| Abstract |
In the past decade, numerous studies demonstrated that both microRNAs (miRNAs) and Promyelocytic Leukemia Protein (PML) were central regulators of different biological processes including cell proliferation, apoptosis and tumorigenesis. Emerging research links miRNAs as well as PML with the regulation of stem cells maintenance. Several miRNAs were reported to sustain embryonic stem cells (ESCs) identity (miR-290-295), while others to induce ESC differentiation (miR-34a, miR-145). At the same time, recent reports using p19 embryonal carcinoma cells and ESCs showed that Nanog and Oct4 – the core pluripotent regulators - associate with PML protein. Moreover, PML was described as a regulator of metabolic pathways in stem cell compartments (hematopoietic stem cells-HSCs) and as an essential component in leukemia-initiating cells maintenance. In an effort to elucidate the molecular mechanism underlying the contribution of PML and miRNAs in stemness phenotype, we examined their role in mESCs. In the first part of this work, we present four new miRNAs (miR-16-1, miR-191, miR-23a, miR-421), which are differentially expressed upon differentiation. miR-191 and miR-16-1 are highly expressed in ESCs and inhibits Activin-Nodal signaling, leading to the suppression of mesoderm and endoderm differentiation. miR-23a, which is also down-regulated in the differentiated state, represses differentiation towards the endoderm and ectoderm lineages. In contrast, miR-421 was characterized as a differentiation-associated regulator that targets the core pluripotency transcription factor Oct4 and the BMP-signaling pathway to promote endoderm and ectoderm differentiation. Our results uncover a regulatory network between the studied miRNAs and both branches of TGF-β/BMP signaling pathways unveiling their importance for ESC lineage decisions.
In the second part of this thesis, we show that PML positively correlates with the undifferentiated state and is a vital regulator of ESC pluripotency. PML knockdown induces up-regulation of multi-lineage differentiation markers and produces enhanced differentiation towards mesodermal lineage, while ectopic expression of PML prevents ESC differentiation in vitro and maintains stem cell phenotypes. Through transcriptome analysis of ESCs WT and ESC PML KD, we identified a large number of differentially expressed genes. Specifically, a group of deregulated genes is involved to the transition from naïve to primed pluripotent state, while a great number of genes associate with both signaling pathways essential for ESC identity as well as cell cycle. Additionally, reprogramming of Pml-/-mouse embryonic fibroblasts (MEFs) showed significantly lower efficiency compared to MEFs WT, highlighting PML as a pivotal mediator of somatic cell reprogramming. We speculate that the potential mechanism is based on regulation of EMT through TGF-β signaling. Our findings reveal that PML acts as an essential regulator in naïve and primed pluripotency as well as somatic cell reprogramming.
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Genetic and epigenetic regulatory networks in mouse embryonic stem cells
