Piscitelli, Silvia (2024) LIN28a modulates ESC differentiation by interacting with PRC2. [Tesi di dottorato]

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Embryonic stem cells (ESCs) are pluripotent stem cells that exhibit the ability to self-renew and differentiate into all cell types of the body. The differentiation of ESCs is regulated at multiple levels including epigenome, transcription, and post-transcriptional regulation. In recent years, new findings have increasingly recognized the significance of RNA metabolism and RNA-binding proteins (RBPs) in ESCs and early differentiation. Among the RBPs that are emerging as key regulator in ESC differentiation, LIN28a is a particularly noteworthy example. LIN28a is well known for its role in the negative regulation of Let-7 microRNA biogenesis in the late stages of differentiation and in cancer. However, many papers have highlighted a critical role of LIN28a in mRNA translation. Recently, it has been highlighted that LIN28a can bind to specific motifs within target mRNAs, exerting both positive and negative regulatory effects on the translation of numerous targets in ESCs. Our study provides evidence that LIN28a plays a crucial role in neural precursor differentiation and provides new insights into its involvement in the modulation of Polycomb Repressive Complex 2 (PRC2). We demonstrate that LIN28a expression is tightly controlled during differentiation of ESCs and that LIN28a protein accumulates and changes localization specifically during differentiation toward the neuroectodermal lineage. To investigate the potential functions of LIN28a in ESC differentiation, we generated Lin28a knockout (KO) ESCs using CRISPR-Cas9 technology. Our results show that the absence of Lin28a is compatible with self-renewal maintenance but impairs proper neuronal differentiation. We also used proteomic approaches to analyze the molecular interactome of LIN28a in undifferentiated and differentiated cells. We found that LIN28a interacts with many proteins related to chromatin organization in undifferentiated ESCs, including core and accessory proteins of PRC2. Our findings suggest that LIN28a plays a pivotal role in the eviction of PRC2 from chromatin, potentially influencing gene transcription during differentiation. We hypothesize that the absence of LIN28a hampers the eviction of PRC2 from chromatin, leading to an alteration of the differentiation program characterized by the sustained level of H3K27me3 at developmental genes. Our data highlighted a new function of LIN28a as epigenetic regulator making this protein a multifunctional player in ESC differentiation.

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