RNA fluorescence in-situ hybridization (RNA-FISH) and algorithms to quantify fluorescence at transcription sites and Xist clouds. I then utilized exonic and intronic RNA-FISH probes combined with immunofluorescence to test mechanisms of antisense lncRNA regulation at the Xist-Tsix locus and used robust analyses to compare data to randomized controls. The results of my experiments and analyses argue against mechanisms that involve Xist and Tsix binding and suggest that diffusing Xist and Tsix do not affect whole-cell antisense transcript levels. Interestingly, my results suggest that nascent transcription levels significantly influence the mechanisms employed: high transcription causes transcriptional interference, whereas medium transcription maximizes chromatin modification, specifically H3K36me3 deposition. This research elucidates lncRNA transcriptional regulation mechanisms at the Xist-Tsix locus and introduces a novel paradigm wherein different lncRNA mechanisms are utilized depending on the transcription level. Furthermore, this dissertation establishes a robust methodology for quantitative lncRNA investigations and concludes with future directions for lncRNA studies for which I have preliminary data or analyses.