Post-Transcriptional Regulation Mediated by Pumilio Proteins in Mouse Embryonic Stem Cells (mESCs)

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology

First Advisor

Lin, Haifan

Abstract

Post-transcriptional regulations play crucial roles in mammalian embryogenesis yet have not been well studied. Previously, our lab discovered that knock-out of Pumilio 1 (Pum1) and Pum2, the two murine members of the RNA-binding Pum protein family, led to developmental delays in mouse embryos starting from early blastocyst stage (embryonic day 3.5) and ultimately embryonic lethality by embryonic day 8.5. However, understanding of the mechanisms underlying these developmental defects was hindered by difficulties to distinguish the direct and secondary effects of Pum1/2 knock-out.In this thesis, I employed targeted protein degradation technologies, including the Halo-PROTAC and dTAG systems, to achieve rapid and orthogonal depletion of Pum1 and/or Pum2 in mouse embryonic stem cells (mESCs). Using this system, I performed temporally controlled Pum1/2 depletion combined with time-course RNA-seq to unambiguously identify approximately 100 direct regulatory target RNAs of Pum proteins in naïve mESCs. The eCLIP experiments further validated this direct regulation by providing high-resolution mapping of Pum1/2 binding sites on the target RNAs. Analysis of mESC differentiation trajectories revealed that dual depletion of Pum1 and Pum2 led to delayed transition from naïve to formative pluripotency, as well as unbalanced cell fate decisions, with reduced neuroectoderm (NE) differentiation and enhanced primordial germ cell (PGC) specification. Further transcriptomic analyses uncovered a key mechanism underlying these defects: Pum1/2 promote degradation of mRNAs encoding Polycomb Repressive Complex 2 (PRC2) components, including Suz12, Jarid2, Eed, and Rbbp7. Depletion of Pum1 and Pum2 resulted in increased H3K27me3 levels during mESC differentiation, indicating elevated PRC2 activity. Analysis of published ChIP-seq data showed that PRC2 and the repressive H3K27me3 marks co-localize in promoter regions of key regulators of NE specification in mESCs. Therefore, over-deposition of H3K27me3 at these genes could be one of the mechanisms underlying impaired NE differentiation in Pum1/2-depleted mESCs. Overall, this thesis establishes essential roles of Pum1 and Pum2 in pluripotency transitions and cell fate decisions of mESCs, and uncovers a novel Pum1/2-PRC2 regulation axis that governs neuroectoderm specification during early embryogenesis.

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