Understanding the role of the transcription factor MGA in primordial germ cell differentiation

Abstract

One of the most crucial cell lineage decisions in mammalian embryos is the differentiation of a few pluripotent epiblast cells into primordial germ cells (PGCs). PGCs are unique for their ability to mature into either sperm or egg, enabling species survival and transmitting genetic and epigenetic information. Remarkably, the spatiotemporal coordination of signal pathways induces a mesoderm T-box factor, T, to initiate the PGC gene expression program, diverging from the mesodermal formation. Although significant progress has been made in identifying the signal pathways, transcription factors, and chromatin remodelers involved in PGC specification, the precise molecular mechanisms leading to this exclusive lineage choice remain unknown. Therefore, it is becoming increasingly evident that novel factors are responsible for this critical cell fate determination and need to be identified. One ideal candidate for investigating the control of gene expression during PGC differentiation is the transcription factor MGA (Max’s giant-associated protein). MGA stands out as a unique transcription factor, harbouring three distinct domains (T-box, bHLH/Zip, DUF4801) and is associated with three diverse families of developmental proteins: T-box factors, MAX-interacting proteins, and Polycomb repressive complex 1.6 (PRC1.6). During early mouse development, MGA expression is observed in the epiblast cells of both pre- and post-implantation embryos, and its abrogation leads to early embryonic lethality. Therefore, considering MGA's structural properties and its essential role in the survival of epiblast cells, which give rise to various fetal cell lineages, including PGCs, it is reasonable to propose that MGA has the potential to influence the development of multiple tissues and exhibit tissue-specific functions. However, despite these properties, the specific role of MGA in PGC differentiation has never been explored. In this study, I aim to reveal the role of the transcription factor MGA during PGC differentiation. For this purpose, I used a well-established in vitro model that mimics early mouse PGC development by progressively differentiating embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs) and subsequently inducing the formation of PGC-like cells (PGCLCs). Then, I employed a combination of genetic and proteomic approaches to explore the function of MGA in the context of PGC differentiation. In Chapter 1, I present the main findings of my study. Using an auxin-inducible degron system, I show that depletion of MGA impairs PGCLC induction, further evidenced by a significant increase in the expression of meiotic genes and reduced expression of PGC markers. Indeed, CUT&RUN sequencing analysis from ESCs towards PGCLCs reveals that MGA dynamically binds to and thereby controls the expression of crucial genes involved in pluripotency, epiblast development, and germ cell formation. Furthermore, motif analysis indicates that MGA works in conjunction with pluripotency and T-box factors. Notably, among all MGA domains, the T-box domain is essential in regulating the expression of PGC-specific genes, as its deletion leads to their premature expression.Interestingly, the MGA interactome analysis uncovers a highly dynamic network of interaction partners during PGCLC differentiation. Indeed, despite the presence of PRC1.6 members and the confirmation of pluripotency factors only in ESCs, the data also reveal an unexpected interplay between MGA and RNA- binding proteins, suggesting that MGA may play a more complex role in regulating gene expression. In Chapter II, I investigate the structure and function of MGA's Domain of Unknown Function 4801 (DUF4801) during PGCLC differentiation. I show that deletion of the DUF4801 domain has a significant impact on MGA's canonical binding sites, resulting in the loss and gain of genes involved in neurogenesis and endoderm fate. Consequently, despite the significant alterations observed in the transcriptome of PGCLCs, the lack of a severe phenotype strongly suggests the involvement of compensatory mechanisms in response to the deletion of the DUF4801 domain. Thus, my data suggest that the DUF4801 domain may have an impact on controlling gene expression and regulating cellular processes.