Dissertation

RNA Metabolism and Transcriptional Control in Pluripotent Stem Cells

Fstkchyan, Yesai Sevak

2021

Overview

Proper developmental progression during embryogenesis depends on a precise, stepwise, sculpting of transcriptional networks during cell fate transitions. During pre-implantation development, waves of epigenetic reprogramming aid in the reorganization of the gene regulatory landscape to allow for the induction of cell-type specific gene programs. Chromatin modifying enzymes play a key role in the repression and activation of transcriptional networks during cell fate transitions. The derivation of mouse embryonic stem cells has yielded a powerful in vitro model that allowed for the elucidation of molecular mechanisms required for proper establishment of gene regulatory networks during embryogenesis. Naïve mESC are representative of the epiblast found in a E3.5 embryo and can be induced to transition to a primed state that represents the pre-streak post-implantation epiblast [epiblast-like cell (EpiLC)]. Although these two share similar characteristics, their epigenome and transcriptional networks differ. Particularly, naïve pluripotent stem cells exabit transposable elements (TEs) activity compared to their primed counterparts. TE are remnants of ancient retroviruses that have stably integrated into out genomes and can be passed to progeny in a mendelian fashion. Moreover, TEs have become domesticated to contain gene regulatory function, however, their mis-regulation can be detrimental to cellular homeostasis. In pluripotent stem cells TEs are repressed through the histone modifications whereas in differentiated progeny are highly methylated. However, approximately 20% of TE are found within open chromatin regions throughout the course of development. How the transcriptional activity of these TEs is control remains unknown.As TEs comprise the bulk of the noncoding RNA compendium in mouse and human genes, they make for an ideal target for the RNA exosome. The RNA exosome is a high conserved multisubunit RNA degradation machine that targets transcripts that are derived from all three RNA polymerases. However, the exosome preferentially degrades noncoding RNA that is produced from gene regulatory regions, such as promoter and enhancers. As TE have the potential to regulate host genes, we hypothesized the RNA exosome co-transcriptional degrades TE found in open chromatin regions. We show that in the absence of the RNA exosome, various classes of TE are upregulated in mESC and EpiLC. Of note, we find MERVL to be amongst the top upregulated TE. MERVL expression is confined in the totipotent 2-cell embryo and becomes subsequently silenced as development progresses. Inability to degrade MERVL results in a reversion of pluripotent stem cells to a 2-cell like cell (2CLC) state. Additionally, we observed the exonization of MERVL LTR into host genes creating chimeric transcripts. Depression of MERVL in the absence of the results in increase RNAPII recruitment to the long terminal repeats (LTRs) and enrichment of H3K27ac, with global changes in methylation. Lastly, we use these data to inform about the etiology of pontocellular hypoplasia (PCH1b) in which mutations in the exosome subunit Exosc3 result in neurological disease. Together, our results indicate that the RNA exosome functions to maintain cellular states to prevent reversion to a previous cell state through co-transcriptional degradation of regulatory RNA.

• Publisher: ProQuest Dissertations & Theses
• Subject: Ablation / Binding sites / Cell cycle / Cellular biology / Developmental biology / DNA damage / DNA methylation / Domestication / Embryos / Epigenetics / Evolution / Experiments / Gene expression / Genetics / Genomes / Insects / Mammals / Mutation / Organisms / Phylogenetics / Proteins / RNA polymerase / Roles / Stem cells / Transcription factors / Viral infections
• ISBN: 9798538130399